KR100376954B1 - Lactobacillus colonized in the intestine - Google Patents

Abstract

Novel Lactobacillus plantarum strain (DSM Accession No. 9843) having the ability to settle in vivo in human intestinal mucosa, and compositions for preventing or treating bacterial infections of the gastrointestinal tract containing the strain together with conventional carriers This is provided according to the present invention.

Description

Lactobacillus colonized in the intestine

The present invention relates to lactobacillus strains having the ability to colonize and settle on intestinal mucosa in vivo after oral ingestion, and their use for the prevention or treatment of bacterial infections, in particular nutrient solutions based on oatmeal fermented by the lactobacillus strains. It relates to the use of said strain in the form of a composition comprising a.

Many have disturbed intestinal microflora. That is, the balance between useful gut bacteria and harmful gut bacteria is broken. Many factors, including stress, the presence of bile salts, and food affect the bacterial flora. Most importantly, however, modern antibiotic treatment can destroy the normal microflora over a long period of time, eliminating the normal fermentation process.

It is widely recognized today that Lactobacillus has a beneficial effect on the intestinal mucosa. However, it is unclear what microbes are involved and the intestinal ecosystem.

A decisive disadvantage other than Lactobacillus used in most conventional foods today is that these microorganisms have a very low survival rate as they pass through the stomach and duodenum. In order to overcome this drawback, acidic dairy products using eosinophilic bacteria have been developed. European Patent Publication (A2) 0 199 535 describes a biologically pure culture of Lactobacillus acidophilus (ATCC Accession No. 53 103) obtained from human stool, which is an in vitro test. It is described as being able to attach to mucosal cells. L. Acidophilus exits well through the upper part of the gastrointestinal tract, but adhesion in vivo has not been demonstrated.

Lactobacillus strains on the market today are selected above all for their ability to grow in conventional primary products such as milk. In order for a strain to exhibit any beneficial effect, it is a prerequisite that it must be able to settle in the gut and compete with existing microflora.

WO 93/01823 discloses a method for the isolation of Lactobacillus strains having the ability to colonize and settle in the intestinal mucosa of the human body, strains obtained in this way, in particular under the Budapest Treaty, DSM (Germany, Braunschweig) Lactobacillus plantarum 299 (DSM 6596) and Lactobacillus cassia subspecies 271 ( Lactabacillus casei ssp ) deposited on July 2, 1991 in Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, rhannosus 271, DSM 6594). This application also relates to compositions for preventing or treating infections in the gastrointestinal tract, including, for example, any of these strains together with a carrier based on oatmeal.

The present invention relates to a Lactobacillus strain capable of colonizing the human intestinal mucosa in vivo, namely Lactobacillus plantarum 299v (DSM 9843) deposited with the International Depository DSM, dated March 16, 1995. .

L. Plantarum 299v was isolated by the method described in WO 93/01823, and strain L. known in the literature. Very similar to Planta Room 299. However, the strain of the present invention was found to be surprisingly superior to known strains in the settling test in the human body.

The present invention also relates to compositions for preventing or treating intragastric tract infections comprising the strain Lactobacillus plantarum 299v with conventional carriers.

Common carriers are, for example, physiologically acceptable substrates fermented by bacteria, as well as various foods, especially those based on starch or milk, and inert solid or liquid substances such as physiological saline or water. Suitable substrates must contain liquid or solid fibers that are not reabsorbed in the gastrointestinal tract and are fermented with Lactobacillus to form short chain fatty acids. As examples of suitable starch-containing substrates, mention may be made of cereals such as oats, wheat, corn, root vegetables such as potatoes and certain fruits such as green bananas.

Preferred compositions according to the invention which also have good nutritional value are nutritious solutions based on oatmeal fermented by Lactobacillus strains. The oats, which are grains, have in many ways proved to be good substrates for fermentation. That is, oats are rich in protein, carbohydrates, fats, dietary fibers, and water-soluble fibers called β-glucans. Oat or oatmeal fats also contain large amounts of surfactant phospholipids, which act as gastric mucosal blockers (“corrosion inhibitors”) to protect the mucosa. Finally, the amino acid composition of oat protein is very close to the amino acid composition required by the human body.

