JPH03215498A - Protein-polysaccharides complex material - Google Patents

Abstract

PURPOSE:To obtain the subject complex material useful for foods or drugs, etc., as functional protein having a high emulsifying activity and an antibacterial activity with safety by boning protein and branched polysaccharide with an aminocarbonyl reaction. CONSTITUTION:Protein is bonded to branched polysaccharide with an aminocarbonyl reaction to afford the objective complex material. Besides, e.g. dextran is used as the polysaccharide and allowed to react preferably in a condition of 50-80 deg.C and 60-80% relative humidity for 2-6 week.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a protein-polysaccharide complex in which a protein and an unactivated branched polysaccharide are bonded by an aminocarbonyl reaction.

[Problems to be solved by conventional techniques and inventions] Conventionally,
Chemical modification of proteins (J, Agric. Food Ch
em. +33, 125 (1985)) and enzyme modification (A
gric. Biol. Chew. ,50. 302
5 (1986)) or denature the protein by heating etc. (Agric. Biol. Che+w
．． ．． 45+ 2775 (1981)), many studies have been conducted to improve the functionality of proteins.

However, conventional chemical modifications, such as acylation, alkylation, amidation, deamidation, and esterification of proteins, as well as the binding of carbohydrates and fatty acids to proteins, involve the use of drugs, so these methods are not recommended from a safety perspective. There are problems in using proteins obtained by chemical modification as food raw materials.

In addition, although proteins generally have emulsifying activity, they become insolubilized when denatured by treatments such as heating, resulting in quality defects when used as emulsifiers and the like.

The present inventors took into account the current situation that most of the chemical and enzymatic modifications that have been carried out to date have been attempts to bind low-molecular substances, and also Since low-molecular-weight emulsifiers such as esters had the disadvantage of being easily affected by pn and salt concentration, in order to obtain excellent polymer-based emulsifiers, high-molecular substances such as Polymer Ii were bonded to proteins. As a result, the branched multi-I! It was discovered that a functional protein that is water-soluble and has excellent emulsifying properties can be obtained by combining these molecules. (Unexamined Japanese Patent Publication No. 1-233300; J,
Agric. Food Chew. 3β,,
42N198B); Agric. Biol.
CheII1. (Vis., 2147 (1989)) However, this method has a branched polymorphism. Since it is essential to activate these compounds, chemicals such as cyanogen bromide, sodium periodate, and cyanuric chloride are used as activators.
As mentioned earlier, safety issues remained.

In addition, the obtained functional protein had a wide molecular weight distribution, making it difficult to standardize the product specifications.

An object of the present invention is to solve the problems of these conventional techniques and to provide a functional protein that is safe and has excellent emulsifying properties.

[Means to solve the problem]

As a result of further research in order to solve the above problems, the present inventors have found that branched polyIJi and proteins undergo aminocarbonyl reaction without activation treatment with drugs that are problematic in terms of safety. The complex is formed through a very naturally occurring and safe reaction, and the resulting complex has excellent emulsifying properties, exhibiting higher emulsifying activity than complexes obtained by activation treatment with drugs, and has a low molecular weight distribution. Also, when lysozyme is used as a protein, it has no antibacterial activity against Durham-negative bacteria, but by making it into the complex of the present invention, it has antibacterial activity against Durham-negative bacteria. The present invention was completed based on the discovery that a new property of expression was also added.

That is, the present invention relates to a protein-polysaccharide complex in which a protein and a branched polysaccharide are bonded together through an aminocarbonyl reaction.

More specifically, it relates to the above complex having emulsifying activity and/or antibacterial activity.

The present invention will be explained in more detail below.

The protein referred to in the present invention may be of either animal origin or plant origin, and includes, for example, ovalbumin, lysozyme, milk protein, fish protein, soybean protein, wheat gluten, and the like.

Examples of branched polyol group II include dextran, dextrin, bullulan, xanthan gum, carrageenan,
Examples include chondroitin sulfate.

These branched polysaccharides do not require activation treatment;
It can be directly subjected to an aminocarbonyl reaction to form a complex with a protein.

The aminocarbonyl reaction in the present invention is carried out, for example, as follows. Protein and branched polysaccharide are mixed in appropriate proportions to form an aqueous solution, which is then freeze-dried. The conditions for the aminocarbonyl reaction of the obtained powder differ depending on the individual protein, but in general, the temperature is 50-80°C and the relative humidity is 60-80°C.
% conditions for every 2 to 6 weeks, the complex of the present invention is formed.

