JPH0358718B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides casein phosphopeptides (casein phosphopeptides) that have a calcium absorption promoting effect.
This invention relates to a method for purifying phosphopeptide (hereinafter abbreviated as CPP). Specifically, the present invention relates to a method for purifying CPP produced by hydrolyzing casein with trypsin to a high purity using metal ions, particularly ferric ions. CPP is a substance whose properties have recently been revealed as a calcium absorption promoting factor [Hiroshi Naito, Chemistry and Biology 18 551-558 (1980), YSLee,
T.Noguchi and H.Naito, British Journal of
Nutrition 43 457-467 (1980)]. Milk has long been known for its nutritional integrity, and among its many benefits, it is considered the best source of calcium among all foods, and CPP is an important factor. It is known that there are two representative types of CPP. That is, there are two types, one derived from β-casein (abbreviated as β-CPP) and one derived from α-casein (abbreviated as α-CPP), and their structures and constants are as shown in Table 1 below. . In particular, β-CPP has been well studied and its structure has been determined. [W. Manson, W.D.
Annan；Archives of Biochemistry and
Biochemistry 145 pp. 16-26 (1971)] However, it is conceivable that α-CPP has a further decomposed structure in its presumed structure.

【table】

[Table] Abbreviations of amino acid residues in this table: Ala: L-alanine, Arg: L-arginine,
Asp: L-aspartic acid, Asn: L-asparagine, Glu: L-glutamic acid, Gln: L-glutamine, Gly: glycine, Ile: L-isoleucine, Leu: L-leucine, Lys: L-lysine,
Met: L-methionine, Pro: L-proline Ser: L-serine Ser: L-phosphoserine Thr: L-threonine, Val: L-valine,
H ₂ N・Asp: N-terminal L-aspartic acid,
H ₂ N・Arg: N-terminal L-arginine, Lys・COOH: c-terminal L-lysine, Arg・
COOH: c-terminal L-arginine. The following methods are known as methods for preparing CPP. After hydrolyzing β-casein with crystalline trypsin to generate CPP, unreacted casein and some impurities were removed by precipitation at pH 4.7, and the supernatant was mixed with barium chloride and ethanol (final concentration 50% (v). /
v))) to recover CPP as a precipitate [for example, RF Peterson, LW Nouman and T.
L. McMeekin, Journal of the American
Chemical Society 80 95-99 (1958)] Hydrolyze β-casein with crystalline trypsin to generate CPP, then treat with trichloroacetic acid, remove insoluble materials, and neutralize with sodium hydroxide (PH5.0) do. After that, it was crudely fractionated using a gel filtration column (Sephadex G-25 Pharmacia), and then adsorbed onto an ion exchange resin column (Dowex x 50 x 2 Dow Chemical Co.) and then added to a pyridine-acetate buffer (0.2N PH2). 5) Elution and purification method [H.Naito
and H.Suzuki, Agricultural Biology
Chemistry 38 1543-1545 (1974)] also uses β-casein, and in this case, it is necessary to purify β-casein from casein in advance, which is disadvantageous as it results in a decrease in yield. In addition, barium salt uses trichloroacetic acid and pyridine, and products using these cannot be used for consumption.
In addition, the amount of CPP processed in the column is small, making it difficult to apply it to industrial production. Patent Application No. 57-52639 (Japanese Unexamined Patent Publication No. 170440-1981) previously filed by the applicant (filed on March 30, 1982)
Basically, we use the method of
This method uses casein as a raw material and replaces barium chloride with calcium salt, allowing CPP to be recovered and used for consumption as is. This is disadvantageous. The present inventors have conducted extensive studies on the method for purifying CPP, and as a result, have found that CPP interacts with various metal ions. Furthermore, they discovered that CPP can be recovered with high purity using certain metal ions, particularly ferric ions, without using any organic solvents, thereby completing the present invention. According to the present invention, casein is hydrolyzed with trypsin to generate CPP, and then metal ions are added to precipitate and recover CPP, so the operation is simple and no organic solvent is used.
It is extremely advantageous industrially. Next, the method of the present invention will be explained. In this method, trypsin or an enzyme containing trypsin is applied to casein to prepare a decomposition product solution containing CPP, and ferric ions are added to this to recover CPP. Casein, which is a raw material used in this production, is best casein such as various acid caseins, sodium caseinate, calcium casein, etc., but unpurified casein such as milk, skim milk, etc. can also be used as a raw material. According to this production method, this raw material casein is dissolved in water, and trypsin or an enzyme agent containing trypsin is added. Although a commercially available pancreatic enzyme (pancreatin) may be sufficient as trypsin or an enzyme agent containing trypsin used in the present invention, crystal grade enzymes are preferred from the viewpoint of yield and the like. By allowing the above-mentioned enzyme to act on the raw material casein, the two react to produce CPP. At this time, the industrial enzyme reaction conditions are that the casein concentration during the reaction is 30 or less, preferably 2 to 30%, and the reaction pH is 6.0.
~9.0, reaction temperature is 15-60℃, preferably 20-50℃
℃ is convenient because CPP is generated well. The amount of enzyme used is equivalent to 0.001% to 2% of crystal trypsin based on the weight of casein as a substrate, and the reaction time is 5 minutes to 100 hours, and the reaction progresses sufficiently to produce CPP.
can be generated. To recover the CPP generated as above,
This is done by adding ferric ions.
Table 2 shows the results of tests with various metal ions. As can be seen from Table 2, in the presence of ethanol, many metal ions including barium
Although it is possible to precipitate CPP, the inventors have shown that CPP can be precipitated with only metal ions without ethanol.
We investigated a method for precipitating and recovering CPP, and found that CPP could be purified as a precipitate using ferric chloride, zinc chloride, lead acetate, and uranium acetate. Considering that CPP is usually ingested as food, the second
Iron (Fe ⁺⁺⁺ ) ions are considered suitable. Next, a more detailed search was conducted regarding the source of ferric ions, and the results shown in Table 3 were obtained. As is clear from Table 3, among ferric salts, pyrophosphate
Salts that are insoluble in water, such as iron, and ferric salts that form complex salts, such as iron ammonium citrate, are
It was found that those which are unsuitable for main purification and produce free ferric ions are preferable. Also, sulfuric acid No. 1
It has also been found that even in substances such as iron, which do not cause CPP precipitation when used alone, CPP precipitation occurs when it is converted to ferric iron by air oxidation or the like.

