# Patent

## Title
Process for preparing peptides with anti-hypertensive properties

## Abstract

Use of a lactic acid bacterium comprising a cell wall proteinase of around 200 kDa to prepare peptides with anti-hypertensive properties and a method for obtaining such a lactic acid bacterium.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
I claim the benefit of the filing dates of the following patent applications: Denmark Application PA 2003 00361, filed Mar. 11, 2003; Denmark Application PA 2002 01194, filed Aug. 9, 2002; and U.S. Provisional Application 60/404,396, filed Aug. 20, 2002. 


FIELD OF INVENTION
The present invention relates to use of a lactic acid bacterium comprising a cell wall proteinase of around 200 kDa to prepare peptides with anti-hypertensive properties and a method for obtaining such a lactic acid bacterium.
DESCRIPTION OF THE BACKGROUND ART
Hypertension (high blood pressure) has been reported to be one of the most important risk factors associated with heart attack in industrialized countries. Hypertension is frequently treated with drugs that strongly inhibit the angiotensin-converting enzyme (ACE). The prevention of high blood pressure in the early stage of the development of the disease, can be an alternative, to the treatment of hypertension with drugs. A large number of food-derived bioactive compounds are currently considered as beneficial for general well being or as health promoting.
In the regulation of blood pressure, angiotensin I-converting enzyme (ACE) plays an important role. ACE acts to increase the blood pressure. In the renin-angiotensin system, ACE converts angiotensin-I to angiotensin-II by hydrolysing His-Leu from its C-terminal. Angiotensin II exhibits a strong vasoconstricting action. Additionally, in the kinin kallikrein system, ACE deactivates bradykinin, which aids vasodilation. ACE inhibitors are therefore useful in reducing blood pressure. Currently several ACE inhibitors already exist. The first reported ACE inhibitors were naturally occurring peptides found in snake venom. Since then, many other ACE inhibitors have also been discovered.
It is known that milk fermented by lactic acid bacteria (LAB) may produce anti-hypertensive effects due to the liberation of peptides from casein in the milk by the proteolytic activity of the lactic acid bacteria. The peptides act as ACE inhibitors.
The article of the company Calpis Food Industry [Yamamoto et al (1994, J. Dairy Sci., 77: 917-922)] discloses that milk fermentation by a Lactobacillus helveticus CP790 strain produced anti-hypertensive effects due to the liberation of peptides from casein in the milk by the proteolytic activity of L. helveticus. The peptides act as ACE inhibitors. The anti-hypertensive activity of these peptides was tested on spontaneously hypertensive rats. Milk fermentation by an isogenic mutant of Lb. helveticus that does not have proteolytic activity does not show any anti-hypertensive effects.
The article [Gobbetti M. et al (2000, Appl Environ Microbiol, 66 (9), 3898-3904.], describes fermented milk containing ACE-inhibitory peptides that were produced by using either Lactococcus lactis subspecies cremoris FT4 or Lactobacillus delbrueckii subspecies bulgaricus SS1 to ferment the milk.
EP821968 (Calpis Food Industry) describes fermented milk containing ACE-inhibitory peptides that were produced by using a Lactobacillus helveticus strain with deposit accession number FERM BP-4835.
EP1016709 (Calpis Food Industry) describes fermented milk containing ACE-inhibitory peptides that were produced by using a Lactobacillus helveticus strain with deposit accession number FERM BP-6060.
WO01/32836 (Valio Ltd) describes fermented milk containing ACE-inhibitory peptides that were produced by using a Lactobacillus helveticus strain with deposit accession number DSM 13137.
Because lactic acid bacteria (LAB) are auxotrophic for a number of amino acids, LAB depend upon a complex proteolytic system to obtain essential amino acids from casein during growth in milk. The hydrolysis of casein into amino acids for use by LAB is initiated by cell wall proteinases that hydrolyse casein into oligopeptides. Oligopeptides are then transported into the bacterial cell via an oligopeptides transport system. Once the oligopeptides are inside the cell, intracellular peptidases hydrolyze them to free amino acids.
The article of University of Wisconsin and Utah State University [Pederson et al (1999, J. of Bacteriology, 181: 4592-4597] describes the DNA and amino acid sequence of a prtH 204 kDa cell wall proteinase from the Lactobacillus helveticus CNRZ32 strain. This article does not describe nor suggest using of the strain to make peptides with anti-hypertensive properties.
The article of the company Calpis Food Industry [Yamamoto et al (2000), Biosci. Biotechnol. Biochem., 64(6): 1217-1222] describes the DNA and amino acid sequence of a prtY 45 kDa cell wall proteinase from the Lactobacillus helveticus CP790 strain. The CP790 strain does not comprises the prth 204 kDa cell wall proteinase [see “Discussion” section of Yamamoto et al (2000)]. The CP790 strain is used in a commercial product of Calpis Food Industry as the Materials and Methods sections reads, “CP790, was isolated from the starter culture of Calpis, a Japanese fermented milk product”.
SUMMARY OF INVENTION
The problem to be solved by the present invention is to provide a method to obtain lactic acid bacteria (LAB) with improved characteristic in particular in relation to make peptides with anti-hypertensive properties.
The solution is based on that the present inventors have identified that lactic acid bacteria comprising a specific cell wall proteinase has such properties. The specific cell wall proteinase is herein termed prtH200. In working example 5 herein is demonstrated in vivo that a lactic acid bacteria having a prtH200 cell wall proteinase, as described herein, is capable of making peptides with improved anti-hypertensive properties.
The prtH200 proteinase, as described herein, correspond to the prtH 204 kDa cell wall proteinase from the Lactobacillus helveticus CNRZ32 strain described in the article of University of Wisconsin and Utah State University [Pederson et al (1999)] (see above). In this article it is not described nor suggested to use the CNRZ32 strain to make peptides with anti-hypertensive properties.
To the knowledge of the present inventor(s), no other references explicitly describe a lactic acid bacterium comprising a cell wall proteinase corresponding to the prtH200 proteinase as described herein.
In working example 3 herein it experimentally demonstrated that the Lactobacillus helveticus strain with deposit accession number DSM 13137, as described in WO01/32836 (Valio Ltd), does not comprise a prtH200 cell wall proteinase gene sequence identifiable by the specific prtH200 PCR primers as described herein.
As explained in the article of the company Calpis Food Industry [Yamamoto et al (2000)] (see above), at least one Japanese fermented milk product Calpis Food Industry comprises a strain (CP790) not having a prtH200 cell wall proteinase as described herein.
The presence, in a lactic acid bacterium, of a gene sequence encoding the prtH200 proteinase may preferably be verified by PCR amplification using suitable designed PCR primers. When the skilled person has suitable designed PCR primers it is easy for him to verify the presence or not of the gene sequence in a lactic acid bacterium using his general knowledge to make a specific suitable PCR amplification protocol.
Consequently, the skilled person may rapidly screen a number of lactic acid bacteria, identify the ones comprising a prtH200 gene sequence, and thereby obtain specific selected lactic acid bacteria with improved industrial relevant characteristic.
Accordingly, a first aspect of the invention relates to a method for obtaining a lactic acid bacterium comprising

    
    
        (i) investigating whether a lactic acid bacterium is a lactic acid bacterium that comprises a gene sequence encoding a cell wall proteinase (termed prtH200) wherein the gene sequences is identifiable by PCR amplification of genomic DNA of the lactic acid bacterium using sets of PCR primers selected from the group consisting of [sense sequence (S); antisense sequence (A)]:
    
    












PrtH200:




(a): (S): 5′ CGATGATAATCCTAGCGAGC 3′,



 



     (A): 5′ TGGCAGAACCTGTGCCTA 3′;



 



(b): (S): 5′ GCCAAGACGCCTCTGGTA 3′,



 



     (A): 5′ TAGGTATAGTTTCCATCAGGA 3′; and



 



(c): (S): 5′ AARGTWCCWTAYGGYYWYAAYTA 3′,



 



     (A): 5′ GCCATDSWDGTRCCDSWCATDTK 3′; and






    
    
        (ii) if the lactic acid bacterium fulfills the criteria of step (i) then the lactic acid bacterium is obtained; or
        (iii) if the lactic acid bacterium does not fulfill the criteria of step (i) then repeat step (i) with another lactic acid bacterium,

with the exception of the situation wherein the lactic acid bacterium is the Lactobacillus helveticus CNRZ32 strain.

    
    


The term “Lactobacillus helveticus CNRZ32 strain” refers to the strain described in the article of University of Wisconsin and Utah State University [Pederson et al (1999)] (see above).
The DNA and amino acid sequence of prtH200 of Lactobacillus helveticus DSM 14998 is shown in SEQ ID NO 1 and SEQ ID NO 2.
Based on the prtH200 sequences and sequences homologues to these suitable PCR primers may routinely be identified in order to identify the prtH200 gene of SEQ ID NO 1 and genes homologous to this.
Accordingly, a second aspect of the invention relates to a method for obtaining a lactic acid bacterium comprising

    
    
        (i) investigating whether a lactic acid bacterium is a lactic acid bacterium that comprises a gene sequence encoding a cell wall proteinase (termed prtH200), wherein the gene sequence is defined as
        
            prtH200 is a DNA sequence encoding an enzyme exhibiting cell wall proteinase activity, which DNA sequence is selected from the group consisting of:
            
                (a) the DNA sequence shown in positions 1-5550 in SEQ ID NO 1;
                (b) a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 50% identical to a corresponding fragment of the DNA sequence defined in (a);
                (c) a DNA sequence that encodes a polypeptide, exhibiting cell wall proteinase activity, comprising a fragment of at least 200 amino acids (aa) that is at least 30% identical to a corresponding fragment of the polypeptide sequence shown in positions 1-1849 of SEQ ID NO 2;
                (d) a DNA sequence which hybridizes with a double-stranded DNA probe comprising the DNA sequence shown in positions 1-5550 in SEQ ID NO 1 at low stringency; and
                (e) a DNA sequence which is a fragment of the DNA sequences specified in (a), (b), (c), or (d); and
            
            
        
        
        (ii) if the lactic acid bacterium fulfills the criteria of step (i) then the lactic acid bacterium is obtained; or
        (iii) if the lactic acid bacterium does not fulfill the criteria of step (i) then repeat step (i) with another lactic acid bacterium,

with the exception of the situation wherein the lactic acid bacterium is the Lactobacillus helveticus CNRZ32 strain.

    
    


As said above, an advantage of lactic acid bacteria comprising a prtH200 proteinase, as described herein, is improved characteristic in relation to make peptides with anti-hypertensive properties.
Consequently, in a third aspect the invention relates to a process for preparing peptides with anti-hypertensive properties, the process comprising fermenting a food material, comprising animal milk proteins or vegetable proteins, with a lactic acid bacterium to obtain a fermented food material which comprises the peptides with anti-hypertensive properties, characterized in that the lactic acid bacterium comprises a gene sequence encoding a cell wall proteinase (termed prtH200) and the presence of the cell wall proteinases is identifiable by PCR amplification of genomic DNA of the lactic acid bacterium using sets of PCR primers selected from the group consisting of [sense sequence (S); antisense sequence (A)]:










PrtH200:




(a): (S): 5′ CGATGATAATCCTAGCGAGC3′,



 



     (A): 5′ TGGCAGAACCTGTGCCTA 3′;



 



(b): (S): 5′ GCCAAGACGCCTCTGGTA 3′,



 



     (A): 5′ TAGGTATAGTTTCCATCAGGA 3′; and



 



(c): (S): 5′ AARGTWCCWTAYGGYYWYAAYTA 3′,



 



     (A): 5′ GCCATDSWDGTRCCDSWCATDTK 3′.






In a fourth aspect, the invention relates to a process for preparing peptides with anti-hypertensive properties, the process comprising fermenting a food material, comprising animal milk proteins or vegetable proteins, with a lactic acid bacterium to obtain a fermented food material which comprises the peptides with anti-hypertensive properties, characterized in that the lactic acid bacterium comprises a gene sequence encoding a cell wall proteinase (termed prtH200), wherein the gene sequence is defined as

    
    
        prtH200 is a DNA sequence encoding an enzyme exhibiting cell wall proteinase activity, which DNA sequence is selected from the group comprising of:
        
            (a) the DNA sequence shown in positions 1-5550 in SEQ ID NO 1;
            (b) a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 50% identical to a corresponding fragment of the DNA sequence defined in (a);
            (c) a DNA sequence that encodes a polypeptide, exhibiting cell wall proteinase activity, comprising a fragment of at least 200 amino acids (aa) that is at least 30% identical to a corresponding fragment of the polypeptide sequence shown in positions 1-1849 of SEQ ID NO 2;
            (d) a DNA sequence which hybridizes with a double-stranded DNA probe comprising the DNA sequence shown in positions 1-5550 in SEQ ID NO 1 at low stringency; and
            (e) a DNA sequence which is a fragment of the DNA sequences specified in (a), (b), (c), or (d).
        