If the reaction conditions are less than 50°C or relative humidity less than 60%, the aminocarbonyl reaction is difficult to occur;
At high temperatures exceeding , browning occurs rapidly, which causes a decrease in the emulsifying activity and antibacterial activity of the protein. Moreover, when the relative humidity exceeds 80%, sootiness occurs and the stability of quality is undesirable. If the reaction is carried out for less than 2 weeks, the reaction will not proceed sufficiently, and if the reaction is carried out for more than 6 weeks, the emulsifying activity of the complex will not improve.

The powder reacted under the above conditions was made into an aqueous solution, and 470n
If the progress of the browning reaction can be confirmed by measuring the absorbance at m, the composite of the present invention can be obtained. Further, the present complex can be used after being concentrated by gel filtration or the like, if necessary.

The thus obtained complex of the present invention is soluble in water and has a higher emulsifying activity than one in which a branched polysaccharide activated by a drug is bound. In addition, it has superior salt resistance and acid resistance in warm conditions compared to existing food emulsifiers, and also has the advantage that its emulsifying properties are improved in alkaline solutions and when heated.

Furthermore, when lysozyme was used as a protein, it had a new function in that by making it into a complex, antibacterial properties against Durham-negative bacteria could not be obtained by using it alone.

〔Effect of the invention〕

According to the present invention, the protein-polysaccharide complex obtained is safe because the aminocarbonyl reaction is used without using any drugs when obtaining the protein-polysaccharide complex.
It can be applied to foods, medicines, cosmetics, etc. Furthermore, the protein-polysaccharide complex of the present invention has excellent acid resistance and salt resistance as well as heat resistance compared to conventional emulsifiers, so it can be used extremely advantageously as an emulsifier. The obtained composite has the advantage that the molecular weight distribution is narrow and the product specifications can be easily matched. In particular, when the protein is lysozyme, the protein-polysaccharide complex appears to have antibacterial activity against Durham-negative bacteria as well, whereas lysozyme alone had no antibacterial activity against Gram-negative bacteria. This invention is groundbreaking in this respect as well, and the present invention will make a significant contribution to the fields of food, medicine, cosmetics, etc. mentioned above.

Next, the present invention will be explained in detail using examples.

(Example) Example 1 Preparation of ovalbumin-dextran complex A mixture of ovalbumin and dextran (manufactured by Wako Pure Chemical Industries, Ltd.; average molecular weight 75,000) (weight ratio 1:1 and l:
5) was dissolved in 100 Id of water per 1 g of egg white albumin, stirred for 30 minutes, and then freeze-dried. The obtained powder was placed in a glass Petri dish and heated at 50'C or 60°C in a desiccator adjusted to approximately 65% humidity with a saturated potassium iodide solution.
The reaction was carried out by keeping the mixture at °C for 3 weeks. As a control, a mixture using glucose instead of dextran (weight ratio 1:1) was similarly prepared.

The obtained complex was dissolved in distilled water, and the absorbance at 470 nm was measured to confirm the progress of the browning reaction. The results are shown in FIG.

From Figure 1, the ovalbumin-glucose mixture is 50%
When reacting with 'C, browning progresses rapidly, whereas
The egg white albumin-dextran mixture did not turn slightly brown under the same conditions, indicating that the aminocarbonyl reaction did not proceed. On the other hand, browning was observed in the ovalbumin-dextran mixture (weight ratio 1:5) reacted at 60°C, indicating that a complex was formed due to the aminocarbonyl reaction.

Example 2 Comparison of emulsifying properties of ovalbumin-dextran complex and oval albumin-glucose complex The emulsifying properties were determined by the method of Pearce et al.
Food. Chem. , 26. 716-723 (19
78)).

That is, oil 1. 0 d and 0.1χ (
w/v) 12000r after shaking the aqueous solution 3.0 well.
Homogenize at pI1 for 1 minute at 20"C to obtain an emulsion. 50 μl or 1
00μ! Take, 0. Diluted with 1χ SDS aqueous solution 5,
The absorbance at 500n+m was measured. The absorbance measured immediately after emulsification was defined as the emulsifying activity, and the emulsifying stability was evaluated based on the half-reduction of the absorbance.

