Strain of bacteria of the species Lactobacillus paracasei...

Details Strain of bacteria of the species Lactobacillus paracasei... Strain of bacteria of the species Lactobacillus paracasei...

2000-09-22

2003-07-29

Marx, Irene (Department: 1651)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Bacteria or actinomycetales

C435S252900, C424S439000

Reexamination Certificate

active

06599504

ABSTRACT:

The present invention relates to a strain of
Lactobacillus paracasei
subsp.
paracasei,
a composition thereof for use in food as well as a product containing said strain.
Definition and Characterisation of the Strain
The novel strain (which in the following for simplicity will be designated LMG-P-17806) is a variant of the species
Lactobacillus paracasei
subsp.
paracasei.
It has the characteristics of the species with a GC-content of 44%. LMG P-17806 has been isolated from samples from the gastrointestinal micro-flora of humans. LMG-P-17806 is a Gram-positive, homofermentative rod-shaped bacteria. It produces L-lactic acid (laevorotatory stereoisomer of lactic acid) and grows optimally at 37° C. The stain is characterized by being tolerant in-vitro against hydrochloric acid and gastric juice by tolerating bile salts without deconjugating them and by having a great ability of assimilating cholesterol. The stain is also characterized by containing three plasmids having a size of 2.2, 4.36 and 9.1 Kb, respectively. Other characteristics are that the strain is fermenting ribose, adonitol, galactose, glucose, fructose, mannose, sorbose, mannitol, sorbitol, N-acetyl-glucosamine, esculin, cellobiose, maltose, lactose, sucrose, trehalose, inulin, melezitose, D-turanose and D-tagatose. On the other hand it does not ferment glycerol, erythritol, D- and L-arabinose, D- and L-xylose, &bgr;-methyl-D-xyloside, rhamnose, dulcitol, inositol, &agr;-methyl-D-mannoside, &agr;-methyl-D-glucoside, amygdalin, arbutin, salicin, melibiose, raffinose, starch, glycogen, xylitol, gentiobiose, D-lyxose D- and L-fucose, D- and L-arabitol and 2- and 5-ketogluconate.
The strain has been characterized by SDS gel electrophoresis, in which it has been compared to six other strains of
Lactobacillus paracasei
subsp.
paracasei,
vide the accompanying figure. In this comparison it has been shown to differ from all other described strains and at the same time as it when being compared to other lactobacilli appears to belong to the designated species. It has also been characterized with regard to ribosomic RNA in a so called Riboprinter®. With this method the strain has been shown to possess 76% similarity with the type strain for
Lactobacillus paracasei
subsp.
paracasei
and 72% similarity to the type strain of
Lactobacillus casei
analyzed at the same occasion.
The strain has been deposited at Belgian Coordinated Collections of Microorganisms—BCCM, LMG collection, and there been given the accession No. LMG P-17806.
The Advantages of the Strain
LMG P-17806 has, when compared to known strains of Lactobacillus, crucial advantages in the use as probiotics in food and naturopathic medicines, i.e., medical food, by a unique combination of good properties;
the strain has good resistance against gastric juice and bile salts, but unlike many other strains it does not deconjugate the bile salts;
it has a great ability to assimilate cholesterol;
the strain is well managing the passage through the stomach;
the strain has an influence on the conditions in the model of large intestine by increasing the production of L-lactic acid therein;
the strain is not more pro-inflammatory than common yogurt bacteria;
the strain prevents intestinal cells from being invaded by pathogenic microorganisms, such as Salmonella typhmurium;
the strain has an antagonistic action against the gastric ulcer bacterium Helicobacter pylori:
the strain forms bacteriocins which are active against clostridiae;
the strain survives well in milk as well as in frozen and dried form;
the strain, unlike most other lactobacilli has a favourable influence on the taste of fermented milk products (does not give any tang).
The present strain of
Lactobacillus paracasei
subsp.
paracasei
can be used as an additive to food or as naturopathic medicine, so called “Medical Food”, or as an additive to naturopathic medicine.
Such medicines can be used for children with the purpose of alleviating atopic problems; for elderly persons in order to correct altered microflora caused by normal alterations by age or an altered secretion of hydrochloric acid; and for persons in general in order to normalize the intestine flora, whereas the content of clostridium bacteria is decreasing, lactobacilli and bufido bacteria being increased and high contents of coliformic bacteria being decreased.
By means of these properties the strain LMG P-17806 differs from previously known strains, which will be shown in the examples below.
Preparation of the Strain
The strain is prepared in the usual way for lactobacilli. A substrate suited for lactobacilli is used. This substrate should for instance contain at least one of the carbohydrates which the strain can ferment according to what is stated above, in combination with proteins, vitamins, minerals and other nutrients which normally are required by lactobacilli. Examples of suitable commercial substrates are yeast extract-glucose broth, MRS (de Man-Rogosa-Sharp broth), Rogosa, milk added with a minor amount of a yeast extract, etc. The strain is cultivated microaerophilicly or in the complete absence of oxygen, suitably at a temperature between +15° C. and +42° C. If the substrate is grafted with 0.1 to 1% of graft a culture time of between 10 and 40 hours is suitable. The strain can, if desired, be concentrated by centrifugation or filtration followed by washing the concentrate in order to remove the culture medium. The concentration can then be frozen or lyophilized in the common way. In this way preparations of between 100 millions and 100,000 millions of living bacteria LMG-P-17806 per g can be prepared. A preparation can then be used as such or be used as an additive to food, for instance to milk or another product which gives LMG P-17806 the possibility to survive and, if desired, to grow.
Investigations
A. Investigation Concerning the Passage of the Strain Through the Gastrointestinal Tract
LMG- P-17806 was cultivated in the way described above and added together with yogurt culture to milk. A fermented product was produced by incubating the milk for five hours at +42° C. A palatable product was obtained which contained fully 100 millions living LMG P-17806 per gram of product. Healthy persons were given 3×200 g product daily for one week. The total intake of LMG P-17806 was between 40 billions and 200 billions.
Faeces samples were examined before the intake, after one week of consumption and one week after the intake had ceased. As is evident from Tables 1 and 2 below, a strong increase in the number of lactobacilli in the test subjects was obtained. Two isolates per test subject were classified as to species on each occasion, i.e. 20 isolates in total. 18 of the isolates for the consumption time appeared to consist of LMG P-17806 according to fenotypical classification. This bacteria strain was not discovered in the samples before or after the intake of LMG P-17806.
In average the contents of the faeces samples during the supply were very high and varied only moderately from 63 millions to 320 millions per gram, i.e. with a factor of 5. Noteworthy was, that the contents were largely the same independent of what contents were measured before the start of the experiment. After the supply had ceased the contents reverted to what seems to be natural for the test subject in question.
Tables 1 and 2 below the content of lactobacilli in faeces determined, in millions per gram, by plating and using the substrate Rogosa.
TABLE 1
5 test subjects with originally low content of
lactobacilli
Test
Before the
During the
After the
subject
experiment
experiment
experiment
1
0.05
120
0.07
2
0.15
320
0.12
3
0.08
97
0.14
4
0.009
63
0.02
5
<0.001
278
0.002
TABLE 2
5 test subjects with high contents of
lactobacilli
Test
Before the
During the
After the
subject
experiment
experiment
experiment
6
1.2
297
1.3
7
0.7
83
0.6
8
0.2
136
0.18
9
4.3
74
3.5
10
0.6
212
0.8
The examination shows that the strain has good survival at the passage through the gastrointestinal tract.
B. Examination Concerning the Formation of L-lactic A

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