JPH02968B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an emulsifier comprising a polypeptide obtained by decomposing whole casein or αs-casein and/or β-casein with a protease. Traditionally, fatty acid monoglycerides, sorbitan fatty acid esters, egg yolk, lecithin, alginic acid, gelatin, and other proteins have been used as emulsifiers in the production of emulsions, and phosphates (molten salts) have also been used as emulsifiers in the production of processed cheese. It is used as. However, commonly used monoglyceride emulsifiers are not suitable for producing food emulsions from the viewpoint of flavor. Furthermore, protein-based emulsifiers generally have a weak emulsifying power and have the disadvantage of impairing flavor. There is a report that a partial hydrolyzate of casein is used as a protein emulsifier (Japanese Unexamined Patent Publication No. 1983-95747, page 1), but this report does not use the peptide itself, which has a specific number of amino acids. There is no mention of its use as an emulsifier, nor is there any suggestion of emulsifying properties of the polypeptide. There are also reports on the use of enzymatically decomposed products of soybean protein as emulsifiers (JP-A-55-39725, JP-A-56-26171, and JP-A-56-42555); The flavor is bad, and the nutritional value is not always satisfactory. In addition, since soybean protein is relatively rigid, it has the disadvantage that it is not susceptible to enzyme action unless it is heated or denatured with alcohol in advance. The present invention has been made in view of the current situation as described above, and provides an emulsion product that does not impair the flavor of emulsion products, has no nutritional defects, and has excellent emulsion stability, especially emulsion stability during heating. The purpose is to provide emulsifiers. Incidentally, the term "emulsion" used here refers to oil-in-water (O/W), water-in-oil (W/O) emulsions, and double emulsions. Examples include foods such as cheese, soups, sauces, creams, juices, mayonnaise, dressings, and spreads, as well as shampoos, cosmetics, liquid nutritional supplements, etc.
Examples of W/O emulsions include foods such as margarine and butter. Therefore, the emulsifier according to the present invention can be applied to the production of these emulsion products. The present invention will be explained in detail below. The emulsifier according to the present invention is made of whole casein or αs
- A reaction mixture obtained by treating casein and/or β-casein with a protease is separated by applying one or more methods such as gel filtration, ion exchange chromatography, high performance liquid chromatography, or electrophoresis. It is characterized by consisting of a polypeptide composed of 5 to 50 amino acids. Casein used as a starting material for preparing the emulsifier of the present invention is different from the above-mentioned soybean protein,
Because it has a relatively loose higher-order structure, it can be used without pretreatment such as heating or alcohol treatment.
It has the advantage of being easily subject to enzymatic degradation even when raw. The above-mentioned whole casein as a starting material is whole milk, skim milk, or acid casein obtained by adjusting the pH of skim milk to around 4.6, or lactic acid casein obtained by fermentation with lactic acid bacteria. Casein can be obtained by fractionating it, for example, by the urea method [for αs-casein, see Zittle et al, J.Dairy
Sci.46, 1183 (1963), for β-casein
Hipp et al, J.Am.Chem.Soc.74, 4822 (1952)
reference〕. Among these raw materials, from the viewpoint of separation and purification of polypeptides obtained by enzymatic decomposition, it is preferable to use αs-casein and/or β-casein obtained by pre-fractionation from whole casein. In the present invention, a polypeptide composed of 5 to 50 amino acids obtained by decomposing whole casein or αs-casein and/or β-casein with a protease is used as an emulsifier. The proteolytic enzyme used is not particularly limited and a wide variety of enzymes can be used, such as chymosin, papain, pepsin, plasmin,
Includes trypsin, α-chymotrypsin, peptidase derived from lactic acid bacteria, and the like. In addition, in order to impart high emulsifying power to the peptide obtained by the above-mentioned enzymatic degradation, the balance between hydrophilicity and hydrophobicity is important. It is preferable to use an enzyme with a certain degree of specificity in terms of decomposition as a proteolytic enzyme, rather than one that enzymatically decomposes the protein randomly. Examples of such enzymes include chymosin and pepsin. In the present invention, the conditions under which these proteolytic enzymes act on the above-mentioned starting materials vary greatly depending on the type of enzyme used, but generally in the case of αs-casein, the conditions are 0.1 to 20% by weight for αs-casein. The enzyme concentration is 1/2 to 1/50,000, the pH is 2 to 12, and the reaction temperature is 3 to 60°C for 1 minute to 1 week. For example, when using chymosin (rennet) as the enzyme and αs-casein as the starting material, αs