In International Application Publication No. WO 89/08405, nutritional compositions are described as suitable for intestinal feeding, where oatmeal is enzymatically degraded, heat treated, enteric with α-amylase, possibly protease, and β-glucanase Obtained by a combination of fermentation with Lactobacillus having the ability to spontaneously attach to. The nutritional composition described in the patent application is combined with the Lactobacillus strain according to the invention, and for patients undergoing normal treatment after major surgery, special treatment and other diseases for patients with disease or systemic rupture that require careful attention, for example A composition superior to oral, intestinal or rectal administration for the treatment of ulcerative colitis. It is also essential to drop the pH value rapidly during fermentation to stop the growth of other bacteria.

It is known that administration of Lactobacillus, which has the ability to form or attach colonies in the intestine, inhibits the colonization of other bacterial flora in the intestine and can reduce the risk of sepsis associated with bacterial infections such as postoperative abdominal surgery. Such treatments appear to be as effective as conventional therapies currently used in the form of antibiotics. Therefore, it would be reasonable for patients undergoing bowel surgery to receive pretreatment with Lactobacillus rather than antibiotics. This means less secondary side effects and less expensive treatments can be established without destroying the normal intestinal microflora.

The present invention also relates to the use of nutritional compositions fermented by Lactobacillus plantarum 299v according to the present invention instead of using antibiotics for the prevention or treatment of bacterial infections associated with surgical procedures.

The composition according to the invention can be administered in any suitable route, preferably orally or rectally, for example in the form of an enema. The composition may also be administered to the intestine via a catheter via the stomach or directly inserted into the intestine. Tests have shown that this effect can be improved, for example, when dietary fiber in the form of oatmeal porridge or β-glucan is fed. Treatment should be performed once or several times a day for 1-2 weeks.

The ability of the strain to colonize the intestine is preferably tested by oral administration followed by demonstration of colony formation on the intestinal mucosa at least 10 days after completion of the administration.

Complementary selection of isolated strains can be carried out before and after colonization tests for several functional and technical properties such as bile resistance, pH resistance, fermentation capacity of the required substrate (preferably oatmeal), flavor production capacity, freeze drying. Resistance to antibiotics and antibiotic resistance can be assessed.

To pass through the gastrointestinal tract, the selected strain must be viable for 30 minutes at pH 1.0 and be able to grow in the presence of 0.1% bile.

Isolation of Lactobacillus Planta Room 299v from Human Body

To isolate strains with the ability to colonize and settle on human intestinal mucosa, Lactobacillus strains were sampled from human mucosa, as described in WO93 / 01823. Biopsy tissue was taken by colonoscopy from the colon and intestinal mucosa pieces were removed from the small intestine (plant and ileum) during surgical operation. Mucosa samples were immediately placed in special medium (0.9% NaCl, 0.1% peptone, 0.1% Tween 80 and 0.02% cystine; all values are weight / volume%), homogenized for 2 minutes in ultrasonic concentration and then Rogosa agar (Difco Laboratories, Detroit Michigan, USA) before stirring for 1 minute. Plates were incubated anaerobically (37 ° C.) at 37 ° C. for 2 days (Gas Pak Anaerobic System, BBL). One to three colonies are randomly drawn from each plate and incubated 5-9 times on pure culture on Rogosa agar and maintained as a dense culture in freeze buffer at -80 ° C. A total of 209 Lactobacillus strains were isolated from about 61 different individuals. All isolates were characterized for their ability to ferment 49 different carbohydrates using API 50 CH, a commercial test kit sold by API (Montalieu Verceu, France). No significant difference was observed in the composition of the Lactobacillus plexus between the small and large intestine.

Representative strains from different groups were evaluated for pH resistance, dietary ability in the presence of bile, and fermentation ability of otomil gruel.

pH resistance was tested by adding 0.1 ml of bacterial suspension (incubated in logon broth and resuspended in centrifugation and physiological salt solution, 10 ⁹ CFU / ml) to 2 ml of phosphate buffer (pH 1.0). Thirty minutes later, the logo was inoculated onto agar plate, and if the growth was visually confirmed after incubation at 37 ° C. for 3 days, the test was determined to be positive. Only a few of the strains tested passed this test.