        
    
    


As explained above, to the knowledge of the present inventor(s), no references explicitly describe the use of a lactic acid bacterium as described herein in a process according to the third and fourth aspect herein. However, it might be that future experiments demonstrate that a lactic acid bacterium, described prior to the filing date of the present application to have been used in a process according to the third and fourth aspect herein, does comprise the prtH200 gene sequences as described herein. In such a hypothetical case the specific lactic acid bacterium will be disclaimed from the scope of the claims of the third and fourth aspect of the present invention. In other words, the third and fourth aspect will include a statement that could read “with the exception of a possible specific lactic acid strain”.
A fifth aspect of the invention relates to a process for preparing peptides comprising

    
    
        (i) obtaining a lactic acid bacterium by a method for obtaining a lactic acid bacterium according to the first and second aspect of the invention;
        (ii) fermenting a material comprising proteins with the lactic acid bacterium obtained in (i) to obtain a fermented material which comprises the peptides.
    
    


An embodiment of the fifth aspect of the invention relates to a process for preparing peptides with anti-hypertensive properties comprising

    
    
        (i) obtaining a lactic acid bacterium by a method for obtaining a lactic acid bacterium according to the first and/or second aspect of the invention;
        (ii) fermenting a food material, comprising animal milk proteins or vegetable proteins, with the lactic acid bacterium obtained in (i) to obtain a fermented food material which comprises the peptides with anti-hypertensive properties.
    
    


The disclaimer discussion above, in relation to the third and fourth aspect of the invention, is not relevant for the fifth aspect and its embodiments. The fifth aspect includes a step of actively investigating the presence, in the lactic acid bacterium, of the prtH200 proteinase discussed herein. In the present context this step is novel.
Peptides produced as described herein, may be used to prepare a functional food product with anti-hypertensive properties.
Accordingly, a sixth aspect the invention relates to a process for preparing a functional food product comprising peptides with anti-hypertensive properties, the process comprising following steps:

    
    
        (i) preparing a fermented food material according to a process for preparing peptides with anti-hypertensive properties, as described herein, and
        (ii) packing it in a suitable way to get a functional food product.
    
    


The term “packing” should be understood broadly. It denotes that once a food material is fermented and a fermented food material is obtained, the fermented food material should be packed in order to could be provided to the consumer. It may be packed in a bottle, a tetra-pack, etc. Preferably, on the package or in corresponding marketing material is indicated that the functional food product has anti-hypertensive properties.
The process of the sixth aspect, illustrates one of the advantages of the processes as described herein. The use of lactic acid bacteria comprising the prtH200 proteinases as described herein provides directly after the fermentation a useful amount of peptides with very good anti-hypertensive properties. Consequently, it is not considered necessary to further purify or up-concentrate the peptides from the fermented food material. The fermented food material may be packed directly and provided to the market as a functional food product.
In a seventh aspect the invention relates to peptides with anti-hypertensive properties obtainable by a process for preparing peptides with anti-hypertensive properties as described herein.
Peptides, produced by fermentation with lactic acid bacteria comprising the prtH200 proteinase as described herein, are different from peptides produced by fermentation with lactic acid bacteria not comprising these proteinases. This may be verified functionally by the improved anti-hypertensive effects of the peptides produced by fermentation with lactic acid bacteria comprising the prtH200 proteinase as described herein.
In an eighth aspect the invention relates to a functional food product comprising peptides with anti-hypertensive properties obtainable by a process for preparing a functional food product as described herein.
In a ninth aspect the invention relates to use of peptides with anti-hypertensive properties of the seventh aspect for the manufacture of a medicament for the treatment of hypertension.
In a tenth aspect the invention relates to use of a functional food product comprising peptides with anti-hypertensive properties of the eighth aspect for the manufacture of a medicament for the treatment of hypertension.
DEFINITIONS
Prior to a discussion of the detailed embodiments of the invention is provided a definition of specific terms related to the main aspects of the invention.
The term “lactic acid bacteria” denotes herein a group of Gram-positive, non-sporing bacteria, which carry out a lactic acid fermentation of sugars.
The term “gene” is herein defined according to is usual meaning as the fundamental physical and functional unit of heredity. A gene is an ordered sequence of nucleotides (e.g. DNA or RNA) located in a particular position on a particular chromosome that encodes a specific functional product (i.e., a protein or RNA molecule).
The “nomenclature of degenerated primers” is according to the standard nomenclature in the art. Y=C or T; R=A or G; M=A or C; K=G or T; S=G or C; W=A or T; H=A or C or T; B=G or T or C; V=G or C or A; D=G or A or T; N=G, A, C or T.
The term a “fragment” in relation to a DNA/amino acid sequence comprising a fragment denotes a continuous partial sequence. For instance, from position 75 to 300 in an amino acid sequence having 600 amino acids.
The term “a corresponding fragment” in relation to identity comparison between two sequences relates to a fragment of corresponding size. Preferably, the size difference, between the two fragments to be compared, is less than 50%. In order words, if one fragment is 100 bp the other is preferably less than 150 bp. More preferably, the size difference, between the two fragments to be compared, is less than 25%, and even more preferably the size difference, between the two fragments to be compared, is less than 5%.
Embodiment(s) of the present invention is described below, by way of example(s) only.



DRAWINGS
FIG. 1: The FIGURE contains pulsed field gel electrophoresis (PFGE) fingerprinting of Lactobacillus helveticus strain CHCC5951 (deposited with accession number DSM 14998) This FIGURE and working example 7 show that a PFGE fingerprint of the CHCC5951 strain identifies at least 12 characterizing bands described infra.



DETAILED DESCRIPTION OF THE INVENTION
prtH200 Cell Wall Proteinase
The activity of a cell wall proteinase is preferably verified while it is present in the lactic acid bacteria. A suitable strategy is to construct a lactic acid bacterium with a lethal mutation in the gene encoding the cell wall proteinase to be analyzed. The proteolytic activity (for a suitable assay see below) of this constructed bacterium could then be compared with the corresponding wildtype bacterium. A measurable decrease in proteolytic activity of the lactic acid bacterium with a lethal mutation as compared with the corresponding wildtype bacterium would experimentally confirm that the gene encoding the cell wall proteinase to be analyzed is a gene encoding a active lactic acid cell wall proteinase.
The skilled person knows how to construct a lactic acid bacterium with a suitable lethal mutation. Reference is made to e.g. Pederson et al (1999) and Yamamoto et al (1994) (see above).
At the filing date of the present invention, the National Center for Biotechnology Information (NCBI) offered at its Internet site the possibility of making a standard BLAST computer sequence homology search.
The DNA and amino acid sequence of prtH200 of Lactobacillus helveticus DSM 14998 as shown in SEQ ID NO 1 and SEQ ID NO 2 has been published at the GeneBank database with accession number AF133727. The database sequence identification is gi|5758038|gb|AF133727.1|AF133727.
Standard protein-protein BLAST [blastp] search using the prtH200 amino acid sequence shown in position 1-1849 in SEQ ID NO 2 as reference sequence gave, among others, following results (in italic is given the database sequence identification. This info unambiguously identifies the published sequence and the skilled person know how to get the sequence based on this):

    
    
        gi|129346|sp|P15293|P2P_LACLC: PII-type proteinase precursor (Lactocepin) (Cell wall-associated serine proteinase). Organism: Lactococcus lactis subsp. cremoris. Identity: a 1600 amino acid fragment with 50% identity of to a corresponding fragment of the prtH200 amino acid sequence of SEQ ID NO 2.
        gi|149582|gb|AAA25248.1|: proteinase Organism: Lactobacillus paracasei. Identity: a 1632 amino acid fragment with 49% identity of to a corresponding fragment of the prtH200 amino acid sequence of SEQ ID NO 2.
        gi|1381114|gb|AAC41529.1|: (L48487) proteinase precursor Organism: Lactobacillus delbrueckii. Identity: a 1682 amino acid fragment with 32% identity of to a corresponding fragment of the prtH200 amino acid sequence of SEQ ID NO 2.
        gi|18568398|gb|AAL76069.1|: (AF468027) cell-envelope proteinase. Organism: Lactobacillus pentosus. Identity: a 415 amino acid fragment with 63% identity of to a corresponding fragment of the prtH200 amino acid sequence of SEQ ID NO 2.
        gi|9963932|gb|AAG09771.1|AF243528—1: (AF243528) cell envelope proteinase. Organism: Streptococcus thermophilus Identity: a 781 amino acid fragment with 30% identity of to a corresponding fragment of the prtH200 amino acid sequence of SEQ ID NO 2.
        gi|482386|pir||A44833: lactocepin (EC 3.4.21.96). Organism: Lactococcus lactis. Identity: a 264 amino acid fragment with 61% identity of to a corresponding fragment of the prtH200 amino acid sequence of SEQ ID NO 2.
    
    


These specific sequences are all representing a DNA sequence that encodes a polypeptide, exhibiting cell wall proteinase activity, comprising a fragment of at least 200 amino acids (aa) that is at least 30% identical to a corresponding fragment of the polypeptide sequence shown in positions 1-1849 of SEQ ID NO 2.
Standard nucleotide-nucleotide BLAST [blastn] search using the prtH200 DNA sequence shown in position 1-5550 in SEQ ID NO 1 as reference sequence gave, among others, following results

    
    
        gi|149580|gb|M83946.1|LBAMPRO. Proteinase (prtP) gene. Organism: Lactobacillus paracasei. Identity: a 102 bp fragment with 84% identity of to a corresponding fragment of the prtH200 DNA sequence of SEQ ID NO 1.
        gi|47197|emb|X14130.1|SLPRT763. plasmid pLP763 prt gene for cell wall-associated serine proteinase. Organism: Streptococcus lactis Identity: a 81 bp fragment with 86% identity of to a corresponding fragment of the prtH200 DNA sequence of SEQ ID NO 1.
        gi|472834|gb|M24767.1|STRWGPROT. Wg2 proteinase gene. Organism: S. cremoris Identity: a 81 bp fragment with 86% identity of to a corresponding fragment of the prtH200 DNA sequence of SEQ ID NO 1.
        gi|149476|gb|J04962.1|LACPRASE. PIII-type proteinase (prtP) and maturation protein. Organism: Lactococcus lactis. Identity: a 81 bp fragment with 86% identity of to a corresponding fragment of the prtH200 DNA sequence of SEQ ID NO 1.
        gi|8568397|gb|AF468027.1|. cell-envelope proteinase (prtP) gene. Organism: Lactobacillus pentosus Identity: a 102 bp fragment with 83% identity of to a corresponding fragment of the prtH200 DNA sequence of SEQ ID NO 1.
    
    


These specific sequences are all representing a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 50% identical to a corresponding fragment of the DNA sequence shown in positions 1-5550 of SEQ ID NO 1.
Other “fingerprint” gene sequences (orfF3, orfF4 and orfF1):
The prtH200 gene sequence discussed herein may be seen as a “fingerprint” of the lactic acid bacteria (LAB).
orfF3:
Preferably, beside a prtH200 gene sequence the LAB also comprises a gene comprising an open reading frame herein termed orfF3. This gene may be seen as an additional fingerprint.
The DNA and amino acid sequence of orfF3 of Lactobacillus helveticus DSM 14998 is shown in SEQ ID NO 3 and SEQ ID NO 4. Working example 3 shows identification of orfF3 based on suitable primers.
Accordingly in a preferred embodiment, a lactic acid bacterium, as described herein, comprises the prtH200 gene and a gene sequence (termed orfF3) encoding an open reading frame wherein the gene sequences is identifiable by PCR amplification of genomic DNA of the lactic acid bacterium using sets of PCR primers selected from the group consisting of [sense sequence (S); antisense sequence (A)]:








orfF3:



(a): (S): 5′ CGAAGGCGATAAGTCAAACTTTGATAATGC 3′,


 


     (A): 5′ CCCGGTTCTGTAAGATAATTTGGATCG 3′; and


 


(b): (S): 5′ ASTCWRRYTTYGATRATGCW 3′,


 


     (A): 5′ BHKYAMSAWARTTTGGATCR 3′.