FIG. 2 shows the results of measuring the emulsifying activity of the three types of complexes obtained in Example 1 and ovalbumin alone maintained at 60°C. From FIG. 2, the ovalbumin-dextran mixture obtained by the aminocarbonyl reaction had significantly higher activity than the ovalbumin-glucose mixture and ovalbumin alone. In particular, the ovalbumin-dextran complex reacted by maintaining it at 60°C for 3 weeks at a weight ratio of 1:5 had high activity.

Example 3 Preparation of various protein-polysaccharide complexes Example 1
An ovalbumin-dextrin complex was prepared in the same manner as in Example 1 except that dextran was replaced with dextrin (the reaction was maintained at a relative humidity of 65χ and a temperature of 60'C for 3 weeks). I got a body.

In addition, a soybean protein-dextran complex and a lysozyme-dextran complex were prepared in the same manner as in Example 1 except that the egg white aluminium in Example 1 was replaced with soybean protein and lysozyme, and the complex of the present invention was obtained. .

Example 4 Purification of ovalbumin-dextran complex and confirmation of complex formation Ovalbumin-dextran complex, which had the highest emulsifying activity in Example 2 (weight ratio 1:5, 3 weeks at 60°C and 65χ relative humidity) Sephacryl S-300 (70X3cm) to further purify the reaction)
Figure 3 shows the results of gel filtration using .

Elution was performed using 50mM acetate buffer (pH 5.0) containing 10n+M NaCl at a flow rate of 0.36ml per minute; 3. It was fractionated in Oml portions.

In FIG. 3, the solid line shows the absorbance at 280 nm (absorption of protein), and the broken line shows the absorbance at 470 nm (absorption of tJ!) after color development using the phenol-sulfuric acid method. The elution position of untreated ovalbumin is shown by an arrow, but the elution position of ovalbumin in the complex of the present invention is shifted to the polymer side compared to the elution position of ovalbumin alone, and this position is also the elution position of dextran. The fact that they almost matched and the two? It was confirmed that an ovalbumin-dextran complex was formed because the release patterns were similar.

Fraction numbers 26-36 in FIG. 3 were dialyzed against deionized water and then freeze-dried to obtain the ovalbumin-dextran complex of the present invention. In order to further confirm the formation of a complex, SDS-polyacrylamide gel electrophoresis was performed, and the results are shown in FIG.

Electrophoresis was performed at 0. Laemmli's method (Nature, 227, 680 (1970)) using a 10χ acrylamide separation gel containing 1χ SDS and a 3χ fixation gel.
). Protein sample (20μf, 0.1χ
) was prepared with a tris-glycine buffer (pH 8.8) containing 1χ SDS and 1χ mercaptoethanol, and electrophoresis was carried out at 0. Using a tris-glycine migration buffer containing 1xSDS, the electrophoresis was carried out at a constant current of 10 mA for 5 hours. The gel was stained with Coomassie Pul 10-250 and fuchsin for carbohydrate staining.

From Figure 4, the ovalbumin-dextran complex shows a single band of protein and carbohydrate staining near the boundary between the fixation gel and separation gel, indicating that ovalbumin is covalently bound to dextran. Recognize. Furthermore, fraction 1 (fraction numbers 26 to 30 in Figure 3) is fraction 2.
(Fraction numbers 31 to 36 in Figure 3) showed a sharper band. On the other hand, the white albumin-dextran complex prepared using dextran activated with cyanogen bromide showed a broad band of polymers in the immobilized gel, indicating that it had a wide molecular weight distribution. This also shows that the composite of the present invention has the advantage that specifications can be more easily met than those using cyanogen bromide or the like. Subsequently, the average molecular weight of the white albumin-dextran complex of the present invention was determined using a low-angle light scattering technique coupled with high-performance liquid chromatography. (J. Hachigric. Food.
Chem. 36, 421 (1988)) As a result, the average molecular weight was about 200,000, and this value was determined by the weight ratio of the binding ratio of ovalbumin to dextran in this complex: l:3, molar ratio t: 1.6-2. It is in good agreement with the estimated value obtained from 2.

Furthermore, the average molecular weight of the zozyme-dextran complex prepared in Example 3 is approximately 150,000, the binding ratio (molar ratio) of lysozyme and dextran is 1:2, and the average molecular weight of the soybean protein-dextran complex is approximately 220,000 to 230,000, and the binding ratio (molar ratio) was 1:1.

