Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2000-03-16
2003-03-18
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C424S093450
Reexamination Certificate
active
06534304
ABSTRACT:
FIELD OF ART
The present invention relates to novel lactic acid bacteria of
Lactobacillus helveticus
that can produce a particular tripeptide with high efficiency when they are cultured in an animal milk medium and that have high extracellular proteinase activity; a fermented milk product containing the lactic acid bacteria; and a method for producing the product.
BACKGROUND ART
Lactobacillus helveticus has been employed for producing fermented milk for a long time as a typical lactic acid bacteria starter for dairy milk products.
Lactobacillus helveticus
has high proteolytic activity, and particularly, its extracellular proteinase having high activity plays an important role in fermentation of animal milk. That is, the extracellular proteinase digests animal milk proteins to produce various peptide fragments. The produced peptides are further subjected to the action of peptidases to become peptides of lower molecular weight. It is known that a part of peptides produced in a medium due to the action of proteinase enzymes is taken into cells of the lactic acid bacteria and utilized as a nitrogen source. It has also been reported that some of the peptides produced in the medium have an inhibitory activity against angiotensin converting enzyme (ACE) which causes hypertension. (J. Dairy Sci. 78:777-783(1995)).
As peptides for inhibiting ACE activity and suppressing rise in blood pressure, various effective peptides have been reported, such as those derived from degradation products of milk proteins, soybean proteins or fish meat proteins. For example, Val-Pro-Pro and Ile-Pro-Pro (abbreviated hereinbelow as VPP and IPP, respectively. These peptides are collectively referred to hereinbelow as lactotripeptides) are known as peptides having ACE inhibitory activity present in a
Lactobacillus helveticus
—fermented milk. These lactotripeptides have been confirmed to have a strong hypotensive effect by experiments using spontaneously hypertensive rat (SHR) (J. Dairy Sci. 78:1253-1257(1995)).
However, the lactotripeptide-containing fermented milk produced by fermenting animal milk with conventional
Lactobacillus helveticus
strains can hardly be taken as it is, because it exhibits high acidity due to a large quantity of lactic acid generated as the fermentation progresses. Dilution of the fermented milk results in extreme decrease in the content of the lactotripeptides.
Thus, it is desired to produce fermented milk with higher content of the lactotripeptides compared to the content of the lactic acid generated in the fermented milk. With an addition of a small amount of such fermented milk to various foods and beverages, products having the function of the lactotripeptides could be prepared easily and provided to consumers in an agreeable form to take. However, none of known lactic acid bacteria strains produce the lactotripeptide with high efficiency.
DISCLOSURE OF THE INVENTION
It is an object of the present invention to provide a novel lactic acid bacteria strain which can produce a large amount of lactotripeptide with high efficiency with respect to the amount of the generated lactic acid.
It is another object of the present invention to provide a fermented milk product which contains the lactotripeptide having activities such as hypotensive activity and expected to have anti-stress effect, and a lactic acid bacteria strain capable of producing a large amount of this lactotripeptide and which can be taken pleasantly as foods or beverages, and a method for producing the same.
According to the present invention, there is provided lactic acid bacteria of
Lactobacillus helveticus
having the following bacteriological properties, said bacteria, when cultured in a medium of animal milk containing 9 wt % solid of non-fat milk, producing tripeptides Val-Pro-Pro and Ile-Pro-Pro in an amount of not less than 60 &mgr;g in terms of Val-Pro-Pro per ml medium, and said bacteria exhibiting extracellular proteinase activity of not lower than 400U/OD
590
:
(Morphological Properties)
1) Shape of Cell; rod,
2) Motility; none,
3)Spore Formation; none,
4) Gram Stain; positive
(Physiological Properties)
1) Catalase Production; negative,
2) Indole Production; negative,
3) Nitrate Reduction; negative,
4) Aerobic Growth; facultative anaerobic,
5) Formation of DL-lactic acid from glucose by homolactic fermentation without formation of gases
6) Carbohydrate Degradation
glucose; +
lactose; +
mannose; +
fructose; +
galactose; +
sucrose; −
maltose; −
xylose; −
rhamnose; −
cellobiose; −
trehalose; −
melibiose; −
raffinose; −
stachyose; −
mannitol; −
sorbitol; −
esculin; −
salicin; −.
According to the present invention, there is also provided the lactic acid bacteria of
Lactobacillus helveticus
wherein said lactic acid bacteria is Lactobacillus helveticus CM4 strain (deposited at National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology on Aug. 15, 1997, deposition number FERM BP-6060).
According to the present invention, there is further provided the lactic acid bacteria of
Lactobacillus helveticus
having a chromosomal DNA which gives a DNA fragment of 15 to 17 kb when said chromosomal DNA is digested with restriction enzymes PstI and EcoRI.
According to the present invention, there is also provided a fermented milk product containing a fermented milk comprising the aforementioned lactic acid bacteria, and a tripeptide selected from the group consisting of Val-Pro-Pro, Ile-Pro-Pro and mixtures thereof.
According to the present invention, there is also provided a method for producing a fermented milk product comprising fermenting a medium containing a food material selected from the group consisting of a peptide, a protein and mixtures thereof including sequence Val-Pro-Pro and Ile-Pro-Pro, with the lactic acid bacteria.
REFERENCES:
patent: 4976975 (1990-12-01), Callanain
patent: 06040944 (1994-02-01), None
patent: 06197786 (1994-07-01), None
Mitsuoka et al., Rinshoukensa 18, 1163 (1974).*
Yasunori Nakamura, et al., Purification and Characterization of Angiotensin I-Converting Enzyme Inhibitors for Sour Milk, 1995 J. Dairy Sci. 78:777-783.
Ishida Yuu
Kawakami Natsue
Yada Hirokazu
Yamamoto Naoyuki
Calpis Co., Ltd.
Darby & Darby
Marx Irene
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