RU2708146C2 - Digestive agent - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to biotechnology, in particular to strains of Lactococcus lactis capable of inhibiting the growth of a pathogenic microorganism and/or improving the integrity of the epithelial intestinal barrier.

EFFECT: invention discloses the use of the strain Lactococcus lactis CNCM I-2807 for producing a fermented milk product which can be used in treating or preventing digestive disorders.

1 cl, 2 tbl, 2 ex

Description

The present invention relates to compositions containing lactic acid bacteria for use in improving digestion. In particular, such compositions comprise a strain of Lactococcus lactis suitable for the treatment and / or prevention of gastrointestinal disorders.

Gastrointestinal infections are extremely common around the world. In the vast majority of victims, full recovery is quick. However, a significant proportion of patients suffering from gastroenteritis develop prolonged gastrointestinal symptoms, such as irritable bowel syndrome (IBS). Indeed, it is estimated that after bacterial or viral gastroenteritis, 3.7-36% of individuals develop IBS (Spiller and Garsed, 2009).

Gastrointestinal infections are also among the most common causes of diarrhea. In severe cases, diarrhea-causing infections can lead to stomach inflammation.

Gastrointestinal infections also include Crohn's disease (CD) and ulcerative colitis (UC), collectively referred to as inflammatory bowel disease (IBD).

Irritable Bowel Syndrome (IBS)

IBS is a chronic functional bowel disorder characterized by diarrhea or constipation, abdominal pain, bloating and passage of mucus into the stool.

Inflammation and infection are thought to play a role in the onset of IBS (Ohman and Simren, 2010). The Enterobacteriaceae family has been observed to be more common in subjects with IBS compared to healthy subjects (Si et al., 2004). However, this observation has not been repeated in other studies (Malinen et al., 2005), which demonstrate that when bacteria of the Enterobacteriaceae family play a role in the pathogenesis of IBS, this is not the only factor. In addition, it has been shown that subjects with IBS carry more enteroaggregative Escherichia coli (i.e. pathogenic Escherichia coli strains) than healthy subjects (Sobieszczanska et al., 2006).

Given that there is an exceptional association between the number of bacteria in the Enterobacteriaceae family and the IBS subgroup, there is increasing evidence that Enterobacteriaceae can cause IBS in the case of post-infectious IBS (PI-IBS) (Ji et al., 2005; Marshall et al ., 2010 and Thabane et al., 2010). These studies have shown that patients suffering from gastroenteritis caused by bacteria of the Enterobacteriaceae family (Escherichia coli, Campylobacter, Shigella) pose a greater risk of getting IBS.

Since the activation of the immune system has been considered recently as suggested, researchers have begun to evaluate the use of anti-inflammatory drugs as IBS therapy as an alleged pathophysiological cause of IBS (Camilleri, 2010). For example, the anti-inflammatory agent Mesalamine has shown very encouraging results in IBS subjects (Corinaldesi et al., 2009). In this study, Mesalamine was shown to alleviate the symptoms of IBS, improving overall well-being.

Further, probiotics, in particular strains of lactic acid bacteria, have been reported to be useful for the treatment and / or prevention of IBS. Examples of such strains are disclosed in International Applications WO 2007/036230, WO 03/010297 and WO 2009/080800.

Irritable Bowel Syndrome (PI-IBS)

PI-IBS is a subgroup of irritable bowel syndrome that develops after a gastrointestinal infection.

PI-IBS has been reported mainly in Europe, especially in the UK. However, in two recent studies, one from China and one from Korea, it was reported that PI-IBS also occurs in eastern countries with a predominance similar to that found in the west.

Pathogenic bacteria have been identified in most studies as etiological factors involved in the development of PI-IBS. Bacteria of the Enterobacteriaceae family, such as Salmonella, Shigella and Campylobacter, have been found among the most commonly isolated bacteria. Other bacteria that cause gastroenteritis, such as Listeria or pathogenic E. coli, could potentially be involved. Recent studies show that other infectious agents may also be involved, including viruses (e.g. rotavirus, adenovirus, calcivirus) and parasites (Giardia lamblia, Blastocystis hominis). The hypothesis is that a decrease in the incidence of intestinal infections in general can reduce the incidence of PI-IBS and possibly the severity of symptoms of PI-IBS.