As said above suitable PCR primers may be identified based on the sequences disclosed herein.
Accordingly, in a preferred embodiment, a lactic acid bacterium, as described herein, comprises the prtH200 gene and a gene sequence encoding an open reading frame (termed orfF3), wherein the gene sequence is defined as

    
    
        orfF3 is a DNA sequence encoding an open reading frame, which DNA sequence is selected from the group comprising of:
        
            (a) the DNA sequence shown in positions 1-2679 in SEQ ID NO 3;
            (b) a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 40% identical to a corresponding fragment of the DNA sequence defined in (a);
            (c) a DNA sequence that encodes a polypeptide comprising a fragment of at least 200 amino acids (aa) that is at least 30% identical to a corresponding fragment of the polypeptide sequence shown in positions 1-893 of SEQ ID NO 4;
            (d) a DNA sequence which hybridizes with a double-stranded DNA probe comprising the DNA sequence shown in positions 1-2679 in SEQ ID NO 3 at low stringency; and
            (e) a DNA sequence which is a fragment of the DNA sequences specified in (a), (b), (c), or (d).
        
        
    
    


The term “open reading frame” denotes a stretch of DNA that contains a signal for the start of translation followed in the correct register by a sufficient length of amino acid encoding triplets to form a protein, followed by a signal for termination of translation, and which may therefore indicate the presence of a protein coding gene.
At the filing date of the present application, a standard protein-protein BLAST [blastp] search using the deduced orfF3 amino acid sequence shown in position 1-893 in SEQ ID NO 4 as reference sequence gave relatively limited conclusive results in relation to published homologous sequences.
However, without being limited to theory, it is believed that an orfF3 gene as described herein encodes a cell wall proteinase. Consequently, in a preferred embodiment the orfF3 gene as described herein encodes a cell wall proteinase.
orfF4:
Preferably, beside a prtH200 gene sequence the LAB also comprises a gene comprising an open reading frame herein termed orfF4.
The DNA and amino acid sequence of orfF4 of Lactobacillus helveticus DSM 14998 is shown in SEQ ID NO 5 and SEQ ID NO 6. Working example 3 shows identification of orfF4 based on suitable primers.
Accordingly in a preferred embodiment, a lactic acid bacterium, as described herein, comprises the prtH200 gene and a gene sequence (termed orfF4) encoding an open reading frame wherein the gene sequences is identifiable by PCR amplification of genomic DNA of the lactic acid bacterium using sets of PCR primers selected from the group consisting of [sense sequence (S); antisense sequence (A)]:










orfF4:




(a): (S): 5′ GGTGTTGCTCCTGAAGC 3′



 



     (A): 5′ ACTCTAGCACCAGCTAATTGAACATCATG 3′.






As said above suitable PCR primers may be identified based on the sequences disclosed herein.
Accordingly, in a preferred embodiment, a lactic acid bacterium, as described herein, comprises the prtH200 gene and a gene sequence encoding an open reading frame (termed orfF4), wherein the gene sequence is defined as

    
    
        orfF4 is a DNA sequence encoding an open reading frame, which DNA sequence is selected from the group comprising of:
        
            (a) the DNA sequence shown in positions 1-4881 in SEQ ID NO 5;
            (b) a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 40% identical to a corresponding fragment of the DNA sequence defined in (a);
            (c) a DNA sequence that encodes a polypeptide comprising a fragment of at least 200 amino acids (aa) that is at least 30% identical to a corresponding fragment of the polypeptide sequence shown in positions 1-1627 of SEQ ID NO 6;
            (d) a DNA sequence which hybridizes with a double-stranded DNA probe comprising the DNA sequence shown in positions 1-4881 in SEQ ID NO 5 at low stringency; and
            (e) a DNA sequence which is a fragment of the DNA sequences specified in (a), (b), (c), or (d).
        
        
    
    


At the filing date of the present application, a standard protein-protein BLAST [blastp] search using the deduced orfF4 amino acid sequence shown in position 1-1627 in SEQ ID NO 6 as reference sequence gave relatively limited conclusive results in relation to published homologous sequences.
However, without being limited to theory, it is believed that an orfF4 gene as described herein encodes a cell wall proteinase. Consequently, in a preferred embodiment the orfF4 gene as described herein encodes a cell wall proteinase.
Preferably, a lactic acid bacterium, as described herein, comprises the prtH200 gene, the orfF3 gene and the orf4 gene as described herein.
orfF1:
Preferably, beside a prtH200 gene sequence the LAB also comprises a gene comprising an open reading frame herein termed orfF1.
The DNA and amino acid sequence of orfF1 of Lactobacillus helveticus DSM 14998 is shown in SEQ ID NO 19 and SEQ ID NO 20.
Accordingly, in a preferred embodiment, a lactic acid bacterium, as described herein, comprises the prtH200 gene and a gene sequence encoding an open reading frame (termed orfF1), wherein the gene sequence is defined as

    
    
        orfF1 is a DNA sequence encoding an open reading frame, which DNA sequence is selected from the group comprising of:
        
            (a) the DNA sequence shown in positions 1-5358 in SEQ ID NO 19;
            (b) a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 40% identical to a corresponding fragment of the DNA sequence defined in (a);
            (c) a DNA sequence that encodes a polypeptide comprising a fragment of at least 200 amino acids (aa) that is at least 30% identical to a corresponding fragment of the polypeptide sequence shown in positions 1-1785 of SEQ ID NO 20;
            (d) a DNA sequence which hybridizes with a double-stranded DNA probe comprising the DNA sequence shown in positions 1-5358 in SEQ ID NO 19 at low stringency; and
            (e) a DNA sequence which is a fragment of the DNA sequences specified in (a), (b), (c), or (d).
        
        
    
    


At the filing date of the present application, a standard protein-protein BLAST [blastp] search using the deduced orfF1 amino acid sequence shown in position 1-1785 in SEQ ID NO 20 as reference sequence gave relatively limited conclusive results in relation to published homologous sequences.
However, without being limited to theory, it is believed that an orfF1 gene as described herein encodes a cell wall proteinase. Consequently, in a preferred embodiment the orfF1 gene as described herein encodes a cell wall proteinase.
Preferably, a lactic acid bacterium, as described herein, comprises the prtH200 gene, the orfF3 gene, the orfF4 gene and the orfF1 gene as described herein.
Pulsed Field Gel Electrophoresis (PFGE) Fingerprinting
Another suitable way of characterizing a LAB as described herein is by use a so-called pulsed field gel electrophoresis (PFGE) fingerprinting technique.
PFGE fingerprinting is a standard technique. The herein preferred protocol is that chromosomal DNA is isolated from a bacterium of interest, completely digested with restriction enzyme SmaI and run on an agarose gel together with a suitable standard MW marker. Working example 7 herein describes a preferred PFGE protocol in further details.
By analyzing the agarose gel, specific DNA bands for a LAB of interest may be identified. FIG. 1 herein, shows this for the herein described very preferred Lactobacillus helveticus strain CHCC5951 (deposited with accession number DSM 14998). FIG. 1 and working example 7 show that a PFGE fingerprint of the CHCC5951 strain identifies at least 12 characterizing bands. These bands are:

    
    
        band no. 1: 283 kbp
        band no. 2: 259 kbp
        band no. 3: 219 kbp
        band no. 4: 138 kbp
        band no. 5: 127 kbp
        band no. 6: 119 kbp
        band no. 7: 106 kbp
        band no. 8: 88 kbp
        band no. 9: 71 kbp
        band no. 10: 59 kbp
        band no. 11: 54 kbp
        band no. 12: 46 kbp
    
    


Identical PFGE fingerprints have been made for a number of strains including the Calpis Food Industry and Valio Ltd strains described in the background art section above.
None of the tested strains comprised a combination of the two bands corresponding to band no. 1 of 283 kbp and band no 3 of 219 kbp. Further none of the tested strains comprised a band corresponding to the band no. 12 of 46 kbp.
These bands encode information that is responsible for the improved characteristic of a LAB as described herein.
Accordingly, in a preferred embodiment, a lactic acid bacterium, as described herein, comprises the prtH200 gene and a combination of the two PFGE fingerprinting bands corresponding to the of 283 kbp and the band of 219 kbp of a PFGE fingerprint of the Lactobacillus helveticus bacterium with the registration number DSM 14998, wherein the PFGE fingerprinting is made by a protocol comprising isolation of chromosomal DNA of the lactic acid bacterium, completely digest the chromosomal DNA with restriction enzyme SmaI and electrophorese the digested DNA on an agarose gel.
The term “PFGE fingerprinting band corresponding to the specific mentioned size band of a PFGE fingerprint of the Lactobacillus helveticus bacterium with the registration number DSM 14998” should be understood in the sense that Lactobacillus helveticus DSM 14998 may be seen as a reference strain. Preferably, there should be made an identical (using the same protocol) PFGE fingerprint of a LAB of interest and the Lactobacillus helveticus DSM 14998. The digested DNA of the LAB of interest and the Lactobacillus helveticus DSM 14998 could thereafter be electrophoresed on the same agarose gel together with a suitable MW marker. By analyzing the electrophoresed agarose gel, the skilled person may then by use of routine skills determine if the LAB of interest comprise the band(s) corresponding to the specific mentioned size band of the Lactobacillus helveticus DSM 14998. As known to the skilled person, there might be some minor variation is size. In the present context, such minor variation should preferably be within ±5 kbp. Accordingly, if the reference band of Lactobacillus helveticus DSM 14998 is e.g. 283 kbp then the corresponding band of an analyzed LAB of interest should preferably be of a size of 283 kbp ±5 kb.
In another preferred embodiment, a lactic acid bacterium, as described herein, comprises the prtH200 gene and the PFGE fingerprinting band corresponding to the 46 kbp band of a PFGE fingerprint of the Lactobacillus helveticus bacterium with the registration number DSM 14998, wherein the PFGE fingerprinting is made by a protocol comprising isolation of chromosomal DNA of the lactic acid bacterium, completely digest the chromosomal DNA with restriction enzyme SmaI and electrophorese the digested DNA on an agarose gel.
Analyzed in the same way it is more preferred that the lactic acid bacterium, as described herein, comprises all of the following PFGE fingerprinting bands:

    
    
        band corresponding to the 283 kbp band of the Lactobacillus helveticus DSM 14998,
        band corresponding to the 219 kbp band of the Lactobacillus helveticus DSM 14998,
        band corresponding to the 46 kbp band of the Lactobacillus helveticus DSM 14998.
    
    


Most preferably, the lactic acid bacterium, as described herein, comprises bands corresponding to all of the twelve PFGE fingerprinting bands given above for the Lactobacillus helveticus DSM 14998.
PCR Amplification
As said above, the presence of the gene sequences, as described herein, may preferably be verified by PCR amplification using PCR primers designed according to the teaching herein. When the skilled person has suitable designed PCR primers it is easy for him to verify the presence or not of these genes in a lactic acid bacterium using his general knowledge to make a specific suitable PCR amplification protocol.
Preferably the PCR amplification protocol (reaction) is made according to the description of Example 1 herein.
Once the PCR have been performed it is routine for the skilled person to investigate whether or not the resulting PCR amplified fragments corresponds to fragments of genes as described herein. Normally this may be identified already based on the size of the PCR fragment, since the skilled person generally roughly knows how big the size of a positive PCR fragment would be. A positive PCR fragment relates to a PCR fragment of a gene as described herein. Alternatively, the PCR fragment may be DNA sequenced and the resulting DNA sequence may then be compared with the sequences disclosed herein. Further, a lactic acid bacterium with a lethal mutation in the gene corresponding to the PCR fragment could be constructed. The proteolytic activity (see below) of this constructed bacterium could then be compared with the corresponding wildtype bacterium and a measurable change in proteolytic activity between the two cells would experimentally confirm whether or not the gene corresponding to the amplified PCR fragment is a gene encoding a lactic acid cell wall proteinase as described herein.
In summary, the skilled person can routinely identify whether or not a specific lactic acid bacterium comprises gene(s) capable of giving corresponding positive PCR fragments using PCR primers designed according to the teaching herein.
PCR is the preferred way to investigate the presence or not in the lactic acid bacteria of the genes as described herein. However, it may be done in other ways such as e.g. by Southern blot analysis.
PCR primers:
As explained above suitable PCR primers in relation to the PrtH200 gene are:










PrtH200:




(a): (S): 5′ CGATGATAATCCTAGCGAGC3′,



 



     (A): 5′ TGGCAGAACCTGTGCCTA 3′;



 



(b): (S): 5′ GCCAAGACGCCTCTGGTA 3′,



 



     (A): 5′ TAGGTATAGTTTCCATCAGGA 3′; and



 



(c): (S): 5′ AARGTWCCWTAYGGYYWYAAYTA 3′,



 



     (A): 5′ GCCATDSWDGTRCCDSWCATDTK 3′.






    PrtH200: (a): (S) is shown in SEQ ID NO 7; PrtH200: (a): (A) is shown in SEQ ID NO 8;
    PrtH200: (b): (S) is shown in SEQ ID NO 9; PrtH200: (b): (A) is shown in SEQ ID NO 10;
    PrtH200: (c): (S) is shown in SEQ ID NO 11; PrtH200: (c): (A) is shown in SEQ ID NO 12.


