JPS59159792A - Production casein phosphopeptide - Google Patents

Abstract

PURPOSE:Bitter-tasting components which have been formed on hydrolysis of casein with tripsin are removed with activated carbon or cation-exchange resins to produce casein phosphopeptide used as a calcium-absorption accelerating factor that can be served directly as food or beverage. CONSTITUTION:Casein is hydrolyzed in a customary manner and the resultant hydrolyzate is passed through a column filled with activated carbon or a cation- exchange resin, preferably in a concentration of 2-15% at 0.2-2.0 SV and casein phosphopeptide is obtained by fractionation of the filtrate.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides casein phosphopeptides that have a calcium absorption promoting effect.
This invention relates to a new method for producing tide (hereinafter abbreviated as CPP).

Specifically, the present invention relates to a method for producing CPP by removing bitterness from tryptic decomposition products in order to provide the CPP produced by tryptic decomposition of casein for consumption.

CPP is a substance whose properties have recently been revealed as a calcium absorption promoting factor. (Hiroshi Naito, Chemistry and Biology 18551-558 (1980), Y, S, Le
e.

T, Noguchi and H, Na1to+
Br1tish Journal of Nutri
tion 43 457-467 (1980))
Milk has long been known for its nutritional integrity, and among its many benefits, it is considered to be the best source of calcium among all foods. It is something. It is known that there are two typical types of CPP. That is, there are two types: one derived from β-casein powder (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. Especially β
-CPP has been well studied and its structure has been determined. (W,
Manson+W, D, 8nnan; 8rch
ives of biochemistry a
ndBiophysics 145 pp. 16-26 (1
(971) ) However, it is possible that α-CPP has a further decomposed structure in its presumed structure.

Table 1 Abbreviations of amino acid residues on the tree surface Ala: L-alanine, Arg: L-arginine.

Asp: l, -aspartic acid, Asn:L-
Asparagine, Glu:L-glutamic acid, Gin:L
- Glutamine, Gly nigericin, lle: L-isoleucine, Leu: L-leucine, Lys: L-lysine,
Met: L-methionine, Pr.

:L-proline Ser: L-serine OH20H (H2N-CH-C00H) Set of 8L-Hoyuhoyari.

OH CH20--P--OH 1 0 (H2N -CH--COO'H) Thr: L-threonine, Val: L-valine.

H2N-A sp: N-terminal L-aspartic acid.

H2N-Arg: N-terminal L-arginine.

Lys-cooH: c-terminal L-lysine, Arg・C0
OH: Each represents c-terminal L-arginine.

The following method is known as the CPP 8-circle layer method.

■ Hydrolyze β-casein with crystalline trypsin to produce CP
After generating P, unreacted casein and some impurities were removed by precipitation at pH 4.7, and barium chloride and ethanol (final concentration 50% (v/v)) were added to the supernatant to precipitate CPP. Collection method [e.g. R, F, Peterson
, L., W., Nouman and T., L., McMee.
Kin, Journal of the A
mericanChemical 5ociety
80 95-99 (1958)) Hydrolyze β-casein with crystalline trypsin to generate CPP, then treat with trichloroacetic acid, remove insoluble matter, and neutralize with sodium hydroxide (pH 5,0) . After that, a gel filtration column (Sephadex G-25 Phar
Macia Co., Ltd.) for crude fractionation, and then an ion exchange resin column (Dowex 50 x 2 Don Chemica Co., Ltd.).
Phosphorus was adsorbed onto pyridine-acetate buffer (
A method of elution and purification (H, N
a1to and H,5uzuki+Agricul
Tural Biological Chemistry
y 38 1543-1545 (1974)) Methods ① and ② both use 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 is used in (2), and trichloroacetic acid and pyridine are used in (2), and products using these cannot be used for consumption. In addition, in case (2), the amount of CPP processed in the column is small, making it difficult to apply to industrial production.

Patent Application No. 57-52639 filed by Junto Honde-(
(filed on March 30, 1981) basically uses the method (2), but uses casein as a raw material, uses calcium salt instead of barium chloride, recovers CPP, and then uses it as it is. Although it can be used for consumption, it is disadvantageous from an industrial perspective because it uses a large amount of organic solvent such as ethanol.

When casein is hydrolyzed with trypsin, CPP is produced, but at the same time a considerable amount of bitter peptides are also produced, and the hydrolyzate itself may be unsuitable for consumption. Therefore, the present inventors have developed an industrially advantageous method for removing bitter peptides without using organic solvents.
We worked hard to find a way to make it possible to directly serve food and drink.