The mechanism underlying PI-IBS has not been clearly identified. Particular attention was paid to the role of persistent moderate inflammation. Spiller and Garsed (2009) found an increase in the number of intraepithelial lymphocytes, T-cells of their own mucosal plate, mast cells and calprotectin-positive macrophages, which did not decrease in patients who develop PI-IBS. Several probiotic strains have been shown to have anti-inflammatory properties.

The intestinal epithelial lining, consisting of epithelial cells and dense compounds, is semi-permeable, allows the passage of small particles, and is also involved in maintaining the barrier function of the mucosa. In vitro and in vivo studies indicate that the activated intestinal immune system in PI-IBS is the result of increased local exposure to antigen associated with increased permeability of the intestinal epithelial layer. Patients with PI-IBS have increased permeability compared to healthy subjects. Decreased intestinal permeability can directly lead to improved symptoms of PI-IBS. Although several studies have shown that several strains of Lactobacilli species can increase the integrity of the epithelial barrier, there is no direct evidence that L. lactis strains have similar properties. In contrast, there is evidence that genetically modified L. lactis strains may be harmful to the epithelium (Shao and Kaushal, 2004).

Inflammatory Bowel Disease (IBD)

IBD is any of two inflammatory bowel diseases: Crohn's disease or ulcerative colitis.

Crohn's disease and ulcerative colitis are chronic idiopathic disorders caused by immunity, with genetic and environmental influences (factors), especially microbial influences. Crohn's disease is often associated with diarrhea, cramps, loss of appetite and weight with local abscesses and scarring. Ulcerative colitis is also associated with diarrhea, but with the release of mucus and blood, cramping abdominal pain and inflammation and swelling of the mucous membrane with ulcer spots. Crohn's disease and ulcerative colitis occur mainly in areas with the highest concentration of predominantly anaerobic bacteria in the distal ileum and colon. Serological responses to microbial antigens are present in 80% of patients with Crohn's disease, and high titers of antibacterial serology are associated with severe illness (Mow et al., 2004).

The ongoing controversy surrounding Crohn’s disease is whether the pathogen is responsible for the chronic recurrent nature of the inflammatory response. Recent significant data directly link functionally abnormal strains of E. coli (belonging to the Enterobacteriaceae family) with the pathogenesis of Crohn's disease (Sartor, 2008). Adherent / invasive E. coli (AIEC) adhere and infect epithelial cells and are retained within intestinal epithelial and macrophage cells (Darfeuille-Michaud et al., 1998). In vitro studies have developed virulence factors and molecular mechanisms that promote epithelial adhesion, invasion and persistence (Darfeuille-Michaud et al., 1998 and Baumgart et al., 2007).

The active IBD study area evaluates the possibility of dysbiosis, which is an abnormal microbial composition or function, as a cause of a chronic inflammatory process in the intestine. Dysbacteriosis can cause intestinal inflammation through several mechanisms (Sartor, 2009). Impaired production of butyrate and other short chain fatty acids can fundamentally affect intestinal epithelial function and mucosal barrier characteristics. The relative balance of beneficial and harmful bacteria can be a decisive factor in the induction of inflammation as opposed to mucosal homeostasis. The ability of an enteric microbiota to produce toxic metabolites may be related to the pathogenesis of ulcerative colitis. Intestinal bacteria can also produce reactive oxygen species that can cause tissue damage, especially in hosts with impaired degradation of oxygen metabolites.

Some studies document the observed changes in intestinal microbiota in patients with IBD (Peterson et al., 2008). Using molecular techniques, a decrease in certain bacterial populations with IBD, especially subsets of clostridia, and an increase in others, including Enterobacteriaceae (Sartor, 2009), have been shown. Swidsinski et al. (2009) noted a high level of Enterobacteriaceae in the microbiota of patients with CD and UC.

Moreover, the determination of microbial functions that are associated with or initiate IBD is complicated by the genetics of the host organism, the state of inflammation, and diet (Peterson et al., 2008). The development of promising studies of human IBD to determine the microbial communities that provoke inflammation was not possible even in genetically sensitive populations.