[Table] Add as shown

【table】

Analyzed in [Table].
CPP precipitates when the concentration of ferric chloride is above 5mM, and as the concentration of ferric chloride increases,
CPP precipitation recovery rate increases. However, conversely, the purity of CPP gradually decreases. As a commercial product, CPP of 50% or more is preferable, so the concentration of ferric chloride added is preferably 5 to 50 mM. The CPP precipitate obtained by the above method is collected by commonly used methods such as decantation, filtration, and centrifugation.
The purity can be further improved by washing the obtained CPP precipitate with a small amount of water, alcohol, etc. The CPP recovered as described above is precipitated by a commonly used drying method, such as hot air drying, fluidized bed drying, vacuum drying, etc., to form a light yellow to yellowish brown, almost odorless and tasteless powder. can get. The purity of the CPP obtained as described above was analyzed by quantifying the nitrogen content by the Kjeldahl method, and the phosphorus content by Allen's wet ashing method and Nakamura's modified method. Atomic ratio (N/
P ratio). The CPP obtained by the method of the present invention has an N/P ratio of 7 to 20, and the bitterness characteristic of casein decomposition products can be virtually ignored, and it can be used as is for food and drink. Furthermore, CPP does not contain aromatic amino acids. Therefore, there is almost no ultraviolet absorption (for example, 280 nm, 254 nm) caused by this, and only absorption derived from the peptide bond (205 to 220 nm) is observed. Another analysis method for this CPP is analysis using a high performance liquid chromatography device. For example, TSK−
It can also be carried out using a column of GEL, G-2000SW (manufactured by Toyo Soda) in a solvent system of PH6.5 and 0.1M phosphate buffer (containing 0.1M sodium chloride). Detection is performed, for example, by ultraviolet absorption at 215 nm. When analyzed by this method, it was found that the
CPP is a component estimated to be derived from α-CPP and β-
Contains both components presumed to be derived from CPP,
The content of CPP is more than 50%. Next, the method of the present invention will be explained with examples. Example 1 10 g of lactic acid casein (produced in New Zealand) was dissolved in water to make a 10% solution (PH8.0). Porcine crystal trypsin (manufactured by Novo) was added to this solution at 0.01% relative to the substrate, and the mixture was reacted at 50° C. for 6 hours at pH 7.5 to 8.5. To this reaction solution, ferric chloride was added to a concentration of 10 mM, and after stirring, the mixture was left at 5° C. overnight. The precipitated CPP fraction was collected by centrifugation and washed with a small amount of water. Afterwards, vacuum dry and CPP fraction
1.2g was obtained. CPP purity was 92%, N/P = 8.9.

Claims (1)

[Claims] 1. A method for purifying casein phosphopeptides, which comprises hydrolyzing casein with trypsin to generate casein phosphopeptides, and then adding ferric ions to the decomposition solution to recover the casein phosphopeptides as a precipitate.