When using the primer set (a) the amplified PrtH200 PCR fragment should preferably be of a size between 400 bp and 800 bp, more preferably of a size between 500 bp and 700 bp. When using the primer set (b) the amplified PrtH200 PCR fragment should preferably be of a size between 200 bp and 500 bp, more preferably of a size between 250 bp and 375 bp. When using the primer set (c) the amplified PrtH200 PCR fragment should preferably be of a size between 400 bp and 800 bp, more preferably of a size between 500 bp and 700 bp.
The most preferred PrtH200 related PCR primers are primer set (a) and primer set (b).
As said above suitable PCR primers in relation to orfF3 are:








orfF3:



(a): (S): 5′ CGAAGGCGATAAGTCAAACTTTGATAATGC 3′,


 


     (A): 5′ CCCGGTTCTGTAAGATAATTTGGATCG 3′; and


 


(b): (S): 5′ ASTCWRRYTTYGATRATGCW 3′,


 


     (A): 5′ BHKYAMSAWARTTTGGATCR 3′.






    orfF3: (a): (S) is shown in SEQ ID NO 13; orfF3: (a): (A) is shown in SEQ ID NO 14;
    orfF3: (b): (S) is shown in SEQ ID NO 15; orfF3: (b): (A) is shown in SEQ ID NO 16.


When using the primer set (a) the amplified orfF3 PCR fragment should preferably be of a size between 1250 bp and 1900 bp, more preferably of a size between 1500 bp and 1725 bp. When using the primer set (b) the amplified orfF3 PCR fragment should preferably be of a size between 1250 bp and 1900 bp, more preferably of a size between 1500 bp and 1725 bp.
The most preferred orfF3 related PCR primers is the primer set (a).
As said above suitable PCR primers in relation to orfF4 is:










orfF4:




(a): (S): 5′ GGTGTTGCTCCTGAAGC 3′



 



     (A): 5′ ACTCTAGCACCAGCTAATTGAACATCATG 3′.






    orfF4: (a): (S) is shown in SEQ ID NO 17; orfF4: (a): (A) is shown in SEQ ID NO 18.


When using the primer set (a) the amplified orfF4 PCR fragment should preferably be of a size between 700 bp and 1150 bp, more preferably of a size between 875 bp and 1025 bp .
Homology/Identity of DNA Sequences
The DNA sequence homology/identity referred to above is determined as the degree of identity between two sequences indicating a deviation of the first sequence from the second.
At the filing date of the present invention, the National Center for Biotechnology Information (NCBI) offered at it Internet site (http://www.ncbi.nlm.nih.gov/) the possibility of making a standard BLAST computer sequence homology search.
BLAST program is described in [Altschul et al (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402].
In the present context, a preferred computer homology search program is a “Standard nucleotide-nucleotide BLAST [blastn]” search as specified, at the filing date of the present application, at the NCBI Internet site with setting filter: Low complexity; Expect: 10, Word Size: 11.
The reference sequence is introduced into the program and the program identifies fragments of published sequences together the identity percentage to a corresponding fragment of the reference sequence.
Using this Standard nucleotide-nucleotide BLAST computer program, a prtH200 sequence as described herein is preferably a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 60% identical to a corresponding fragment of the prtH200 DNA sequence shown in position 1-5550 of SEQ ID NO 1, more preferably a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 70% identical to a corresponding fragment of the prtH200 DNA sequence shown in position 1-5550 of SEQ ID NO 1, and even more preferably a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 80% identical to a corresponding fragment of the prtH200 DNA sequence shown in position 1-5550 of SEQ ID NO 1.
With the identity percentages given above, it is preferred that the fragment is at least 100 bp pairs (bp), more preferably that the fragment is at least 200 bp pairs (bp), even more preferably that the fragment is at least 400 bp pairs (bp), and most preferably that the fragment is at least 1500 bp pairs (bp).
Using this Standard nucleotide-nucleotide BLAST computer program, a orfF3 sequence as described herein is preferably a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 60% identical to a corresponding fragment of the orfF3 DNA sequence shown in position 1-2679 of SEQ ID NO 3, more preferably a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 70% identical to a corresponding fragment of the orfF3 DNA sequence shown in position 1-2679 of SEQ ID NO 3, and even more preferably a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 80% identical to a corresponding fragment of the orfF3 DNA sequence shown in position 1-2679 of SEQ ID NO 3.
With the identity percentages given above, it is preferred that the fragment is at least 100 bp pairs (bp), more preferably that the fragment is at least 200 bp pairs (bp), even more preferably that the fragment is at least 400 bp pairs (bp), and most preferably that the fragment is at least 1500 bp pairs (bp). p Using this Standard nucleotide-nucleotide BLAST computer program, a orfF4 sequence as described herein is preferably a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 60% identical to a corresponding fragment of the orfF4 DNA sequence shown in position 1-4881 of SEQ ID NO 5, more preferably a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 70% identical to a corresponding fragment of the orfF4 DNA sequence shown in position 1-4881 of SEQ ID NO 5, and even more preferably a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 80% identical to a corresponding fragment of the orfF4 DNA sequence shown in position 1-4881 of SEQ ID NO 5.
With the identity percentages given above, it is preferred that the fragment is at least 100 bp pairs (bp), more preferably that the fragment is at least 200 bp pairs (bp), even more preferably that the fragment is at least 400 bp pairs (bp), and most preferably that the fragment is at least 1500 bp pairs (bp).
Alternatively, the homology/identity may suitably be determined by means of computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, Aug. 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711)(Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453).
Using GAP with the following settings for DNA sequence comparison, GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the preferred identity percentages given above in relation to the BLAST program are also the preferred identities when using GAP.
Homology to Amino Acid Sequences
Similar to the nucleotide homology analysis, in the present context, a preferred computer homology search program is a “Standard protein-protein BLAST [blastp]” search as specified, at the filing date of the present application, at the NCBI Internet site with settings Composition-based statistics: yes, filter: Low complexity; Expect: 10, Word Size: 3, Matrix: BLOSUM 62, Gap Costs: Existence 11 Extension 1.
Using this standard protein-protein BLAST computer program, a prtH200 sequence as described herein is preferably a DNA sequence that encodes a polypeptide, exhibiting cell wall proteinase activity, comprising a fragment of at least 200 amino acids (aa) that is at least 40% identical to a corresponding fragment of the prtH200 polypeptide sequence shown in positions 1-1849 of SEQ ID NO 2, more preferably a DNA sequence that encodes a polypeptide, exhibiting cell wall proteinase activity, comprising a fragment of at least 200 amino acids (aa) that is at least 50% identical to a corresponding fragment of the prtH200 polypeptide sequence shown in positions 1-1849 of SEQ ID NO 2, even more preferably a DNA sequence that encodes a polypeptide, exhibiting cell wall proteinase activity, comprising a fragment of at least 200 amino acids (aa) that is at least 65% identical to a corresponding fragment of the prtH200 polypeptide sequence shown in positions 1-1849 of SEQ ID NO 2, and most preferably a DNA sequence that encodes a polypeptide, exhibiting cell wall proteinase activity, comprising a fragment of at least 200 amino acids (aa) that is at least 80% identical to a corresponding fragment of the prtH200 polypeptide sequence shown in positions 1-1849 of SEQ ID NO 2.
With the identity percentages given above, it is preferred that the fragment is at least 300 amino acids (aa), more preferably that the fragment is at least 400 amino acids (aa), even more preferably that the fragment is at least 800 amino acids (aa), and most preferably that the fragment is at least 1200 amino acids (aa).
Using this standard protein-protein BLAST computer program, a orfF3 sequence as described herein is preferably a DNA sequence that encodes a polypeptide comprising a fragment of at least 200 amino acids (aa) that is at least 40% identical to a corresponding fragment of the orfF3 polypeptide sequence shown in positions 1-893 of SEQ ID NO 4, more preferably a DNA sequence that encodes a polypeptide comprising a fragment of at least 200 amino acids (aa) that is at least 50% identical to a corresponding fragment of the orfF3 polypeptide sequence shown in positions 1-893 of SEQ ID NO 4, even more preferably a DNA sequence that encodes a polypeptide comprising a fragment of at least 200 amino acids (aa) that is at least 65% identical to a corresponding fragment of the orfF3 polypeptide sequence shown in positions 1-893 of SEQ ID NO 4, and most preferably a DNA sequence that encodes a polypeptide comprising a fragment of at least 200 amino acids (aa) that is at least 80% identical to a corresponding fragment of the orfF3 polypeptide sequence shown in positions 1-893 of SEQ ID NO 4.
With the identity percentages given above, it is preferred that the fragment is at least 300 amino acids (aa), more preferably that the fragment is at least 400 amino acids (aa), even more preferably that the fragment is at least 800 amino acids (aa), and most preferably that the fragment is at least 1200 amino acids (aa).
Using this standard protein-protein BLAST computer program, a orfF4 sequence as described herein is preferably a DNA sequence that encodes a polypeptide comprising a fragment of at least 200 amino acids (aa) that is at least 40% identical to a corresponding fragment of the orfF4 polypeptide sequence shown in positions 1-1627 of SEQ ID NO 6, more preferably a DNA sequence that encodes a polypeptide comprising a fragment of at least 200 amino acids (aa) that is at least 50% identical to a corresponding fragment of the orfF4 polypeptide sequence shown in positions 1-1627 of SEQ ID NO 6, even more preferably a DNA sequence that encodes a polypeptide comprising a fragment of at least 200 amino acids (aa) that is at least 65% identical to a corresponding fragment of the orfF4 polypeptide sequence shown in positions 1-1627 of SEQ ID NO 6, and most preferably a DNA sequence that encodes a polypeptide comprising a fragment of at least 200 amino acids (aa) that is at least 80% identical to a corresponding fragment of the orfF4 polypeptide sequence shown in positions 1-1627 of SEQ ID NO 6.
With the identity percentages given above, it is preferred that the fragment is at least 300 amino acids (aa), more preferably that the fragment is at least 400 amino acids (aa), even more preferably that the fragment is at least 800 amino acids (aa), and most preferably that the fragment is at least 1200 amino acids (aa).
Alternatively, the homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, Aug. 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453.
Using GAP with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1, the preferred identity percentages given above in relation to the BLAST program are also the preferred identities when using GAP
Hybridization
The hybridization referred to above is intended to comprise an analogous DNA sequence which hybridizes to a double-stranded DNA probe. Suitable experimental conditions for determining hybridization at low, medium, or high stringency between a nucleotide probe and a homologous DNA or RNA sequence involve presoaking of the filter containing the DNA fragments or RNA to hybridize in 5×SSC (Sodium chloride/Sodium citrate, Sambrook et al. 1989) for 10 min, and prehybridization of the filter in a solution of 5×SSC, 5×Denhardt's solution (Sambrook et al. 1989), 0.5% SDS and 100 mu g/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing 10 ng/ml of a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13), P-dCTP-labeled (specific activity>1×10 cpm/ mu g ) probe for 12 hours at 45° C. The filter is then washed twice for 30 minutes in 2×SSC, 0.5% SDS at a temperature of at least 55° C. (low stringency), more preferably at least 60° C. (medium stringency), still more preferably at least 65° C. (medium/high stringency), even more preferably at least 70° C. (high stringency), even more preferably at least 75° C. (very high stringency).
Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using an X-ray film.
A Fermentable Material Comprising Proteins
The term “a material comprising proteins” in relation expressions such as “fermenting a material comprising proteins with a lactic acid bacteria” denotes herein any material comprising proteins wherein the lactic acid bacterium may growth and thereby make it possible to obtain a fermented material that comprises peptides. Peptides should be understood as peptides obtained by lactic acid bacteria cell wall proteinase based hydrolysis the proteins.
For example, a material comprising proteins may be a suitable standard lactic acid bacteria fermentation media such as M17 broth or MRS broth. Preferably the media comprises animal milk proteins preferably enumerated by, for example milk protein components, such as whole or defatted animal milk or milk casein.
Food Material
The food material should comprise animal milk proteins or vegetable proteins.
Preferably it comprises animal milk proteins preferably enumerated by, for example milk protein components, such as whole or defatted animal milk or milk casein.
Food material with vegetable proteins may preferably be enumerated by, for example corn, corn protein, wheat, wheat protein, soybean, defatted soybean or soybean protein.
Lactic Acid Bacterium
The term “lactic acid bacteria” denotes herein a group of Gram-positive, non-sporing bacteria, which carry out a lactic acid fermentation of sugars.
Among others, it includes species of lactic acid bacteria belonging to genus Lactobacillus, such as Lactobacillus helveticus, Lactobacillus delbruekii subsp. bulgaricus, etc., lactic acid bacteria belonging to genus Lactococcus, such as Lactococcus lactis, lactic acid bacteria belonging to genus Streptococcus, such as Streptococcus salivarius subsp. thermophilus, lactic acid bacteria belonging to genus Leuconostoc, such as Leuconostoc lactis, lactic acid bacteria belonging to genus Bifidobacterium, such as Bifidobacterium longum or Bifidobacterium breve, and lactic acid bacteria belonging to genus Pediococcus.
The lactic acid bacteria may be used as a mixture with other microorganisms, e.g. yeasts.
Numerous different lactic acid bacteria are publicly available to the skilled person. Reference is e.g. made to Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ); and the Internet taxonomy browser of NCBI.
In order to identify a suitable specific lactic acid bacterium, it is routine work for the skilled person to simply e.g. obtain an adequate amount of different public available bacteria and identify one or more specific strains, which comprise the gene sequence(s) as discussed herein. Preferably, this is done by a PCR amplification protocol as described herein.
Preferably, the lactic acid bacterium is a bacterium of the phylium Firmicutes, more preferably of the class Bacilli, even more preferably of the order Lactobacillales. Within this order a preferred lactic acid bacterium is a bacterium of the family Lactobacillaceae, more preferably of the genus Lactobacillus. Most preferably it is a Lactobacillus helveticus strain. For further details in relation to taxomony reference is made to (Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 1: The Archea and the Deeply Branching and Phototrophic Bacteria).
A sample of a particular preferred Lactobacillus helveticus strain CHCC5951 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) under the accession number DSM 14998 with a deposit date of 15th May 2002. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
Accordingly, a particular preferred embodiment relates to a process as described herein wherein the lactic acid bacterium is Lactobacillus helveticus with the registration number DSM 14998.
In this relation, a separate aspect of the invention relates to a Lactobacillus helveticus bacterium with the registration number DSM 14998 or a mutant thereof.
Using as starting material the deposited the deposited DSM 14998 strain, the skilled reader can by conventional mutagenesis or re-isolation techniques obtain further mutants or derivatives which retain the ability to be suitable for preparing peptides with anti-hypertensive properties.
Further a sample of a Lactobacillus helveticus strain CHCC4080 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) under the accession number DSM 14997 with a deposit date of 15th May 2002. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
Proteolytic Activity of the Lactic Acid Bacteria
The prtH200, orfF3 and orfF4 gene sequences as described herein may be seen as fingerprints highly suitable to identity useful lactic acid bacteria as described herein. Without being limited to theory, theoretically it may be that some strains could exist that despite comprising the fingerprint gene sequence(s) as described herein are not exhibiting the advantageous properties as described herein.
Accordingly, once having identified, in a lactic acid bacterium, the presence of fingerprint gene sequence(s) as described herein, it may be advantageous to test the proteolytic activity of the lactic acid bacterium. In the present context, a preferred lactic acid bacterium has preferably a proteolytic activity as described below.
In the present context, a lactic acid bacterium is considered to have proteolytic activity when it is capable of synthesizing an active cell wall proteinase. In other words, capable of proving a proteinase, which is active outside the intracellular part of the bacterium. Further, the proteinase should have a specificity making it capable of degrading proteins (e.g. casein comprised in milk) to obtain peptides with anti-hypertensive properties.
Preferably, the proteolytic activity of a bacterium is ascertained by a protocol comprising the steps:

    
    
        (i) fermenting overnight 200 ml of a food material with the bacterium,
        (ii) extracting the produced peptides, and
        (iii) measuring the anti-hypertensive properties of the extracted peptides by an assay measuring the peptidic concentration required to inhibit 50% of the ACE activity.
    
    


The ACE inhibition activity assay is herein also termed DL50. The lower the DL50 value is the better is the anti-hypertensive effect of the peptides comprised in the fermented food material.
In step (i) of the protocol, the food material is preferably fresh milk. Further, the bacterium is preferably inoculated to the food material in the form of an overnight stock culture of (1% v/v) and maintained overnight at a suitable temperature. A suitable temperature is a temperature that is suitable for growth of the bacterium. The skilled person knows how to identify this for a particular lactic acid bacterium. For Lactobaccillus species a suitable temperature is 37° C. and for Lactococcus species a suitable temperature is 30° C.
In working example 2 herein is provided a detailed preferred protocol for the fermenting and extracting steps and a detailed preferred protocol for the DL50 ACE activity assay.
Preferably, the lactic acid bacterium has a proteolytic activity making it capable of, in a protocol comprising the steps:

    
    
        (i) fermenting overnight 200 ml of a food material with the bacterium,
        (ii) extracting the produced peptides, and
        (iii) measuring the anti-hypertensive properties of the extracted peptides by an assay measuring the peptidic concentration required to inhibit 50% of the ACE activity (DL50),

producing peptides with an angiotensin-converting enzyme (ACE) inhibition activity (DL50) of from 0.25 to 5.0 (mg/ml).

    
    


More preferably, the lactic acid bacterium is capable of producing peptides with an angiotensin-converting enzyme (ACE) inhibition activity (DL50) of from 0.25 to 4.0 (mg/ml), and even more preferably the proteolytic lactic acid bacterium is capable of producing peptides with an angiotensin-converting enzyme (ACE) inhibition activity (DL50) of from 0.25 to 3.5 (mg/ml).
The lower DL50 range may be, instead of 0.25 mg/ml, 1.0 mg/ml.
Fermentation
In the process of the present invention, the food material is fermented by lactic acid bacteria under operating conditions, which may be varied depending on the types of the food material and/or the combination of the lactic acid bacteria. Preferably, if the food material is not already an aqueous solution, food material is dissolved in a suitable aqueous solution, which is then admixed with lactic acid bacteria and cultivated by way of fermentation.
The culturing of the lactic acid bacteria may be performed by adding pre-cultured lactic acid bacteria starter to the food material medium, which may have been previously heat-sterilized and cooled to the predetermined temperature for incubation. The inoculation amount of the lactic acid bacteria starter may preferably be 105 to 107 cells of lactic acid bacteria/ml medium. The temperature for incubation is usually 20 to 50° C. and preferably 30 to 45° C. The incubation time is usually 3 to 48 hours and preferably 6 to 24 hours. Particularly, it is preferred to perform cultivation in the medium having pH in a range of 3.5 to 7, more preferably 5 to 6, in order to perform cultivation of lactic acid bacteria efficiently. Further, it is preferred to perform pH-stat cultivation maintaining pH in a range of 4 to 7. The incubation may be terminated, without restriction, when the number of lactic acid bacteria exceeds 108 cells/ml.
A preferred embodiment relates to a process, as described herein, wherein the fermenting of the food material is performed under conditions, which produce from 0.5 to 25 mg peptides with anti-hypertensive properties per 100 ml of the food material, more preferably which produce from 1 to 5 mg peptides with anti-hypertensive properties per 100 ml of the food material.
Subsequent Purification of the Anti-hypertensive Peptides from the Fermented Food Material
As stated above, the use of the lactic acid bacteria, as described herein, provides directly after the fermentation a useful amount of peptides with very good anti-hypertensive properties.
However, in some circumstances it may be preferred to perform a subsequent purification of the anti-hypertensive peptides from the fermented food material. This may for instance be when the peptides are to be used in a pharmaceutical tablet, which requires a very high concentration of the anti-hypertensive peptides.
Accordingly, an embodiment of the invention relates to a process for preparing peptides with anti-hypertensive properties of as described herein, wherein the fermented food material is further processed in a way that purify or up-concentrate the peptides with anti-hypertensive properties.
For instance, the fermented food material containing peptides with anti-hypertensive properties may be centrifuged, and the resulting supernatant may be subjected to purifying treatment with a reverse-phase resin, for obtaining a sample in which the content of the peptides with anti-hypertensive properties is increased.
The centrifugation may preferably be performed, for example, at 2,000 to 20,000 rpm for 1 to 20 minutes. The centrifugation may also be performed in a centrifugator.
The purifying treatment with a reverse-phase resin may be performed by absorption and elution of the peptides with a reverse-phase resin, and/or by reverse-phase chromatography, thereby increasing purity of the peptides.
For further technical details in relation to this reverse-phase resin protocol reference is made to EP821968.
Alternatively, the fermented food material is further processed in a way wherein a nanofiltration is performed on the fermented food material. This may be done in order to remove lactic acid or monovalent ions from the fermented food material.
For further technical details in relation to this nanofiltration protocol reference is made to WO01/32905.
A Functional Food Product Comprising Peptides with Anti-hypertensive Properties
As said above, the use of lactic acid bacteria comprising the prtH200 proteinases as described herein provides directly after the fermentation a useful amount of peptides with very good anti-hypertensive properties. Consequently, it is not considered necessary to further purify or up-concentrate the peptides from the fermented food material. The fermented food material may be packed directly and provided to the market as a functional food product or a food product additive, e.g. in a freeze-dried form.
In Example 6 this is demonstrated. In short, the results of Example 6 show that fermented milk in itself without any further treatment has good blood pressure reducing effects. Further freeze-dried fermented milk could be suspended in neutral milk and thereby give a suitable functional food product. The freeze-dried fermented milk could therefore be seen as a suitable food additive product.
Accordingly, an embodiment of the invention relates to a process for preparing a functional food product comprising peptides with anti-hypertensive properties, the process comprising following steps:

    
    
        (i) preparing a fermented food material according to a process for preparing peptides with anti-hypertensive properties as described herein,
        (ia) drying the fermented food material, and
        (ii) packing it in a suitable way to get a functional food product.
    
    


Step (ia) is preferably freeze drying.
In other words, since there is no need for further treatment of the final functional food product it may also be characterized by it comprises a substantial part of the lactic acid bacteria that were present during the fermentation. Example 6 clearly demonstrates that such a product works fine and since lactic acid bacteria are know to be beneficial in a number of contexts it might actually be an advantage that such lactic acid bacteria are present in the functional food product.
Accordingly, an embodiment of the invention relates to a process for preparing a functional food product comprising peptides with anti-hypertensive properties, the process comprising following steps:

    
    
        (ii) preparing a fermented food material according to a process for preparing peptides with anti-hypertensive properties as described herein,
        (ia) keeping at least a part of the lactic acid bacteria present during the fermentation in the fermented food material, and
        (ii) packing it in a suitable way to get a functional food product.
    
    


The term “keeping at least a part of the lactic acid bacteria present during the fermentation in the fermented food material” should be understood in view of above explaining that it is not considered necessary to remove the lactic acid bacteria. Some of the bacteria may be removed. Quantitatively, it may be expressed as keeping at least a 5% part of the lactic acid bacteria present during the fermentation in the fermented food material or keeping at least a 20% part of the lactic acid bacteria present during the fermentation in the fermented food material.
As shown in example 6 the lactic acid bacteria kept in the functional food product may be dead or alive, since a heat-treated fermented food material also had good blood reducing properties.
It may be preferred to perform a subsequent purification of the anti-hypertensive peptides from the fermented food material.
Accordingly, an embodiment of the invention relates to a process for preparing a functional food product comprising peptides with anti-hypertensive properties, the process comprising following steps:

    
    
        (iii) preparing a fermented food material according to a process for preparing peptides with anti-hypertensive properties as described herein,
        (ia) the fermented food material of step (i) is further processed in a way that purify or up-concentrate the peptides with anti-hypertensive properties according to a process as described above,
        (ib) the purified or up-concentrated peptides of step (ia) is then added to a food material, and
        (iv) packing it in a suitable way to get a functional food product.
    
    


Preferably, the food material of step (ib) is a fermented food material prepared according to a process as described herein. This corresponds to a situation where one wants a relatively high concentration of the peptides with anti-hypertensive properties in the functional food product.
Use and Preferred Doses of the Peptides with Anti-hypertensive Properties
The peptides with anti-hypertensive properties, obtained by a process of the present invention, are usually a mixture of peptides, and may contain other peptides. For use as foods and drinks, the fermented food material containing the petides and/or purified products thereof may be used directly. Alternatively, the agent may be powdered by freeze drying, spray drying or drum dryer drying, before use.
A preferred effective amount of the anti-hypertensive peptides of the present invention varies depending upon the age and condition of a person, and is in a range of 0.05 to 10 mg/kg body weight/day. It is preferable to administer 0.3 to 3.0 mg/kg body weight/day. If the dose is not less than 0.05 mg/kg body weight/day, sufficient effect may be expected. If the dose is not more than 10 mg/kg body weight/day, the effect may be exhibited efficiently.
Use for Cholesterol Lowering Therapy
The article (Teo, K. et al, Circulation (2000) 102:1748-1754) describes that ACE inhibition drugs (EnalApril) may have a positive effect in a cholesterol lowering therapy in particular in reducing coronary atherosclerotic disease.
Accordingly, a separate aspect of the invention relates to use of peptides with anti-hypertensive properties obtained by a process of the present invention for the manufacture of a medicament or a functional food product for use in a cholesterol lowering therapy in particular in relation to reducing coronary atherosclerotic disease.
Claim Presentation of Aspects and Embodiments
Aspects and embodiments of the invention may be presented in a so-called claim format. Some aspect and embodiments of the invention are given below is such a claim format.