- Casein concentration 1.5% by weight, rennet concentration 0.4
It is preferable to act for 3 hours at a pH of 6.4 (weight%) and a temperature of 30°C. Incidentally, by this enzymatic decomposition, more than 80% of αs-casein produces a polypeptide having 23 to 24 amino acids from the N-terminus, and the remainder produces αs-I casein. The degree of decomposition when a proteolytic enzyme is applied to αs-casein can be simply expressed as:
It can be measured by quantifying the amount of nitrogen soluble in TCA (trichloroacetic acid), but a more accurate method is the electrophoresis method by Hill et al [J.Dairy
Res. 41: 147 (1974)] to analyze the migration pattern of the enzymatic degradation reaction mixture, and this measurement makes it possible to know what percentage of αs-casein has been degraded into what kind of peptides. . To separate the desired polypeptide from the reaction mixture obtained by the enzymatic digestion described above, gel filtration,
Techniques such as ion exchange chromatography, high performance liquid chromatography, and separation electrophoresis may be applied, or these techniques may be combined. The polypeptide used in the present invention is an isolated polypeptide consisting of 5 to 50 amino acids, preferably 10 to 30 amino acids, as described above, and has a well-balanced composition of hydrophilic and hydrophobic groups. is preferable. In other words, the balance between hydrophilicity and hydrophobicity within a polypeptide is important in order to increase the emulsifying power of a polypeptide, and in order to impart this balanced amphipathic structure to a polypeptide, the number of amino acids must be increased from 5 to 5.
It is necessary to make it 50 pieces. In other words, if the number of amino acids is less than 5 or more than 50, it will be difficult to maintain the above balance. Polypeptides with hydrophilic and hydrophobic amphipathic properties are similar to known emulsifiers, with hydrophobic groups binding to fat globules, while hydrophilic groups exit into water to form a stable emulsion. Become. Next, the emulsifying properties of the polypeptide in the present invention will be described. The emulsifying property of O/W emulsion for cheese is exemplarily shown. 25 g of dry powder of a polypeptide having 23 (or 24) amino acids prepared as shown in the example below for 1 kg of cheddar cheese with a maturity of 4 months crushed using a chiyotsupar.
A mixture of dissolved in 180 ml of water is supplied to an emulsifying pot, emulsified by stirring at 800 rpm at 40°C, and then heat sterilized (at 90°C for 30 minutes) to obtain a stable emulsion. I looked into gender. For comparison, processed cheese was obtained in the same manner using phosphate (molten salt) and soda casein, which are conventionally used as emulsifiers, instead of polypeptide, and their emulsion stability was also investigated. As a result, when polypeptide was added, approximately 3
After stirring for several minutes, the fat in the cheese was uniformly dispersed in the protein-water phase, resulting in a stable emulsified system, but in the case of the cheese containing soda and casein, no matter how much stirring was continued, the fat separated by heating remained. It did not disperse or emulsify in the protein-aqueous phase. Furthermore, when phosphate was added, an emulsified system was obtained after stirring for about 4 minutes. Next, the oil-off value of cheese prepared by adding polypeptide and phosphate was measured by the following method, and the results were as follows. Measurement of oil-off value: Place the cheese sample on paper and place it in a constant temperature bath at 30°C (humidity
The measured value is the fat leaching area/sample area in the cheese sample after being held at 90%) for 24 hours. The smaller this number is, the better the emulsion stability is. Oil-off value Cheese added with polypeptide 1.2 Cheese added with phosphate 2.3 As seen in the above results, the emulsion stability of the processed cheese prepared using the polypeptide according to the present invention as an emulsifier is higher than that of the processed cheese prepared using the conventional method. Remarkably superior. The emulsifier of the present invention can be applied to emulsify various oils and fats in addition to the milk fat mentioned above, such as vegetable oils such as coconut oil, safflower oil, castor oil, cottonseed oil, corn oil, palm oil, palm kernel oil, rapeseed oil, soybean oil,
Peanut oil, sunflower oil, olive oil, cocoa butter, etc., and animal oils such as lard, head, mutton tallow, and whale oil. In order to use the polypeptide as an emulsifier of the present invention to emulsify these oils and fats, the polypeptide is dissolved in water, added to the oil and fat before the emulsification operation, and an emulsion is formed by applying an emulsifier such as a high-speed emulsifier. .