Example 5 Comparison of emulsifying properties between the rabbit albumin-dextran complex of the present invention and the ovalbumin-dextran complex activated with cyanogen bromide Fraction of the egg white albumin dextran complex of the present invention fractionated in Example 4 Figure 5 shows the results of measuring the emulsifying properties of Fraction 1, Fraction 2 (Figure 3) and the egg white albumin-dextran complex bound with dextran activated with cyanogen bromide using the same method as in Example 2. .

The complex of the present invention had several times higher emulsifying activity than the complex bound to dextran activated with cyanogen bromide. This also shows that the composite of the present invention has strong emulsifying power. Moreover, the activity of a simple mixture was as low as that of egg white albumin alone.

Furthermore, the absorbance half-life of ovalbumin alone is approximately 3
0 seconds, and about 1 minute for the complex bound to dextran activated with cyanogen bromide, while for the complex of the present invention it takes about I
The emulsion stability was also excellent. Furthermore, the absorbance half-life of three commercially available emulsifiers (see Example 8) was approximately 5 minutes.

Incidentally, since there was almost no difference in emulsifying properties between Fraction 1 and Fraction 2, they were used together from now on.

Example 6 Effect of pl+ on emulsification properties The effect of pl+ on the emulsification properties of the ovalbumin-dextran complex purified in Example 4 was investigated using 1/15M phosphate buffer (pH 7.4) as the aqueous phase and 1/15M phosphate buffer (pH 7.4) as the aqueous phase. 15M carbonate buffer (pH
10), l/15M citrate buffer (pl+3)
FIG. 6 shows the results of measurements using three types.

From Figure 6, the excellent emulsifying properties of this complex remain stable even in an acidic solution of p113, and are further improved in an alkaline solution of p1+10, the emulsifying activity is approximately doubled, and the half-life of absorbance is extended. Emulsion stability was also increased. The superiority of this composite can also be seen from the fact that most commercially available emulsifiers have extremely low activity in acidic solutions.

Moreover, this effect appeared similarly for all the protein-polysaccharide complexes prepared in Example 3.

Example 7 Effect of heat treatment on emulsification properties The white albumin-dextran complex purified in Example 4 was
Heated at °C and compared the emulsifying properties with the untreated one.
Shown in the figure. From FIG. 7, it can be seen that when this complex is heated at 100°C, the emulsifying activity increases about 1.6 times, and the half-life of absorbance, which indicates emulsion stability, is also clearly extended. No protein precipitation due to heat denaturation was observed, and the above results indicate that this complex has the advantage of being heat sterilized and usable.

Furthermore, all of the protein complexes prepared in Example 3 had similar effects.

Example 8 Comparison of emulsifying activity with commercially available emulsifiers The emulsifying activity of aqueous solution, acid resistance, and salt resistance was measured using the method used in Example 2, and the results of comparison with three types of commercially available emulsifiers are shown in Table 1. Both were used as 0.1x (w/v) aqueous solutions, and for acid resistance, acetic acid was added for 1x, and for salt resistance, NaCl was added for 3x and emulsified. As a commercially available emulsifier, Quillayanin C-
100 (manufactured by Maruzen Kasei), monoglyceride (manufactured by Taiyo Kagaku), sucrose fatty acid ester S-1570 (manufactured by Mitsubishi Kasei)
The protein-multi-Ig complex of the present invention is the ovalbumin-dextran complex and the soybean protein-dextran complex prepared in Example 1 and Example 3 at a weight ratio of 1:5.60''. C, 3-week reaction) was used.

(Margins below the text) As described above, it can be seen that the protein-polysaccharide complex of the present invention has better acid resistance and salt resistance than commercially available emulsifiers.

Example 9 Antibacterial activity of lysozyme-dextran complex The antibacterial activity of the lysozyme-dextran complex prepared in Example 3 was measured by antibacterial spectrum, and the results are shown in Table 2. The bacterial species used were 8. hydro old 1a
, V. parahaemolyticus, E.
All three types of coli are Durham-negative bacteria.

For each bacterium, 100 ppm of lysozyme and lysozyme-dextran complex (calculated as lysozyme) was added to an agar medium having the composition shown in Tables 2 to 4, and incubated at 30°C for 30 minutes.
Cultured for 1 day.