    1. A method for obtaining a lactic acid bacterium comprising
    
        (i) investigating whether a lactic acid bacterium is a lactic acid bacterium that comprises a gene sequence encoding a cell wall proteinase (termed prtH200) wherein the gene sequences is identifiable by PCR amplification of genomic DNA of the lactic acid bacterium using sets of PCR primers selected from the group consisting of [sense sequence (S); antisense sequence (A)]:
    
    












PrtH200:




(a): (S): 5′ CGATGATAATCCTAGCGAGC3′,



 



     (A): 5′ TGGCAGAACCTGTGCCTA 3′;



 



(b): (S): 5′ GCCAAGACGCCTCTGGTA 3′,



 



     (A): 5′ TAGGTATAGTTTCCATCAGGA 3′; and



 



(c): (S): 5′ AARGTWCCWTAYGGYYWYAAYTA 3′,



 



     (A): 5′ GCCATDSWDGTRCCDSWCATDTK 3′; and






    
    
        (ii) if the lactic acid bacterium fulfills the criteria of step (i) then the lactic acid bacterium culture is obtained; or
        (iii) if the lactic acid bacterium does not fulfill the criteria of step (i) then repeat step (i) with another lactic acid bacterium.
    
    
    2. The method for obtaining a lactic acid bacterium of claim 1, wherein step (i) also comprises investigating whether the lactic acid bacterium comprises a gene sequence (termed orfF3) encoding an open reading frame wherein the gene sequences is identifiable by PCR amplification of genomic DNA of the lactic acid bacterium using sets of PCR primers selected from the group consisting of [sense sequence (S); antisense sequence (A)]:










orfF3:



(a): (S): 5′ CGAAGGCGATAAGTCAAACTTTGATAATGC 3′,


 


     (A): 5′ CCCGGTTCTGTAAGATAATTTGGATCG 3′; and


 


(b): (S): 5′ ASTCWRRYTTYGATRATGCW 3′,


 


     (A): 5′ BHKYAMSAWARTTTGGATCR 3′.






    3. A method for obtaining a lactic acid bacterium comprising
    
        (i) investigating whether a lactic acid bacterium is a lactic acid bacterium thatcomprises a gene sequence encoding a cell wall proteinase (termed prtH200), wherein the gene sequence is defined as
        
            prtH200 is a DNA sequence encoding an enzyme exhibiting cell wall proteinase activity, which DNA sequence is selected from the group consisting of:
            
                (a) the DNA sequence shown in positions 1-5550 in SEQ ID NO 1;
                (b) a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 50% identical to a corresponding fragment of the DNA sequence defined in (a);
                (c) a DNA sequence that encodes a polypeptide, exhibiting cell wall proteinase activity, comprising a fragment of at least 200 amino acids (aa) that is at least 30% identical to a corresponding fragment of the polypeptide sequence shown in positions 1-1849 of SEQ ID NO 2;
                (d) a DNA sequence which hybridizes with a double-stranded DNA probe comprising the DNA sequence shown in positions 1-5550 in SEQ ID NO 1 at low stringency; and
                (e) a DNA sequence which is a fragment of the DNA sequences specified in (a), (b), (c), or (d);
            
            
            (ii) if the lactic acid bacterium fulfills the criteria of step (i) then the lactic acid bacterium is obtained; or
            (iii) if the lactic acid bacterium does not fulfill the criteria of step (i) then repeat step (i) with another lactic acid bacterium.
        
        
    
    
    4. The method for obtaining a lactic acid bacterium of claim 3, wherein step (i) also comprises investigating whether the lactic acid bacterium comprises a gene sequence encoding an open reading frame (termed orfF3), wherein the gene sequence is defined as
    
        orfF3 is a DNA sequence encoding an open reading frame, which DNA sequence is selected from the group comprising of:
        
            (a) the DNA sequence shown in positions 1-2679 in SEQ ID NO 3;
            (b) a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 40% identical to a corresponding fragment of the DNA sequence defined in (a);
            (c) a DNA sequence that encodes a polypeptide comprising a fragment of at least 200 amino acids (aa) that is at least 30% identical to a corresponding fragment of the polypeptide sequence shown in positions 1-893 of SEQ ID NO4;
            (d) a DNA sequence which hybridizes with a double-stranded DNA probe comprising the DNA sequence shown in positions 1-5550 in SEQ ID NO 3 at low stringency; and
            (e) a DNA sequence which is a fragment of the DNA sequences specified in (a), (b), (c), or (d).
        
        
    
    
    5. The method for obtaining a lactic acid bacterium of any of claims 1 to 4, wherein the lactic acid bacterium has a proteolytic activity making it capable of, in a protocol comprising the steps:
    
        (i) fermenting overnight 200 ml of a food material with the bacterium;
        (ii) extracting the produced peptides, and
        (iii) measuring the anti-hypertensive properties of the extracted peptides by an assay measuring the peptidic concentration required to inhibit 50% of the ACE activity (DL50),

producing peptides with an angiotensin-converting enzyme (ACE) inhibition activity (DL50) of from 0.25 to 5.0 (mg/ml).

    
    
    6. The method for obtaining a lactic acid bacterium of any of the preceding claims, wherein the lactic acid bacterium is a bacterium of the phylium Firmicutes, more preferably of the class Bacilli, even more preferably of the order Lactobacillales.


7. The method for obtaining a lactic acid bacterium of claim 6, wherein the bacterium, of the order Lactobacillales, is a bacterium of the family Lactobacillaceae, more preferably of the genus Lactobacillus, and even more preferably a Lactobacillus helveticus bacterium.

    8. The method for obtaining a lactic acid bacterium of claim 7, wherein the bacterium is a Lactobacillus helveticus bacterium with the registration number DSM 14998 or a mutant thereof.
    9. A process for preparing peptides with anti-hypertensive properties, the process comprising fermenting a food material, comprising animal milk proteins or vegetable proteins, with a lactic acid bacterium to obtain a fermented food material which comprises the peptides with anti-hypertensive properties, characterized in that the lactic acid bacterium comprises a gene sequence encoding a cell wall proteinase (termed prtH200) wherein the gene sequences is identifiable by PCR amplification of genomic DNA of the lactic acid bacterium using sets of PCR primers selected from the group consisting of [sense sequence (S); antisense sequence (A)]:












PrtH200:




(a): (S): 5′ CGATGATAATCCTAGCGAGC3′,



 



     (A): 5′ TGGCAGAACCTGTGCCTA 3′;



 



(b): (S): 5′ GCCAAGACGCCTCTGGTA 3′,



 



     (A): 5′ TAGGTATAGTTTCCATCAGGA 3′; and



 



(c): (S): 5′ AARGTWCCWTAYGGYYWYAAYTA 3′,



 



     (A): 5′ GCCATDSWDGTRCCDSWCATDTK 3′.






    10. The process for preparing peptides with anti-hypertensive properties of claim 10, wherein the lactic acid bacterium also comprises a gene sequence (termed orfF3) encoding an open reading frame wherein the gene sequences is identifiable by PCR amplification of genomic DNA of the lactic acid bacterium using sets of PCR primers selected from the group consisting of [sense sequence (S); antisense sequence (A)]:










orfF3:



(a): (S): 5′ CGAAGGCGATAAGTCAAACTTTGATAATGC 3′,


 


     (A): 5′ CCCGGTTCTGTAAGATAATTTGGATCG 3′; and


 


(b): (S): 5′ ASTCWRRYTTYGATRATGCW 3′,


 


     (A): 5′ BHKYAMSAWARTTTGGATCR 3′.






    11. A process for preparing peptides with anti-hypertensive properties, the process comprising fermenting a food material, comprising animal milk proteins or vegetable proteins, with a lactic acid bacterium to obtain a fermented food material which comprises the peptides with anti-hypertensive properties, characterized in that the lactic acid bacterium comprises a gene sequence encoding a cell wall proteinase (termed prtH200), wherein the gene sequence is defined as
    
        prtH200 is a DNA sequence encoding an enzyme exhibiting cell wall proteinase activity, which DNA sequence is selected from the group consisting of:
        
            (a) the DNA sequence shown in positions 1-5550 in SEQ ID NO 1;
            (b) a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 50% identical to a corresponding fragment of the DNA sequence defined in (a);
            (c) a DNA sequence that encodes a polypeptide, exhibiting cell wall proteinase activity, comprising a fragment of at least 200 amino acids (aa) that is at least 30% identical to a corresponding fragment of the polypeptide sequence shown in positions 1-1849 of SEQ ID NO 2;
            (d) a DNA sequence which hybridizes with a double-stranded DNA probe comprising the DNA sequence shown in positions 1-5550 in SEQ ID NO 1 at low stringency; and
            (e) a DNA sequence which is a fragment of the DNA sequences specified in (a), (b), (c), or (d).
        
        
    
    
    12: The process for preparing peptides with anti-hypertensive properties of claim 11, wherein the lactic acid bacterium also comprises a gene sequence encoding an open reading frame (termed orfF3), wherein the gene sequence is defined as
    
        orfF3 is a DNA sequence encoding an open reading frame, which DNA sequence is selected from the group comprising of:
        
            (a) the DNA sequence shown in positions 1-2679 in SEQ ID NO 3;
            (b) a DNA sequence comprising a fragment of at least 75 base pairs (bp) that is at least 40% identical to a corresponding fragment of the DNA sequence defined in (a);
            (c) a DNA sequence that encodes a polypeptide comprising a fragment of at least 200 amino acids (aa) that is at least 30% identical to a corresponding fragment of the polypeptide sequence shown in positions 1-893 of SEQ ID NO4;
            (d) a DNA sequence which hybridizes with a double-stranded DNA probe comprising the DNA sequence shown in positions 1-2679 in SEQ ID NO 3 at low stringency; and
            (e) a DNA sequence which is a fragment of the DNA sequences specified in (a), (b), (c), or (d).
        
        
    
    
    13. A process for preparing peptides comprising
    
        (i) obtaining a lactic acid bacterium by a method for obtaining a lactic acid bacterium according to any of claims 1 8;
        (ii) fermenting a material comprising proteins with the lactic acid bacterium obtained in (i) to obtain a fermented material which comprises the peptides.
    
    
    14. The process for preparing peptides of claim 13, wherein the process is a process for preparing peptides with anti-hypertensive properties comprising
    
        (i) obtaining a lactic acid bacterium by a method for obtaining a lactic acid bacterium according to any of claims 1 8;
        (ii) fermenting a food material, comprising animal milk proteins or vegetable proteins, with the lactic acid bacterium obtained in (i) to obtain a fermented food material which comprises the peptides with anti-hypertensive properties.
    
    
    15. The process for preparing peptides of any of claims 9 to 14, wherein the food material comprises animal milk proteins.
    16. The process for preparing peptides of claim 15, wherein an animal milk protein is casein.
    17. The process for preparing peptides of claim 15, wherein the food material is milk or milk based material.
    18. The process for preparing peptides of any of the claims 9 to 17, wherein the lactic acid bacterium has a proteolytic activity making it capable of, in a protocol comprising the steps:
    
        (iv) fermenting overnight 200 ml of a food material with the bacterium;
        (v) extracting the produced peptides, and
        (vi) measuring the anti-hypertensive properties of the extracted peptides by an assay measuring the peptidic concentration required to inhibit 50% of the ACE activity (DL50),

producing peptides with an angiotensin-converting enzyme (ACE) inhibition activity (DL50) of from 0.25 to 5.0 (mg/ml).