As a result, over the past few years, various mouse models of the chronic intestinal inflammatory process resembling IBD have been developed, and information has been provided on IBD and microbiota imbalance. Various IBD models in mice, including genetically modified animals with defects in the immune system or mucosal glycans, which were grown in the usual way with microbiota and then treated with antibiotics, or which were sterile and then colonized by intestinal microbiota from healthy donors, show that microbiota plays an important role in identifying pathologies (Garrett et al., 2007; Kang et al., 2008 and Sartor, 2008). It is important to emphasize that, although rodent colitis patterns typically depend on the presence of intestinal microbiota, not all microbes cause the disease (Kim et al., 2007). Studies on animal models show that some bacterial strains are harmful, while others have a protective effect, and many of them are neither aggressive nor beneficial (Sartor, 2009).

The L. Glimcher group developed a robust model of ulcerative colitis in mice, whose pathological features and response to anti-TNF treatment resemble this disease in humans. They found that the lack of certain regulatory genes in the natural immune system (obtained by double knockout of the T-bet and RAG2 genes; named TRUC for ulcerative colitis T-bet RAG2) resulted in aggressive, spontaneous and contact-transmitted UC and increased susceptibility to colitis in immunologically intact hosts (Garrett et al., 2007).

These in vivo results are consistent with clinical observations: Klebsiella and Proteus species are more likely to be observed in stool of patients with UC than in healthy subjects (Dorofeyev et al., 2009). There are also numerous reports of increased antibody titers against Enterobacteriaceae family bacteria in patients with IBD (Cooper et al., 1988; Ibbotson et al, 1987 and Tiwana et al, 1998).

In conclusion, the balance of beneficial and harmful bacterial species determines homeostasis as opposed to inflammation. The involvement of Enterobacteriaceae bacteria in these mechanisms has been demonstrated. For the treatment and prevention of recurrence of IBD, this balance can be manipulated, in particular, using probiotics.

In addition, it was shown that during active periods of IBD, inflammation of the gastrointestinal tract causes increased permeability of the epithelium, which leads to damage to the barrier function of the epithelium (Madsen et al, 2001).

The use of anti-inflammatory substances (also called biologists) for the treatment of inflammatory bowel diseases has finally increased, and they are currently used by gastroenterologists and doctors in caring for patients with IBD (as a review: Bosani et al., 2009). Alternatively, probiotics were used to alleviate IBD, and anti-inflammatory mechanisms have been proposed as a mechanism of action (Vanderpool et al., 2008).

Further evidence is growing that some probiotic and symbiotic bacteria improve weakening of the intestinal barrier (decreased permeability) in vitro (Resta-Lenert and Barratt, 2006 and Miyauchi et al., 2008) and in vivo and help alleviate the symptoms of IBS and IBD.

It follows from the foregoing that pathogens can cause gastrointestinal infections (including IBS, PI-IBS and IBD) and that compounds or probiotics with anti-pathogenic activity can prevent and / or cure these disorders. Further, the integrity of the epithelial barrier associated with anti-pathogenic activity can further prevent inflammation.

Lactococcus lactis bacteria are lactic acid bacteria commonly found in dairy products, such as cheese or fermented dairy products (including yoghurts). Some Lactococcus lactis bacteria have been described as having activity against pathogenic bacteria of the Enterobacteriaceae family, such as Salmonella enteritidis and Escherichia coli (Olasupo et al., 2003; Ganzle et al., 1999).

The authors of the present invention showed in vitro that two strains of Lactococcus lactis subsp. lactis, namely, strains DN 030 066 and DN 030 087, improve the integrity of the epithelial barrier compared to strains of bacteria known for their anti-inflammatory properties, and further, that strain DN 030 066 has superior antimicrobial activity compared to strains known for their antimicrobial activity.

Strain DN 030 066 was deposited, in accordance with the Budapest Treaty, at CNCM (National Collection of Microorganism Cultures, 25 Rue du Docteur Roux, Paris) on October 24, 1995, numbered 1-1631. This strain is disclosed in International Application WO 97/16529.

In CNCM, strain 1-1631 has the following characteristics:

- Morphology: Gram-positive microorganism, mainly small diplococci;

- Fermentation of the following sugars (results obtained on API 50 CH strip - API MRS at 30 ° C for 48 hours): ribose, galactose, glucose, mannose, mannitol, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, cellobiose , maltose, lactose, trehalose, amidone, beta gentibiosis.