Proliferation in the presence of bile was tested by propagating Lactobacillus isolates by anaerobic incubation for 3 days at 37 ° C. in the presence of 0.1% and 0.15% beef bile in Rogosa agar plates, respectively. About 80% of the strains could grow in the presence of 0.1% bile, while only 18% could grow in the presence of 0.15% bile.

As a result of these tests, the following 19 different Lactobacillus strains were selected for later testing: L. Salivarius 132, L. Salibarius 280, L. L. reuteri 108, L. Loot 47, L. Kazei Subjong Psuplantarum 136, L. L. jenseni , L. L. gasseri 140, L. Zenseni-L. Gasseri 292, L. Ashdophilus-L. Crispatus 308, L. Planta Room 283, L-Planta Room 299, L. Planta Room 299v, L. Casei subspecies ramnosus 98, l. Casei subspecies ramnosus 271, l. Algilis 294, Lactobacillus subspecies 96, Lactobacillus sp. Strain 99, Lactobacillus sp. Strain 138, Lactobacillus sp. Strain 227 and Lactobacillus sp. Strain 282.

Strain designation is described by Josnsson et al., Appl. Environ. Microbiol. vol, 59, No. 1., 15-20, 1993, which may be incorporated by reference.

To assess the colonization capacity of 19 selected strains in the human intestine, a mixture of the selected strains in fermented oatmeal soup was orally administered to 13 healthy volunteers. The daily intake was about 5 × 10 ⁸ CFU for each strain and dosing continued for 10 days. One day before dosing, one and eleven days after the end of dosing, biopsy tissue was taken from the upper mill and russ rectum. Randomly extracted lactobacil isolates (10 per logo plate) were identified according to phenotype through API 50 CH test, and described in [ S, International Jonrnal of Systematic Bacteriology, 40: 189-193, 1990], and genotyping was determined by the results of restriction enzyme analysis and plasmid DNA of chromosomal DNA. The results are summarized in Table 1.

Table 1

Lactobacillus strains found in biopsy tissue taken from intestinal mucosa of 13 volunteers 11 days after oral administration of a mixture containing 19 different lactobacillus strains in lactically fermented oatmeal soup

Five of the strains administered were found 11 days after the end of the dosing, which was the same as found one day after discontinuation. All recovered strains could be isolated from the rectum and the plant, but not necessarily from the same individual. It should be emphasized that because of the separation process, only strains in the predominant position of the Lactobacillus layer are expected to be recovered, that is, only those strains drawn from the countable Logo company plates are expected to be recovered.

Re-isolated strains 11 days after the end of dosing at concentrations of about 3 x 10 ³ to 10 ⁵ CFU / g (mucosa) in the small intestine on the mucosa and 10 ³ to 3 x 10 ⁷ CFU / g (mucosa) in the large intestine Found in concentration.

Description of Lactobacillus Strain 299v

Strain 299v isolated from healthy human intestinal mucosa and sour dough was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, March 16, 1995, Accession No. DSM 9843 It became.

Phenotype description

Strain 299v consists of Gram-positive, catalase-negative bacilli growing on Rogosa agar at pH 5.5. The ability of this strain to ferment different carbohydrates is shown in Table 2. The test was performed using API 50 CH according to the manufacturer's instructions.

TABLE 2

Ability to form acids from different carbohydrates

Phenotypicly, strain 299v can be identified as Lactobacillus plantarum (only raffinose deviated from the test pattern for L. plantarum ATCC 14917 ^T. This strain is the type strain for L. plantarum , Ie, the strain defining this species).

Genotype Description

Restriction Endonuclease Assay (REA, supra) According to, et al., Et al., The cleavage pattern of chromosomal DNA was examined in relation to the result of cleavage with EcoRI. REA-pattern means the pattern formed by electrophoresis on the agar gel of DNA cut | disconnected with the restriction enzyme according to the following method. By characterizing the strain by the REA pattern, it is possible to identify used isolates that were not previously possible. Closely related Lactobacillus, which differs in the REA-pattern, also differs in their ability to adhere to intestinal epithelial cells. REA can be described schematically as follows.