    
    
    19. The process for preparing peptides of any of the claims 9 to 18, wherein the lactic acid bacterium is a bacterium of the phylium Firmicutes, more preferably of the class Bacilli, even more preferably of the order Lactobacillales.
    20. The process for preparing peptides of claim 19, wherein the bacterium, of the order Lactobacillales, is a bacterium of the family Lactobacillaceae, more preferably of the genus Lactobacillus, and even more preferably a Lactobacillus helveticus bacterium.
    21. The process for preparing peptides of claim 20, wherein the bacterium is a Lactobacillus helveticus bacterium with the registration number DSM 14998 or a mutant thereof.
    22. The process for preparing peptides of any of the claims 9 to 21, wherein the fermenting of the food material is performed under conditions, which produce from 0.5 to 25 mg peptides with anti-hypertensive properties per 100 ml of the food material.
    23. The process for preparing peptides of any of the claims 9 to 22, wherein the fermenting of the food material is performed at 20 to 50° C. for 3 to 48 hours.
    24. The process for preparing peptides of any of the claims 9 to 23, wherein the fermenting of the food material is performed under conditions wherein the pH is in a range from pH 3.5 to 7.
    25. The process for preparing peptides of any of the claims 9 to 24, wherein the fermented food material is further processed in a way that purify or up-concentrate the peptides with anti-hypertensive properties.
    26. The process for preparing peptides of claim 25, wherein the fermented food material is centrifuged and the obtained supernatant, comprising the peptides with anti-hypertensive properties, is isolated.
    27. The process for preparing peptides of claim 26, wherein the peptides are purified from the supernatant with a reverse-phage resin.
    28. The process for preparing peptides of claim 25, wherein a nanofiltration is performed on the fermented food material.
    29. A process for preparing a functional food product comprising peptides with anti-hypertensive properties, the process comprising following steps:
    
        (i) preparing a fermented food material according to a process of any of claims 9 to 24, and
        (ii) packing it in a suitable way to get a functional food product.
    
    
    30. The process for preparing a functional food product of claim 29, comprising following further intermediate steps [between steps (i) and (ii)]:
    
        (ia) the fermented food material of step (i) of claim 20 is further processed in a way that purify or up-concentrate the peptides with anti-hypertensive properties according to a process of any of claims 25 to 28,
        (ib) the purified or up-concentrated peptides of step (ia) is then added to a food material.
    
    
    31. The process for preparing a functional food product of claim 30, wherein the food material of step (ib) of claim 21 is a fermented food material prepared according to a process of any of claims 9 to 24.
    32. Peptides with anti-hypertensive properties obtainable by a process for preparing peptides with anti-hypertensive properties of any of claims 25-28.
    33. A functional food product comprising peptides with anti-hypertensive properties obtainable by a process for preparing a functional food product of any of claims 29 to 31.
    34. Use of peptides with anti-hypertensive properties of claim 32 for the manufacture of a medicament for the treatment of hypertension.
    35. Use of a functional food product comprising peptides with anti-hypertensive properties of claim 33 for the manufacture of a medicament for the treatment of hypertension.
    36. A Lactobacillus helveticus bacterium with the registration number DSM 14998 or a mutant thereof.


EXAMPLES
If not otherwise mentioned, individual steps were performed using standard methods as e.g. described in the general textbooks (Maniatis, T., Fritsch, E. F., Sambrook, J. “Molecular Cloning. A laboratory manual”. Cold Spring Harbor Laboratories, 2nd Edition/3 Volume, 1989; Ausubel, F. M., et al. (eds.)“Current Protocols in Molecular Biology”. John Wiley and Sons, 1995.
Example 1
PCR Amplification Reaction
Template DNA was obtained by phenol-chloroform extraction as previously described (Marmur (1961, Journal of Molecular Biology, 3, 208-218). The final preparation was genomic template DNA in a TE buffer+RNAse.
The PCR reaction was prepared as follows:

    (i) 1.0 μl of template DNA
    
        1.0 μl of forward primer (5 pmol/μl)
        1.0 μl of reverse primer (5 pmol/μl)
        1.0 μl 2.5 mM dNTD (mixture of dATP, dCTP, dGTP, dTTP)
        5.0 μl Mg buffer (20 mM MgS04)
        0.5 μl DNA polymerase (Pwo, 100 U)
        10.5 μl H20
    
    
    (ii) The thermal program for the PCR amplification was 30 cycles of 1 min at 94° C., 90 s at 50° C. (when primers Tm was around 55° C.), 90 s at 55° C. (when primers Tm was around 62° C.), 90 s at 45° C. (when primers Tm was around 50° C.) and 1 min at 72° C . The samples were cooled to 4° C. after the 30 cycles were completed.
    (iii) The PCR products were run on a 1.5% agarose gel at 60 V, excised from the gel under UV light and purified using a QIAquick Gel Extraction Kit according to the manufacturer's instructions (Qiagen, Cat. No. 28704).


Example 2
Proteolytic (ACE) Activity Assay
Preparation of Stock Culture
Lactobacillus species were streaked on MRS agar and incubated anaerobically for 48 h at 37° C. A single colony was picked, inoculated into MRS broth and grown overnight at 37° C. Lactococcus species were streaked on M17 agar and incubated aerobically for 48 h at 30° C. A single colony was picked, inoculated into M17 broth and grown overnight at 30° C. Stock cultures were prepared from these overnight cultures and were stored at −80° C. in 20% glycerol.
Preparation of Fermented Milk and Extraction of Peptides
Fermentation is performed by inoculate 200 ml of fresh milk with an overnight stock culture of example 1 (1% v/v) and maintain overnight at 37° C. or 30° C. dependent on the strain used.
From the fermented milk, extraction of the peptides may be achieved by using the following protocol:

    
    
        Centrifuge at 3000 g for 10 min at room temperature.
        Withdraw the supernatant and adjust to pH 8.3 (optimal pH for ACE activity test) with NaOH.
        Centrifuge the obtained supernatant at 3000 g for 10 min at room temperature.
        Withdraw the supernatant (whey), which comprises the peptides.
        Determine the concentration of peptides in the whey by the Lowry test (mg peptide/ml whey) (Lowry et al, 1951. J. Biol. Chem., 193:265-275).
    
    


The whey may be used directly for ACE assay or freeze at −20° C. The whey comprising the peptides is termed “peptide solution” in example 3.
ACE Activity Assay
The peptide pools of milk fermented are tested for ACE activity in vitro. The DL50 (mg/ml) is the peptidic concentration, which inhibits 50% of ACE activity. The lower this value is, the better the anti-hypertensive effect of the fermented milk. The ACE activity of the extracted peptides is measured by the following protocol:
The essence of the assay is that ACE degrades a hippuryl-L-histidyl-L-leucine (HHL) substrate and adding a colour agent develops a colour. If peptides are present the peptides inhibit ACE and less HHL substrate is degraded. This means less colour is developed after addition of the colour agent.
Solution preparation:
Incubation buffer: 188 mmol/l boric acid pH 8.3, 1.375 mmol/l potassium chloride. (Dissolve 2.91 g of boric acid and 25.63 g potassium chloride in 200 ml of distilled water. Adjust the pH to 8.3 with 1 mol/l potassium hydroxide and dilute to 250 ml with distilled water. Store at room temperature).
Substrate solution: 5.8 mmol/l hippuryl-L-histidyl-L-leucine (HHL). (Dissolve 250 mg hippuryl-L-histidyl-L-leucine in about 90 ml incubation buffer and fill up to 100 ml with the same buffer. Store at 40° C. The substrate solution can be used for at least 2 weeks).
Stop solution: 100 mmol/l HEPES pH 9, 2.5 mmol EDTA. (Dissolve 23.83 g HEPES and 0.93 g EDTA in 800 ml distilled water. Adjust to pH 9 with 1 mol/l sodium hydroxide and dilute to 1 1with distilled water. Store at room temperature).
Colour reagent: 136 mmol/l cyanuric chloride in 1,4-dioxane. (Dissolve 12.50 g cyanuric chloride in about 400 ml of 1,4-dioxane and fill up with 1,4-dioxane to 500 ml. Store at room temperature in dark-brown glass bottle).
Assay: (all solutions are equilibrated to room temperature)

    
    
        Make a dilution series of the peptide solution with incubation buffer. The series consist of 6 dilutions going from the undiluted peptide solution to a blank (only incubation buffer)
        For each of the dilutions, place 10 μl of peptide solution, 40 μl of substrate (HHL) solution (2.5 g/l) and 2.5 μl of ACE (0.25 Units/ml) in a glass tube.
        The positive control comprises 2.5 μl ACE, 10 μl of incubation and 40 μl of substrate (HHL)
        The negative control comprises 12 μl of incubation buffer and 40 μl of substrate (HHL)
        Incubate at 37° C. for 1 hour.
        Stop the reaction by adding 300 μl of stop solution, followed by 150 μl of colour reagent
        Mix vigorously.
        Allow to stand for 5 minutes and centrifuge at 3300 g for 30 min at room temperature to remove denatured protein and excess cyanuric chloride.
        Transfer 300 μl of supernatant of each sample to microtiter plate hole.
        Read at 405 nm against water as a blank.
    
    


The ACE inhibition percentage is expressed by the formula:




  
    ACE
    ⁢
    
      
    
    ⁢
    inhibition
    ⁢
    
      
    
    ⁢
    activity
  
  =
  
    
      
        
          OD
          ⁢
          405
        
        ⁢
        
          
        
        ⁢
        nm
        ⁢
        
          
        
        ⁢
        positive
        ⁢
        
          
        
        ⁢
        control
      
      -
      
        
          OD
          ⁢
          405
        
        ⁢
        
          
        
        ⁢
        nm
        ⁢
        
          
        
        ⁢
        sample
      
    
    
      
        
          OD
          ⁢
          405
        
        ⁢
        
          
        
        ⁢
        nm
        ⁢
        
          
        
        ⁢
        positive
        ⁢
        
          
        
        ⁢
        control
      
      -
      
        
          OD
          ⁢
          405
        
        ⁢
        
          
        
        ⁢
        nm
        ⁢
        
          
        
        ⁢
        negative
        ⁢
        
          
        
        ⁢
        control
      
    
  




Each dilution has its own ACE inhibition percentage value that gives a curve expressing the ACE inhibition percentage in function of the peptide concentration of the whey. DL50 (peptidic concentration that inhibits 50% of ACE activity) is obtained by reading the peptidic concentration at the intersection point between the curve and the corresponding 50% ACE inhibition point on the axe.
Example 3
Investigation of Presence of the prtH200, orfF3 and orfF4 Gene Sequences in Different Bacteria
Different lactic acid bacterial strains were investigated for the presence of the prtH200, orfF3 and orfF4 gene sequences. PCR reactions were performed as described in Example 1. Table 1a shows peptides and PCR primers used to investigate for presence of a prtH200 gene encoding a proteinase. Table 1b shows peptides and PCR primers used to investigate for presence of a orfF3 gene. Table 1c shows peptides and PCR primers used to investigate for presence of a orfF4 gene.
In these tables are also given the estimated length of these amplified sequences. The estimated length was determined based on prtH200 SEQ ID NO 1, orfF3 SEQ ID NO 3 and orfF4 SEQ ID NO 5.







TABLE 1a







(prtH200 proteinase)












Esti-





mated


Primer

length


set

of PCR


number
Sequence
fragment





(a)
(S): 5′ CGATGATAATCCTAGCGAGC3′,
620 bp



 



(A): 5′ TGGCAGAACCTGTGCCTA 3′



 


(b)
(S): 5′ GCCAAGACGCCTCTGGTA 3′,
313 bp


 



(A): 5′ TAGGTATAGTTTCCATCAGGA 3′



 


(c)
(S): 5′ AARGTWCCWTAYGGYYWYAAYTA 3′,
624 bp


 



(A): 5′ GCCATDSWDGTRCCDSWCATDTK 3′
















TABLE 1b







(orfF3 proteinase)












Estimated



Primer set

length of


number
Sequence
PCR fragment





(a)
(S): 5′ CGAAGGCGATAAGTCAAACTTTGATAATGC 3′,
1605 bp



 



(A): 5′ CCCGGTTCTGTAAGATAATTTGGATCG 3′



 


(b)
(S): 5′ ASTCWRRYTTYGATRATGCW 3′,
1587 bp


 



(A): 5′ BHKYAMSAWARTTTGGATCR 3′.





sense sequence (S),


antisense sequence (A)













TABLE 1c







(orfF4 proteinase)












Esti-





mated


Prim-

length


er

of


set

PCR


num-

frag-


ber
Sequence
ment





(a)
(S): 5′ GGTGTTGCTCCTGAAGC 3′
950



 



(A): 5′ ACTCTAGCACCAGCTAATTGAACATCATG 3





sense sequence (S),


antisense sequence (A)






Table 2 shows the results of the PCR based investigations.















TABLE 2






Ref
p:
p:
p:
o3:
o3:
o4


Species
(CHCC)
(a)
(b)
(c)
(a)
(b)
(a)







Lb. helveticus
5951
+
+
+
+
+
+


Lb. helveticus
4080
−
−
−
−
−
−


Lb. helveticus
3610
+
+
+
−
−
?


Lb. helveticus
 637
−
−
−
+
?
+


Lb. helveticus
3552
−
−
−
−
?
+


Lb. helveticus
DSM
−
−
?
+
?
+



13137 





“p:” is prtH200 primer sets.


“o3:” is orfF3 primer sets.


“o4:” is orfF4 primer sets.


“+” denote a positive PCR fragment.


“−” denotes no PCR fragment or a negative PCR fragment.


“?” denotes not tested.


