Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Separation or purification
Reexamination Certificate
1999-01-14
2001-03-20
Weber, Jon P. (Department: 1651)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Separation or purification
C530S421000, C530S414000, C530S417000
Reexamination Certificate
active
06204362
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method of purifying whey separated from lactic acid fermentation liquid by electrodialysis. The whey contains angiotensin-converting enzyme inhibiting peptides. The peptides obtained in the purified whey according to the present invention can be used for anti-high blood pressure agent or foods, when the whey is further treated for oral administration.
The angiotensin-converting enzyme, hereinafter referred to as ACE, is mainly present in lungs and vascular endothelial cells, and acts on angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu SEQ ID No:1) to remove a dipeptide (His-Leu) at its c-terminal and to form angiotensin II, which has a strong blood pressure increasing activity. The ACE also has an ability to decompose and to inactivate bradykinin which decreases blood pressure. Thus, the ACE acts to increase blood pressure by producing angiotensin II on the one hand, while decomposing bradykinin to increase blood pressure. Accordingly, when the angiotensin-converting enzyme is inhibited, high blood pressure could decrease, and many drugs which contain the angiotensin-converting enzyme inhibitor have been developed and used for anti-high blood pressure.
Certain peptides were recently found to be useful, being as low in toxicity and highly safe anti-high blood pressure agents, and natural and synthetic peptides are reported to be possible anti-high blood pressure drugs (Japanese Patent Publication No.120,225/1991). It is also known that peptides containing Ile-Pro-Pro (hereinafter referred to as IPP) or Val-Pro-Pro (hereinafter referred to as VPP) as its basic peptide structure have ACE inhibiting properties, and that these peptides can be produced in large amounts by culturing certain lactic acid bacteria, or lactic acid bacteria and yeast (Japanese patents Nos.2,782,142 and 2,782,153). Drugs or foods consisting of the peptides are proposed to be highly safe and useful in a small amount for decreasing high blood pressure in forms of oral administration, when the cultured liquid is treated for purification and separation.
It may be possible to use the fermentation liquid as obtained in accordance with the processes described in the above patents as the ACE-inhibiting drugs or foods containing the ACE inhibiting peptides (hereinafter referred to as ACEI peptides). However, it may have poor palatability for oral intake, and is not appropriate to drink without further purification processes, because they contain lactic acid and some other substances to some extent. Accordingly, it is desirable to remove substances other than the ACEI peptides from the liquid. Drugs or foods in a dry form including the peptides in more concentrated form than the liquid are more useful. Proteins and the ACEI peptides having IPP or VPP as its basic peptide structure, which are produced by culturing lactic acid bacteria or lactic acid bacteria and yeast are partly hydrolyzed to form IPP and VPP in the cultured broth.
BRIEF DESCRIPTION OF THE INVENTION
The fermentation liquid is subjected to treatments for solid-liquid separation, including centrifugation and filtration to obtain whey as a supernatant, which contains a major portion of the ACEI peptides. The acids and other impurities in the whey may be removed by subjecting the whey to one or a combination of the following treatments including electrodialysis, a treatment with ion exchange resins, hollow fiber membrane dialysis, reverse osmosis treatment, and hydrophobic column chromatography. The purified whey is converted to dry matter containing ACEI peptides.
Electrodialysis is generally considered to be one of the most advantageous techniques to purify the whey, however, generally known dialysis has disadvantages in that it took a long period for the dialysis, because of slow movement of ions due to polarization or fouling. Another disadvantage of electrodialysis is low efficiency in recovering the end product ACEI peptides, because proteins and other compounds having large molecule sizes resolved in the whey may decrease permeability of acids, water, and other ions through the ion exchange membrane. On the other hand, ACEI peptides, such as IPP and VPP, having small molecular sizes easily penetrate through the membrane.
We have now found that when an anion exchange membrane having a permeability of diffusion coefficient in a range of 3.0 to 9.0×10
−6
cm/sec., more preferably in a range of from 5.0 through 7.0×10
−6
cm/sec., is used for purification and concentration of the whey, which is obtained from lactic acid fermentation liquid by separation, and contains ACEI peptides, the lactic acid and other ions are smoothly removed, and the ACEI peptides can be recovered in very high yield for a very short period of treatment; nevertheless, the membrane is called loose-type membrane and has rather large holes, through which charged substances having large molecular weights easily penetrate.
Accordingly, there is provided a method of purifying whey separated from lactic acid fermentation liquid containing ACEI peptides by electrodialysis, which comprises using an anion exchange membrane having a permeability of diffusion coefficient in a range of 3.0 to 9.0×10
−6
cm/sec., and more preferably in the range of 5.0 through 7.0×10
−6
cm/sec. in the dialysis.
It is an aspect of the present invention to provide an efficient method of electrodialysis to remove lactic acid and ions other than ACEI peptides from whey.
It is another aspect of the present invention to provide a method of purifying whey by electrodialysis to remove lactic acid and ions other than ACEI peptides in a short period of time.
DETAILED DESCRIPTION OF THE INVENTION
The lactic acid fermentation liquid containing ACEI peptides can be obtained by culturing lactic acid bacteria, or lactic acid bacteria and yeast in accordance with the description in Japanese Patents Nos. 2,782,142 and 2,782,153. Whey can be obtained from the fermentation liquid as supernatant by solid-liquid separation, including centrifugation, filtration or decantation of the liquid. Whey thus obtained contain large amounts of ACEI peptides as well as acids, other ions and proteins, and subjected to electrodialysis for purification. If desired, the solutions may be partly concentrated under diminished pressure before subjecting to dialysis.
In the method of purifying the whey by electrodialysis according to the present invention, an anion exchange membrane having a permeability of diffusion coefficient in the range of 3.0 to 9.0×10
−6
cm/sec., and more preferably, in the range of 5.0 to 7.0×10
−6
cm/sec. is used. The diffusion coefficient was determined by the following processes; 0.5 normal sodium chloride solution-membrane-3 normal sodium chloride solution are stirred in a vessel, differences in the concentration of the sodium chloride is measured, and diffusion coefficient of the used membrane was determined by the formula of:
Diffusion coefficient(cm/sec.)=transferred amount of sodium chloride(mol/sec.)×area of membrane(cm
2
)×&Dgr;C(mol/cc)
Cation permeable ion exchange membrane which can be used in the present invention may not have any restrictions, and suitable membranes may include those having a permeability of diffusion coefficient in the range of 3.0 to 5.0×10
−6
cm/sec., and those in the range of 4.0 to 6.0×10
−6
cm/sec, and other membranes giving similar results may be used.
There are no more operating conditions to be added to conventional electrodialysis particularly according to the present invention, generally known electrodialysis units can be used, and the current applied to the electrodes is controlled to maximize current efficiency. The rate of conductivity of the whey to the current density may be that generally used, and the dialysis is performed at ambient temperatures up to about 50° C., for 3 to 10 hours. During dialysis, acids including lactic acids, and other charged substances will be removed from the whey
Kitamura Shuji
Ueyama Takashi
Calpis Co., Ltd.
Darby & Darby
Weber Jon P.
LandOfFree
Method of purifying whey of lactic acid fermentation by... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method of purifying whey of lactic acid fermentation by..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of purifying whey of lactic acid fermentation by... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2529298