Table 5 shows the antibacterial activity against each bacteria.

- in the table indicates that no antibacterial effect was observed, ++
indicates that the antibacterial effect was particularly strong.

Table 2 A. Hydrophila culture medium Table 3 ■. parahaemolyticus Table 4 E, coli medium ^. byrophila V, parahaemolyticus {+ ++ E. coli ÷+ As mentioned above, lysozyme alone does not show antibacterial activity against Durham-negative bacteria for any bacterial species, but it has been shown that lysozyme exerts antibacterial activity by binding dextran through an aminocarbonyl reaction. Recognize.

Thus, it is presumed that the aminocarbonyl reaction forms a protein-polysaccharide complex, which has emulsifying activity and has a structure that easily binds to the cells of Durham-negative bacteria.

That is, it is thought that the lysozyme-dextran complex binds to the povosaccharide on the outer wall of the Durham-negative bacterial cell, destabilizing the outer wall lipopolysaccharide and destroying the bacterial cell.

Example 10 Bread making effect of egg white albumin-dextran complex When bread was baked using the egg white albumin-dextran complex prepared in Example 1 at the mixing ratio shown in Table 6 in a conventional manner in a home bakery, Compared to the control, the volume of the bread increased significantly, resulting in fluffy baked goods.

(Margins below this page) Table 6 Bread composition ratio Strong flour 140g Sand it! 8.5g skim milk 3g salt 2.5g hatah
5.5g water 105d dry yeast 1.5g egg white albumin dextran complex of the present invention 0.2 g Example 11 Production of dressings to which egg white albumin-dextran complex was added When a dressing was prepared using a conventional method using the compounding ratio shown in Table 7, a dressing with emulsifying properties that was highly stable against acids and salts and difficult to separate was obtained.

Table 7 Dressing ratio water 40χ sand $J!
15χ Vinegar 15χ Vegetable oil 24χ Xanthan gum 0.2χ Salt 5χ Ovalbumin-dextran complex of the present invention 0.3χ Sodium glutamate 0.5χ Example 12 Production of mayonnaise containing lysozyme-dextran complex Lysozyme-dextran prepared in Example 3 When mayonnaise was prepared using the complex according to the conventional method at the mixing ratio shown in Table 8 and stored at room temperature for one week, spoilage was observed in the control, but no spoilage was observed with the lysozyme dextran complex of the present invention. .

Table 8 Mayonnaise blending ratio Oil 72χ Egg yolk
10χ Vinegar 12χ Salt 1.5χ Mustard powder 0.2χ Water 4.0χ Lizuzyme dextran complex of the present invention 0.3χ [Effects of the invention] According to the present invention, branches that have not been activated by proteins, drugs, etc. A protein that has excellent emulsifying activity is produced by combining Shata IHR with aminocarbonyl reaction! This protein-polysaccharide complex is excellent in terms of safety and can be suitably used in the production of foods, medicines, cosmetics, etc. It also has antibacterial activity, so it can be used as a preservative.

[Brief explanation of drawings]

Figure 1 shows the degree of browning of the ovalbumin-carbohydrate mixture during the aminocarbonyl reaction, Figure 2 shows the change in emulsifying activity of each ovalbumin carbohydrate mixture during the aminocarbonyl reaction, and Figure 3 shows the degree of browning of the ovalbumin-carbohydrate mixture during the aminocarbonyl reaction. Figure 4 shows the elution pattern of the ovalbumin-dextran complex reacted for 3 weeks on a Cephacryl S-300 column, Figure 4 shows the SDS-polyacrylamide electrophoresis of the ovalbumin-dextran complex, and Figure 5 shows the elution pattern of the ovalbumin-dextran complex reacted for 3 weeks. Figure 6 shows the effect of pH on the emulsifying properties of the ovalbumin-dextran complex.
FIG. 7 is a diagram showing the influence of heat treatment on the emulsification properties of the ovalbumin-dextran complex.

Claims (1)

[Scope of Claims] 1. A protein-polysaccharide complex in which a protein and a branched polysaccharide are bonded together by an aminocarbonyl reaction. 2. The protein-polysaccharide complex according to claim 1, which has emulsifying activity. 3. The protein-polysaccharide complex according to claim 1, which has antibacterial activity.