“Lb. helveticus DSM 13137” is described in WO01/32836 (Valio Ltd).






The positive fragments of Table 2 were within the expected size given in Table 1. They were all DNA sequenced and confirmed to comprise a DNA sequence corresponding to an expected DNA sequence.
The specific strain with ref number 5951 comprises prtH200, orfF3 and orfF4 gene sequences according to the present invention.
Example 4
ACE Activity
Cultures of the strains mentioned in Example 3 were tested in proteolytic (ACE) activity test as described in Example 2. Table 3 gives the results.










TABLE 3








Anti-hypertensive effect with DL 50



Culture used
(mg/ml). Average of three determinations









Lb. helveticus 5951
2.99



Lb. helveticus 4080
2.34



Lb. helveticus 637
3.32










Example 5
In vivo Test of Anti-hypertensive Properties
Materials and Methods
Strains and Cultivation
Strains were streaked on MRS agar and incubated anaerobically for 48 h at 37° C. A single colony was picked, inoculated into MRS broth and grown overnight at 37° C. Stock cultures were prepared from this overnight culture and were stored at −80° C. in 20% glycerol. The strains were precultivated in milk overnight and inoculated 1% (v/v) in fresh milk for the fermentation.
Preparation of the Samples
Concentrated fermented milk:
The milk was fermented with the strains of single or mixed cultures for 16 hours with an inoculation level of 1% (v/v). The total product was freeze-dried. A second fermentation, with the same conditions as the first one, was centrifuged. The pellet was discarded and the whey was filtered through a 0,45 mm filter and frozen. The whey was used to solubilize the freeze-dried powder to concentrate it with a factor 5 before feeding the rats.
Spontaneously Hypertensive Rats:
Spontaneously Hypertensive Rats (SHR) was obtained from IFFA CREDO (a Charles River company), Lyon, France.
All treatments (fermented products) were administered between 09:00 h and 09:15 h by gavages at a dose of 2 ml of product.
Experimental Design:
Three groups were formed:
Group 1 (n=16): receiving treatment 1 (milk; n=16)
Group 2 (n=12): receiving successive treatment 2-7, each administration separated by a 3-day washout period
Group 3 (n=12): receiving successive treatment 8-12, each administration separated by a 3-day washout period
Investigated Parameters:
Systolic blood pressure was determined by plethysmography in conscious SHR before as well as 5 and 24 hours after gavages, at the different times described below.
Prior to the experiments all SHR were acclimated to the animal facilities for 9 weeks. Moreover, all animals were accustomed to gavage and measurement of systolic blood pressure 3 days before the first gavage.
Systolic blood pressure of Group 1 was determined parallel to those of groups 2 and 3 and serves as control.
The day of the gavage systolic blood pressure was determined before as well as 6 and 24 hours after gavage.
Results
In table 4 is shown the variation of systolic blood pressure determined 5 and 24 hours after administration of different substances.















TABLE 4










Calpis









commercial
CHCC
CHCC
CHCC



Milk
Tap water
Enalapril
product
4080
5951
637























 5 hours
−4
−4
−30
−13
−14
−18
−8


24 hours
−4
−2
−17
+2
−1
−19 
−8





Milk: this sample is non fermented milk, 9.5% reconstituted skim milk.


Enalapril: Enalapril is a medicine usually used to treat patients with high blood pressure. It was added to the milk at the high concentration of 20 mg/kg, to test the capacity of the rats to react to an anti-hypertensive compound.


Calpis commercial product: It is a commercial product of Calpis Food Industry. The name of the product is called Calpis. It is liquid fermented milk with a mixture of Lb. helveticus and Saccharomyces cerevisiae strains.


CHCC 4080: Concentrated fermented milk with CHCC 4080


CHCC 5951: Concentrated fermented milk with CHCC 5951


CHCC 637: Concentrated fermented milk with CHCC 637






The results demonstrate that CHCC 5951 is the lactic acid bacteria strain that is capable of making peptides with the best anti-hypertensive properties. Table 2 (above) shows that this strain comprises the prtH200, orfF3 and orfF4 genes as described herein. At 24 hours the peptides produced by use of the CHCC 5951 strain had an effect comparable to the medicament Enalapril.
CHCC 4080 comprises none of the genes. CHCC 637 comprises orfF3 and orfF4 but not prtH200.
As said above, a commercial product of Calpis Food Industry are made by using the CP790 lactic acid bacterium and this strain does not comprise prtH200 [Yamamoto et al (2000)].
Example 6
Further in vivo Tests using the CHCC5951 Strain
Strains and Cultivation and SHR Rats: The same as in Example 5
Preparation of the Samples
Fermented milk:
The milk was fermented with the strains of single or mixed cultures for 16 hours with an inoculation level of 1% (v/v). The total product was freeze-dried. A second fermentation, with the same conditions as the first one, was centrifuged. The pellet was discarded and the whey was filtered through a 0,45 mm filter and frozen. The whey was used to solubilize the freeze-dried powder to concentrate it with different factors before feeding the rats.
Experimental Design:


    
    
        Animals: Spontaneously Hypertensive Rats (22 week old).
        Groups:
        
            1) Placebo (Milk)
            2) Sample 1 (Fermented milk administered at 3 different whey concentrated 3 doses. Dose 1: factor 1, Dose 2: factor 2.5, Dose 3: factor 5)
            3) Sample 2 (freeze-dried fermented milk suspended in neutral pH milk)
            4) Sample 3 (just the fermented milk product)
            5) Sample 4 (Fermented milk that was heat treated after fermentation)
            6) Sample 5 (milk that had not been fermented but it included the live bacteria)
        
        
    
    


All treatments were administered between 10:00h and 10:05h by gavage at a dose of 2 ml of product.
Investigated Parameters: Systolic blood pressure was determined for 24 hours after gavage by telemetry (Data Sciences Int.) in conscious SHR at different time points In brief, the mean (over a 1 minute period) of systolic, diastolic blood pressures were recorded every 15 minutes, the 24 hours before administration and the 48 hours after administration. From these tracings, 24 hours mean of systolic and diastolic blood pressures were calculated in each group. Furthermore, the variations induced by each substance compared to placebo of the same parameters, i.e. over 24 hours, and during the 3 to 6, the 12 to 15 and the 21 to 24 hours after gavage, were calculated.
Prior to the experiments all SHR were acclimated to the animal facilities for 9 weeks. Moreover, all animals were accustomed to gavage the 3 days before the first administration of the substance.
Statistics.
All results are expressed as mean±standard error of mean.
Result table.







TABLE 1







Systolic blood pressure after gavage










Period
















Groupe
0-24
3-6
1
2
0-24
3-6
12-15
2












Variation vs. untreated 1















Placebo 1
184 ± 6
184 ± 6
183 ± 5
190 ± 8






Sample 1


Dose 1
178 ± 4*
172 ± 4*
176 ± 5*
184 ± 7
—
—
—
—


Dose 2
178 ± 4*
175 ± 5*
172 ± 4*
184 ± 5
—
−9.6 ± 2.7
—
—


Dose 3
178 ± 3*
174 ± 4*
175 ± 3*
186 ± 4
—
—
—
—









Variation vs. untreated 2















Placebo 2
176 ± 9
169 ± 9
179 ± 9
161 ± 9






Sample 2
169 ± 9*
163 ± 9*
172 ± 7*
166 ± 9*
—
−5.9 ± 2.7
—
—


Sample 3
170 ± 8*
168 ± 9
169 ± 7*
165 ± 8*
—
−0.6 ± 3.1
—
—


Sample 4
170 ± 8
167 ± 9
172 ± 8
168 ± 8
—
−1.3 ± 4.4
—
—


Sample 5
180 ± 10
180 ± 14
181 ± 8
178 ± 8*
5.2 ± 1.7
11.4 ± 6.4
2.6 ± 2.2
7.1 ± 2.8





*p < 0.05 vs. Placebo


Untreated 1 and untreated 2 are the controls of study 1 and study number 2, respectively.













TABLE 2







Diastolic blood pressure after gavage.










Period
Variation vs. Placebo















Groupe
0-24
3-6
12-15
21-24
0-24
3-6
12-15
21-24





Untreated
126±
127±
124±
129±






Sample 1


Dose 1
120±
118±
118±
124±
—
—
—
−5.3 ± 2.4


Dose 2
122±
120±
120±
129±
—
—
—
−0.3 ± 2.3


Dose 3
123±
120±
121±
128±
—
—
—
−1.4 ± 1.6


Untreated
131 ± 5
125 ± 5
134 ± 5
125 ± 5


Sample 2
123±
119±
125±
119±
—
—
—
−5.7 ± 3.3


Sample 3
126±
127±
125±
121±
—
2.7 ± 2.6
—
−4.1 ± 2.3


Sample 4
127±
126±
128±
123±
—
1.2 ± 3.5
—
−2.5 ± 2.6


Sample 5
133±
135±
133±
130±
3.2 ± 2.3
9.7 ± 5.5
—
 4.8 ± 3.3





*p < 0.05 vs. Placebo


Untreated 1 and untreated 2 are the controls of study 1 and study number 2, respectively






The results demonstrate that there is no need to up-concentrate the fermented milk product. Sample 3 is just the fermented milk product and it has a corresponding blood pressure reducing effects as for the freeze dried concentrated fermented samples 1 (Dose 1, 2 , 3 ) and sample 2.
Sample 2 is freeze-dried fermented milk suspended in neutral pH milk. It reduces the blood pressure. This demonstrates the wide application of a fermented product as described herein since it may be dissolved in different liquids to get a required final appropriate use. Characteristic such as different pH are not affecting the blood reducing pressure activity of the product.
Sample 4 with heat treatment after fermentation reduce also the blood pressure. Substantially all bacteria are killed in this sample. Accordingly, this demonstrates that there is no need to have live bacteria in the final product.
Sample 5 has not been fermented but it includes the live bacteria. It does not reduce the blood pressure. It demonstrates that the fermentation step is required.
Example 7
Pulsed Field Gel Electrophoresis (PFGE) Fingerprinting
Lactobacillus helveticus strain CHCC5951 (deposited with accession number DSM 14998) was grown overnight at 37° C. in standard MRS media. Chromosomal DNA was isolated by use of a Qiagen kit. The isolated chromosomal DNA was completely digested with restriction enzyme SmaI (done according to manufacture instructions).
Electrophoresis conditions for the digested DNA were: CHEF Mapper XA System, Pulse times 2 to 30 sec linearly ramped over 24 hrs at 5.3 V/cm. 1.1% agarose, 1⁄2×TBE, 14° C.
The program used to run the gel was:













PFGE programme no.













Parameter
1
2
3
4
5
6
















Initial
2
3
2
2
3
1


switch


time in sec


final
30
80
60
40
18
10


switch


time in sec


ramp
linear
−1.53
−1.24
linear
linear
linear


factor


run time in
24
24
24
24
24
21


hrs









The bands were compared on computer by using the Gel Compar II program.
Band sizes were determined by measuring band migration distances on three independent pulsed field electrophoreses and correlating the distances to the migration of a standard with known band sizes. The sizes are means of the three measured values ±5 kbp.
The resulting agarose gel is shown in FIG. 1. The sizes of the indicated 12 bands are:

    
    
        band no. 1: 283 kbp
        band no. 2: 259 kbp
        band no. 3: 219 kbp
        band no. 4: 138 kbp
        band no. 5: 127 kbp
        band no. 6: 119 kbp
        band no. 7: 106 kbp
        band no. 8: 88 kbp
        band no. 9: 71 kbp
        band no. 10: 59 kbp
        band no. 11: 54 kbp
        band no. 12: 46 kbp
    
    


REFERENCES
Below are mentioned references that are considered relevant in relation to the present invention.

    Yamamoto et al , (1994), J. Dairy Sci., 77: 917-922
    Gobbetti M. et al (2000), Appl Environ Microbiol, 66 (9), 3898-3904
    EP821968
    EP1016709.
    WO01/32836
    Pederson et al (1999), J. of Bacteriology, 181: 4592-4597
    Yamamoto et al (2000), Biosci. Biotechnol. Biochem., 64(6): 1217-1222
    EP058074

## Claims

1. A process for preparing peptides with anti-hypertensive properties, the process comprising fermenting a food material, comprising animal milk proteins or vegetable proteins, with a Lactobacillus helveticus strain to obtain a fermented food material which comprises said peptides with anti-hypertensive properties, characterized in that the Lactobacillus helveticus strain is the Lactobacillus helveticus strain with the registration number DSM 14998.

2. The process for preparing peptides of claim 1, wherein the food material comprises an animal milk protein.

3. The process for preparing peptides of claim 2, wherein the animal milk protein is casein.

4. The process for preparing peptides of claim 2, wherein the food material is milk or milk based material.