Strain DN 030 087 was deposited, in accordance with the Budapest Treaty, at CNCM (National Collection of Microorganism Cultures, 25 Rue du Docteur Roux, Paris) on February 19, 2002 under the number 1-2807.

In CNCM, strain 1-2807 has the following characteristics:

- Morphology: Gram-positive microorganism, mainly small diplococci;

- Fermentation of the following sugars (results obtained on API 50 CH strip - API MRS at 37 ° C for 72 hours): ribose, galactose, glucose, mannose, mannitol, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, cellobiose , maltose, lactose, trehalose, amidone, beta gentibiosis.

Accordingly, the present invention provides a strain of Lactococcus lactis selected from the group consisting of strains DN 030 066 (CNCM 1-1631) and DN 030 087 (CNCM 1-2807) for use in the treatment or prevention of digestive disorders.

In an embodiment, said use is used to inhibit the growth of pathogenic microorganisms, preferably pathogenic bacteria of the Enterobacteriaceae family, and / or to improve the integrity of the intestinal epithelial barrier.

Said pathogen is preferably selected from the group consisting of the genera Escherichia, Salmonella, Listeria, Campylobacter, Shigella, Proteus (e.g. P. mirabilis) and Klebsiella, preferably from the group consisting of E. coli, L. monocytogenes and S. enteritidis species.

The specified digestive tract disorder may be, in particular, gastric or intestinal discomfort; gastric or intestinal infection; diarrhea, constipation; IBS PI-IBS or IBD (i.e., Crohn's disease and / or ulcerative colitis).

The present invention also provides a mutant strain of Lactococcus lactis obtained from strain DN 030 066 or strain DN 030 087 by mutagenesis or genetic transformation thereof to inhibit the growth of pathogenic microorganisms, preferably pathogenic Enterobacteriaceae, and / or improve the integrity of the intestinal epithelial barrier, in the treatment or prevention of disorders digestion as defined above.

The specified mutant strain of Lactococcus lactis can be obtained by mutating one or more endogenous genes (a) of the strain DN 030 066 or strain DN 030 087, for example, in order to change some of its properties (for example, its ability to ferment sugar, its resistance to acidity, its survival during transport in the gastrointestinal tract or its survival after acidification). It can also be a mutant strain resulting from the genetic transformation of strain DN 030 066 or strain DN 030 087 by one or more genes of interest (ohms) of interest, for example, in order to provide the physiological features with the specified strain.

These strains can be used as whole bacteria, which can be dead or alive, preferably alive.

The present invention also provides a cell fraction or metabolite obtained from strain DN 030 066 or strain DN 030 087 to inhibit the growth of a pathogenic microorganism, preferably pathogenic bacteria of the Enterobacteriaceae family, and / or to improve the integrity of the intestinal epithelial barrier in the treatment or prevention of digestive disorders, such as defined above.

Said cell fraction may be, for example, a cell wall fraction, a cytoplasmic fraction or a culture supernatant, or a fraction thereof.

The specified metabolite can be, for example, short chain fatty acids, peptides (e.g. bacteriocins) and / or compounds formed during the fermentation of strains DN 030 066 or DN 030 087.

The present invention also provides a composition comprising

- a strain of Lactococcus lactis selected from the group consisting of strains DN 030 066 (CNCM 1-1631) and DN 030 087 (CNCM 1-2807), as defined above, or

a mutant strain of Lactococcus lactis as defined above, or

- cell fraction or metabolite obtained from strain DN 030 066 or strain DN 030 087, as defined above,

for inhibiting the growth of a pathogenic microorganism, preferably a pathogenic bacterium of the Enterobacteriaceae family, and / or improving the integrity of the intestinal epithelial barrier, in the treatment or prevention of digestive disorders, as defined above.

The composition may include at least one other strain of Lactococcus lactis other than the strains defined above and / or at least one lactic acid bacteria of a species other than Lactococcus lactis, such as lactic acid bacteria of the genus Lactobacillus or Bifidobacterium.

The composition may be in any form suitable for administration, in particular oral administration. It includes, for example, solids, semi-solids, liquids and powders. A liquid composition is generally preferred for easier administration, for example, in the form of drinks.