The amount of polypeptide added varies depending on the type of fat or oil to be emulsified, but is usually in the range of 0.01 to 30% by weight based on the fat or oil. For example, in the production of processed cheese, when the raw material cheese is semi-hard, it is 0.2 to 30% by weight, preferably 1 to 10% by weight of the cheese.
is within the range of As mentioned above, the emulsion prepared using the polypeptide of the present invention as an emulsifier has excellent emulsion stability, and in particular has good stability against heat, so when applied to food processing, the emulsion can be easily destroyed during the sterilization process. This method is particularly advantageous for improving the quality and hygiene of emulsion foods because phase conversion does not occur and the high-temperature emulsion can be directly filled into containers. In addition, polypeptides do not adversely affect the flavor of the product even when used in significant amounts, and have the advantage of being nutritionally enriched rather than nutritionally disadvantageous. Examples are shown below. Example 1 Preparation of polypeptide: 15g of αs-casein in 0.1M acetic acid buffer (PH6.4)
1 and heated to 30°C. After adding 0.7 mg of rennet to the resulting solution and reacting for 3 hours,
Rennet was inactivated by heating at 80°C for 10 minutes.
After the resulting reaction solution (enzyme digestion solution) was cooled to room temperature, about 2 g of a polypeptide having the primary structure shown below was obtained from the reaction solution by applying gel filtration and ion exchange chromatography. Arg−Pro−Lys−His−Pro−Ile−Lys−His
−Gln−Gly−Leu−Pro−Gln−Glu−Val−Leu
−Asn−Glu−Asn−Leu−Leu−Arg−Phe(−
Phe) Next, the application of the thus obtained polypeptide to various emulsified foods will be illustrated. Emulsifiability of polypeptide in margarine: Using the polypeptide obtained above as an emulsifier, margarine having the formulation shown in Table 1 below was produced in a conventional manner using a votator. For comparison, margarine was produced in the same manner using monoglyceride and lecithin as emulsifiers instead of the above polypeptide, and the emulsion stability of both margarines was examined.

【table】

[Table] The average diameter of water droplets in the dispersed state of both margarines was measured using a microscope and the results are as follows. Present invention 1.9μ Comparison example 4.3μ The above average diameter of water droplets indicates the state of emulsion stability of margarine, and the smaller the diameter value, the better the emulsion stability. Emulsifying property of polypeptide in cream: Using 1% by weight of the polypeptide obtained above as an emulsifier, a cream having the formulation shown below was manufactured according to the following procedure. For comparison, creams were produced in the same manner without the addition of an emulsifier and using 1% by weight of soda casein (Na-casein) and 0.4% by weight of sucrose fatty acid ester (DKF160) in place of the polypeptide. Cream composition: Salad oil 34 (wt%) Skimmed milk powder 4 Water 66 Cream manufacturing procedure: Skim milk powder of the above composition is dispersed in water with each emulsifier and heated to 60℃, followed by salad oil heated to 60℃. Using a Polytron, add the mixture to
The cream was formed by stirring at a rotation speed of 1000 rpm. The size of oil droplets in each of the obtained creams was observed under a microscope to examine the emulsification state. The results are shown in Table 2.

[Table] As shown in Table 2, the size of oil droplets in the casein using soda casein was as large as in the casein without emulsifier, and the emulsification state was poor, but in the casein using the polypeptide according to the present invention, the oil droplet size was as large as in the casein without emulsifier. The oil droplets are so small and the emulsification state is good that it is comparable to the case of currently used sucrose fatty acid esters. Emulsifying properties of polypeptides for cheese-like products: The emulsifying properties of polypeptides when producing cheese-like products by adding butter oil to natural cheese were investigated as follows. 1.5 of the polypeptide in a formulation with the composition shown below.
Add 29% by weight of the % solution and 23% by weight of the 3.8% solution, and cut the mixture at 40°C using a food cutter to approx.
The mixture was emulsified by stirring at 2000 rpm, and the emulsification state of the resulting emulsion was examined. Composition of raw material formulation: Fat 68 (wt%) Fat 13% by weight from natural cheese Fat 55% by weight from butter oil Protein 13 Ash 2.3 Other 0.5 Water 16.2 Results show that 29% by weight of a 1.5% solution of polypeptide was added The size of the fat globules in the emulsion was 2.4μ, and the size of the fat globules in the emulsion with 23% by weight of a 3.8% polypeptide solution was 1.9μ. In addition, in a control in which no polypeptide was added, butter oil was separated and no emulsion was obtained. Example 2 Preparation of polypeptide: 20 g of β-casein was dissolved in 0.01M phosphate buffer 1 of pH 6.4, 0.4 g of pepsin was added thereto, and the mixture was reacted at 37° C. for 10 hours. As a result of adjusting the pH to 4.6 with hydrochloric acid, a precipitate was generated, which was removed by centrifugation. Adjust the pH of the supernatant liquid with sodium hydroxide.