As a result, it was found that bitter peptides can be adsorbed and removed extremely effectively by passing the liquid through a column made of activated carbon or cation exchange resin. According to Kempo, almost the entire amount of CPP can be recovered without being adsorbed by the column, and there is no need to use organic solvents (and the operation is simple).

Next, the configuration of the present invention will be explained.

The best casein used as a raw material for this production is casein such as various acid caseins, sodium caseinate, calcium casein, etc., but unrefined casein such as milk, skim milk, etc. can also be used as a raw material. If you follow this manufacturing method,
This raw material casein is dissolved in water, and trypsin or an enzyme containing trypsin is added. Although a commercially available pancreatic enzyme (vancreatin) is 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 enzyme to act on the raw material casein,
Both react to produce CPP. At this time, the industrial enzyme reaction conditions include casein concentration during reaction of 30 or less, preferably 2 to 30%, reaction temperature of 6.0 to 9.0, and reaction temperature of 15 to 60'C1, preferably 20 to 50%. ℃ is good
It is convenient because PP is generated.

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 processing time is 5%.
The reaction can proceed sufficiently in minutes to 100 hours to produce cpP.

The fractionation operation of the present invention is extremely simple, and all that is required is to fill a column with an adsorbent and pass the tryptic decomposition product of casein through the column.
It is preferable to remove some impurities.

Table 2 shows the screening results for bitter adsorbents.
Selection of adsorbent As is clear from the results above, activated carbon and cation exchange resin were excellent in adsorbing bitter taste. Anion exchange resin is
Bitter adsorption ability was weak, and CPP tended to be adsorbed. Cation exchange resin does not adsorb CPP at all, and activated carbon
It was found that although some CPP was initially adsorbed, it eluted as the amount of solution increased, and that it selectively adsorbed bitter peptides without actually adsorbing much CPP.

In the adsorbent used in the present invention, granular activated carbon that can be packed into a column is used as activated carbon. For example, Bitzhag activated carbon CAL (Organo) 9 granular white gKL (Takeda Pharmaceutical Company)
, Kuraray Coal GLC (Kuraray Chemical), etc. are used. As a cation exchange resin, Amberlite IR-
120A (Organo), Dowenx HCR-W2 (Muromachi Kagaku), etc. are used.

The above adsorbent is packed into a column, and a liquid containing CPP and bitter peptide is passed therethrough. The liquid passing concentration is 2 to 15% for easy operation, and the liquid passing rate is superficial velocity (SV) of 0.2 to 15%.
It is preferable to do this in the range of 2.0.

When this method is carried out, it is possible to process an amount equivalent to about 1 to 2 kg of starting casein using a 1β column.

The purified fraction containing CPP thus obtained has almost no bitter taste and can be directly consumed. In addition, the adsorption column can be regenerated by acid-alkali and calcination, and can be used repeatedly.

Next, the present invention will be explained in detail with reference to Examples.

Example 1 1 kg of lactic acid casein produced in New Zealand was dissolved in water to make a 10% solution (pH 8.0). To this solution, 0.01% of porcine crystal trypsin (manufactured by Novo) was added to the substrate.
The reaction was carried out at 50° C. for 6 hours at H7.5 to 8.5.

Although CPP was produced in the reaction solution, a strong bitter taste was also felt. After the reaction, the - of the solution was adjusted to 4.5, and the resulting precipitate was removed by centrifugation. Although some impurities were removed, the bitterness remained strong. The obtained supernatant was subjected to Kuraraycol GLC.
The column (200 ml) was supplied with 5V=0.2. Approximately 2
A passed liquid with no bitter taste was obtained until it was passed through the β solution, but after that, a bitter taste gradually started to be felt, so the flow was discontinued, and both parts without bitterness were made into powder by spray drying.

90 g (required % yield based on casein) of powder was obtained. The yield of CPP was 100%, and no bitter taste was felt at all.

Example 2 Using the upper layer obtained in Example 1, DOWEX HCR-
The liquid was passed through a W2 column (100 mJ).

The speed was 5V=2. I didn't feel any bitterness until I passed about 2I1 of the solution. When the temperature exceeded 21, a bitter taste was felt, so the liquid flow was stopped and the passed liquid was freeze-dried. powder 5
Obtained 0g. The casein yield was 29.5%, the cpp yield was 100%, and no bitterness was felt at all.

Patent applicant: Meiji Seika Co., Ltd. Agent: Riki Arai (and 2 others)

Claims (1)

[Claims] 1. After hydrolyzing casein with trypsin to generate casein phosphopeptide, the decomposed solution is passed through an activated carbon or cation exchange resin column to adsorb and remove bitter components, and the casein phosphopeptide fraction is obtained. A method for producing casein phosphopeptide, characterized in that it is obtained.