(1) Isolate chromosomal DNA from the strains involved in the study.

(2) DNA is cut with restriction enzymes.

(3) The cleaved DNA fragments are separated by size by agarose gel electrophoresis.

(4) Band patterns of different strains are registered and interpreted with a laser densitometer and associated program. Differences in REA-pattern between strains can be represented mathematically by principal component analysis.

1 shows the REA-patterns of Lactobacillus plantarum 299v and Lactobacillus plantarum 299. Lane s represents standard high molecular weight DNA markers (AEH, BRL, Bethesda Research Laboratories, Life Technologies, Inc.). Strain 299v cannot be distinguished from strain 299 by conventional phenotypic testing. Genetically, strains 299 and 299v are very close.

Tests were also performed on the contents of the plasmids. S. Molin G & S, Systematic and Applied Microbiology 11: 320-325, 1989]. Strain 299v contains four plasmids of size 4MDal, 9MDal, 20MDal and 35 MDal, respectively.

Culture of Lactobacillus 299v

Inoculum from the -81 ° C. freezer is added to 50 ml of Lactobacillus culture medium (LCM, Efthymiou & Hansen, J. Infect, Dis., 110: 258-267, 1962) or Logo.

Incubate it for about 40 hours at 37 ℃.

Inoculate 50 ml in 500 ml LCM.

Incubate it for about 40 hours at 37 ℃.

Inoculate 500 ml into 5 l.

Incubate at 37 ° C. for about 25-30 hours.

Centrifuge for 10 minutes at 10,000 rpm.

Wash once in physiological salt solution.

Dissolve the pellet in about 1 l of physiological salt solution.

This amount seems to be sufficient for 400 to 500 l of oatmeal porridge. Culture medium is not optimized. Logo was more effective than LCM because its buffering function was better. 2% glucose was added to the LCM. The same method can be used to produce other Lactobacillus strains.

Manufacture of Oatmeal Porridge

Fermented oatmeal porridge was made in three stages.

(i) 1295 g of oatmeal (MP-450, Nord-Mills, Protein content 14.2% and ash content 2.1%), 129.5 g of enzyme mixture (Nord Malt, ) And 5390 g of tap water were mixed and heated to 95 ° C. with gentle stirring. Oatmeal porridge was cooled to 50 ° C., 1% β-glucanase (weight / volume) was added (GV-L; Grindsted Products A / S, Braband, Denmark) and incubated at 50 ° C. for 2 hours.

(ii) The porridge was inoculated with fresh Lactobacillus and fermented at 37 ° C. for 15-20 hours. pH was 3.4-3.9.

(iii) The fermented porridge was lyophilized. The mixture was supplemented with 20% (w / w) soy flour (51% protein, 5.5% ash content, 1% fat). The nutrient mixture contained 2 × 10 ⁷ CFU / g and was stored at −18 ° C. In the same manner as above, non-fermented oatmeal porridge was prepared without the fermentation process.

The ability to flavor oatmeal porridge by fermentation was assessed by a panel of experts consisting of four experts evaluating oatmeal porridge fermented by different strains. Flavors were evaluated in scores from 5 to 1, with 5 meaning "very good" and 1 meaning "unpleasant." The evaluation score for the strain Lactobacillus plantarum 299v was 5.

1 is a photograph showing the REA-pattern of two strains Lactobacillus plantarum 299v and 299.

Claims (4)

Lactobacillus plantarum 299v (DSM 9843), a Lactobacillus strain having the ability to colonize the human intestinal mucosa in vivo. A composition for preventing or treating infection of the gastrointestinal tract, comprising the strain Lactobacillus plantarum 299v with a conventional carrier. The composition for oral, enteric or rectal administration according to claim 2, which is an oatmeal based nutrient solution fermented by Lactobacillus strains. A nutritional composition for replacing antibiotics for the prevention or treatment of bacterial infections associated with surgical operations obtained by fermenting a substrate with Lactobacillus plantarum 299v according to claim 1.