After adjusting the temperature to 7.8, 10 mg of trypsin was added, and the reaction was further carried out at 37°C for 10 hours. Next, the enzyme was inactivated by heating at 80°C for 10 minutes, and then cooled to room temperature. DEAE the obtained decomposition liquid
- The solution was passed through a Sephacel column and subjected to ion exchange chromatography using a concentration gradient of sodium chloride to obtain a fraction containing a polypeptide having the structure shown below. Val-Leu-Pro-Val-Pro-Glu-Lys In addition, as a result of measuring the peptide concentration by amino acid analysis, the concentration of the above peptide was 0.02%. Measurement of emulsifying activity: The fraction containing the above polypeptide was concentrated under reduced pressure at 70°C to a concentration of 2%, and then the pH was reduced to 3 with hydrochloric acid.
Adjusted to. Add soybean oil to a final concentration of 20% and use a high-speed homogenizer (Polytron).
Homogenization was carried out for 3 minutes using the highest rotation speed. Immediately, it was diluted with a 0.1% SDS solution, the turbidity at 500 nm was determined, and it was expressed as emulsifying activity (m ² /g). The more uniformly the fat globules are dispersed, the higher the emulsifying activity is. As a comparative example, undegraded β-casein was used instead of the above polypeptide. The emulsifying activity at PH3 is 7.3 m ² /g for undegraded β-casein.
whereas for polypeptide it was 10.4m ² /g.
It had excellent emulsification ability under acidic conditions. Example 3 Preparation of polypeptide: 1 kg of lactic acid casein was dispersed in 10 parts of water and dissolved while adjusting the pH to 6.4 with sodium hydroxide. After adding 7 g of chymosin to this and reacting at 42° C. for 10 minutes, CaCl ₂ was added to give a concentration of 70 mM. The supernatant liquid 8 obtained by removing the resulting curd was heated at 70°C for 30 minutes to inactivate chymosin, and then cooled to 15°C. Next, hydrochloric acid was added to adjust the pH to 2, 1 g of pepsin was added, and the mixture was reacted for 20 minutes. After returning the pH to 7 with sodium hydroxide, the mixture was heated at 80°C for 10 minutes to deactivate oxygen. Until the liquid level reaches 2
The residue was concentrated under reduced pressure at 70°C and passed through a Q-Sepharose column. Ion exchange chromatography using a concentration gradient of sodium chloride was performed, and the eluate was desalted by gel filtration using Sephadex G-15 and freeze-dried. In this way, 8 g of a polypeptide having the structure shown below was obtained. Met−Ala−Ile−Pro−Pro−Lys−Lys−Asn
−Gln−Asp−Lys−Thr−Glu−Ile−Pro−Thr
−Ile−Asn−Thr−Ile−Ala−Ser−Gly−Glu−
Pro−Thr−Ser−Thr−Pro−Thr−Ile−Glu−
Ala−Val−Glu−Ser−Thr−Val−Ala−Thr−
Stability of the Leu-Glu-Ala-Ser-Pro-Glu-Val polypeptide in dressing: Using the polypeptide obtained above as an emulsifier, a dressing was prepared with the formulation shown in Table 2, and its emulsion stability was investigated. Ta. In addition, as a comparative example, whole casein was blended instead of polypeptide. Table 3 Weight% Water 27.1 Vinegar 6.5 Cornstarch 3.1 Salt 1.0 Sugar 6.2 Seasoning 0.6 Polypeptide 5.5 Rapeseed oil 50 Total 100 An aqueous solution of the formulation shown in Table 3 without rapeseed oil was heated to 90°C, cooled to room temperature, and homogenized. Homogenized with a mixer. Next, rapeseed oil was slowly added dropwise to this, and the mixture was homogenized again. This was incubated in warm water at 60° C. for 1 hour and centrifuged at 2800 _×g for 10 minutes. The separated hot water was discarded. Emulsion stability was expressed as (weight after centrifugation)/(weight before centrifugation)%. It was 87.4% for total casein, while it was 94.2% for the above polypeptide.

Claims (1)

[Scope of Claims] 1. Obtained by separating whole casein from a reaction mixture obtained by acting on a protease using one or more of gel filtration, ion exchange chromatography, high performance liquid chromatography, or electrophoresis. An emulsifier consisting of a polypeptide composed of 5 to 50 amino acids. 2. Gel filtration, ion exchange chromatography,
An emulsifier consisting of a polypeptide composed of 5 to 50 amino acids obtained by separation using one or more of high-performance liquid chromatography or electrophoresis. 3. The emulsifier according to claim 1 or 2, wherein the polypeptide is composed of 10 to 30 amino acids.