Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2000-03-22
2002-01-22
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S243000, C435S252100, C435S252400, C435S252900, C435S253600, C435S854000, C435S855000, C435S856000
Reexamination Certificate
active
06340585
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel synthetic medium suitable to cultivate lactic acid bacteria of the genus Bifidobacteria or Lactobacillus which contains particular nucleotides and deoxynucleotides. In particular, the present invention pertains to the use of said medium for the isolation of bioactive molecules or functional metabolites.
BACKGROUND OF THE INVENTION
Lactobaccili are widely distributed in nature and are largely used for industrial fermentation process, for example, the preparation of dairy products. In recent years the study of their metabolism has been greatly enhanced since particular strains have been found to exert a positive effect on the maintenance of the healthy state of organisms. Their complex nutrient requirements are usually satisfied by natural sources of synthetic growth media, containing matrices of undefined and complex composition, such as yeast extract and peptones of various origins.
Some semi-synthetic and completely chemically defined media have been developed for lactic acid bacteria for different purposes, such as the investigation of the nutritional requirements of bacterial cells, the identification of the role of specific components by detection of the effects after removal thereof from the medium or the isolation of mutants auxotrophic for certain substances. Growth media with a defined chemical composition were also used to determine the requirements of Lactobaccili for nucleotides and to attribute their essential or non-essential role with regard to different DNA precursors.
In the past few decades several studies were performed by means of defined media, on the strain
Lactobacillus johnsonii
ATCC 11506 (formerly known as
Lactobacillus acidophilus
R-26), firstly proposed by Hoff-Jorgensen as an experimental organism for determining the presence of DNA residues in biological samples (Hoff-Jorgenson, “A microbiological assay for deoxyribonucleosides and deoxyribonucleic acid”, Biochem J. 50 (1952), 400-403). Ives and Ikeda report in “Life on the salvage path: the deoxynucleoside kinases of
Lactobacillus acidophilus
R26”, Progr. Nucl. Acid. Res. (1998), 207-252, that this strain requires the presence of at least one deoxyribonucleoside in the growth medium due to the functional absence of ribonucleotide reductase activity.
Further, it could be shown that in
Lactobaccillus delbrueckii subsp. lactis
ATCC 7830 (formerly known as
L. leichmannii
ATCC 7830), in contrast to strain R-26, the requirement for deoxyribonucleosides could be replaced by Vitamin B12.
The latter strain was subjected to several investigation in order to elucidate the nucleotide requirements of Lactobaccili and the effects of supplementation of the medium with DNA molecules (Jeener & Jeener, Exptl. Cell Res. 3 (1952), 675-680; Okazaki & Okazaki, J. Biochem. 35 (1959), 434-445; Hoff-Jorgensen, Meth. Enzymol. 3 (1957), 781-785; Mc Nutt Meth. Enzymol. 2 (1955), 464-468; Lovtrup & Shugar J. Bacteriol. 82 (1961), 623-631.
Thymidine was often indicated as a key factor for the growth of
Lactobacillus acidophilus
and
L. leichmannii.
Further, in later studies the removal of uracil was demonstrated to deeply affect RNA synthesis and cell division in lactic acid bacteria.
In J. Bacteriol. 73 (1957), 670-675 Siedler et al., reported an optimization of Hoff-Jorgensen's medium by studying the ability of uracil, vitamin, vitamin B6 and acid-hydrolized casein to reproduce the positive effect of yeast extract on
L. acidophilus
development in a semi-defined medium.
Recently, Imbert & Blondeau disclosed in Curr. Microbiol. 37 (1998), 64-66, a chemically defined medium for examining the ability of some Lactobacillus species to grow after iron chelation. Furthermore, the interaction between manganese and iron was examined. The supplement of chelated iron did not affect bacterial growth in the presence of manganese, while a slightly positive effect was observed following to the addition thereof to the same medium deprived of manganese especially for
L. acidophilus
ATCC 4346T after aerobic incubation.
It is known that most pathogenic bacteria require iron for their growth. In contrast, lactic acid bacteria have been generally recognized as exceptions among the living organisms in that they do not show such an indispensable iron requirement. This is considered to represent an ecological advantage against pathogens in natural environments.
Few publications exist reporting the average content of metal in lactic acid bacteria. In general, a strong variability among the Lactobacillus species has been found exemplified by a comparison between the iron content of
Lactobacillus plantarum
and
Escherichia coli
cells in which a lower level in the former species was confirmed (Archibald et al., FEMS Microbiol. Lett. 19 (1983), 29-32).
Recently, particular strains of the genus Lactobacillus and Bifidobacteria have attracted great attention since properties beneficial to the host organism have been attributed to them. So far it is only known that these strains exhibit the properties reported, yet the reason for these properties was not elucidated.
In this respect EP 0 577 903 discloses the use of lactic acid bacteria, especially a Lactobacillus strain which upon ingestion reveals beneficial effects to organisms infected by
Helicobacter pylori.
Accordingly, the Lactobacillus is obviously capable of producing metabolites that are capable preventing further growth and/or adhesion of Helicobacter to gastric and/or intestinal mucosal structures. From the point of view of identifying these metabolites, it would be desirable to have a medium from which compounds produced by the lactic acid bacteria may be isolated.
In order to isolate said compounds, the bacterial cells shall be cultivated to a reasonable extent in the medium. Yet media providing a sufficient growth of lactic acid bacteria are normally not defined and comprise complex matrices, such as yeast extract and peptones, from which a desired, still unknown compound cannot be isolated.
On the other hand, known defined media are normally specific for a given bacterial strain, and moreover do not provide for sufficient growth of the microorganism.
SUMMARY OF THE INVENTION
Consequently, a problem of the present invention is how to provide a novel defined medium, which allows for a sufficient growth of plurality of different bacterial strains?
This problem was solved by providing a synthetic medium for cultivating lactic acid bacteria belonging to the genus Lactobacillus or Bifidobacteria comprising a carbon source, buffer, a nitrogen source, trace elements, antioxidants and vitamins characterized in that it contains two free bases, one ribonucleoside and two 2′-deoxynucleosides, each in an amount sufficient to promote growth of the microorganisms.
During the extensive studies leading to the present invention, a chemically defined growth medium for
Lactobacillus johnsonii
was developed, which was surprisingly found to be suitable for the cultivation of other Lactobacilli and/or Bifodobacteria as well. In the experiments, particular attention was paid to the nucleotide composition of the medium and several sources of DNA precursors were examined for the ability to support Lactobacillus/Bifidobacteria growth.
DETAILED DESCRIPTION OF THE INVENTION
To this end a defined medium for
L. johnsonii
was supplemented with free bases (adenine, cytosine, guanine, thymine, uracil and inosine), ribonucleosides (adenosine, cytindine, guanosine, uridine) and deoxyribonucleosides (2′-deoxyadenosine, 2′-deoxycytidine, 2′-deoxyguanosine, 2′-deoxyuridine and thymidine). The different Lactobacilli investigated had the ability to grow in the defined medium in the simultaneous presence of all the five free bases, all four ribonucleosides and all the five deoxyribonucleosides. Whereas, the minimal requirement for substantial growth was found to be a combination of at least two free bases, one nucleosides and two deoxyribonucleosides.
It could be shown that both adenine and guanine could
Elli Marina
Marchesini-Huber Barbara
Reniero Roberto
Zink Ralf
Flood Michele
Nestec S.A.
Tate Christopher R.
Winston & Strawn
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