The composition may include from 10 ⁵ to 10 ¹³ colony forming units (cfu) of at least one strain of L. lactis or a mutant strain of L. lactis, as defined above, preferably at least 10 ⁶ cfu, more preferably, at least 10 ⁷ cfu, even more preferably at least 10 ⁸ cfu, and most preferably at least 10 ⁹ cfu per gram dry weight of the composition. In the case of a liquid composition, this generally corresponds to 10 ⁷ -10 ¹² colony forming units (cfu), preferably at least 10 ⁵ cfu, more preferably at least 10 ⁶ cfu, even more preferably at least 10 ⁷ cfu and most preferably at least 10 ⁹ cfu / ml.

The composition may be a food product or a pharmaceutical product.

The composition may also be a dairy product or a fermented product, preferably a fermented dairy product, such as a fermented milk product, or a fermented whey product.

The fermented product can be presented in the form of a liquid or presented in the form of a dry powder obtained by drying the fermentation liquid.

The introduction of a fermented dairy product has the additional advantage of low lactose, which is an additional advantage for correcting digestive disorders, as defined above.

The specified fermented product may be a fresh product. A fresh product that has not undergone a deep heat treatment step has the advantage that the present bacterial strains are live.

The specified fermented milk product may be a yogurt or sour milk in an established, mixed or drinking form or cheese.

The specified dairy product may also be a fermented vegetable product, such as fermented soy, cereals and / or fruits in a fixed, mixed or drinking form.

In another embodiment, said composition is a baby food, infant formula, or infant formula.

In another embodiment, said composition is a nutraceutical or nutritional supplement.

The nutritional compositions of the invention also include nutritional supplements and functional foods.

As used herein, “nutritional supplement” refers to a product made from compounds commonly used in food products, but which are in the form of tablets, powder, capsules, servings, or in any other form, typically not related to food , and which have a beneficial effect on health.

As used herein, “functional foods” are foods that also have beneficial effects on health. In particular, nutritional supplements and functional foods can have a physiological effect - protective or therapeutic - in relation to a disease, for example, in relation to a chronic disease.

The composition is preferably intended for use by people, including, in particular, infants, the elderly and subjects suffering from imbalance in metabolism, for example, subjects suffering from obesity.

The present invention also provides a method for producing a mutant strain of Lactococcus lactis capable of inhibiting the growth of a pathogenic microorganism, preferably pathogenic Enterobacteriaceae, and / or improving the integrity of the intestinal epithelial barrier, comprising the step of mutagenesis or genetic transformation of a strain of Lactococcus lactis selected from the group consisting of strains DN 030 06666 (CNCM 1-1631) and DN 030 087 (CNCM 1-2807).

Methods of mutagenesis or genetic transformation of a strain of Lactococcus lactis are well known to any person skilled in the art.

The present invention also provides a strain of Lactococcus lactis, characterized in that it is a strain deposited in CNCM under the number 1-2807.

The specified strain is able to inhibit the growth of a pathogenic microorganism, preferably pathogenic Enterobacteriaceae, as defined above, and / or to improve the integrity of the intestinal epithelial barrier.

The present invention also provides a composition comprising a Lactococcus lactis CNCM 1-2807 strain.

The present invention also provides a Lactococcus lactis CNCM 1-2807 strain or composition comprising said strain for use as a medicine.

The present invention also provides a method of treating or preventing a digestive disorder, as defined above, in a subject in need thereof, comprising administering a L. lactis strain or composition, as defined above, to said subject.

In addition to the above features, the invention includes other features that will be identified from the following description, which relates to examples illustrating anti-pathogenic activity and transepithelial electrical resistance activity (TEER) of strains DN 030 066 and DN 030 087.

EXAMPLE 1: EVALUATION OF ANTI-PATHOGENIC ACTIVITIES OF THE STRAIN L. LACTIS DN 030 066

Antimicrobial activity against Salmonella enteritidis B1241, Listeria, and pathogenic E. coli was determined in overlapping tests. Three strains of Lactococcus lactis were evaluated in this test: the strain in accordance with the present invention DN 030 066, Lactococcus lactis subsp. lactis DSMZ 20729 and Lactococcus lactis subsp. cremoris DSMZ 4645, previously described as producer strains of bacteriocin (nisin and lacticin, respectively) (Bartoloni et al., 2004 and Park et al, 2003).

Strains of L. lactis were plated from frozen effluents on fresh plates with agar medium using a replicator. Cups were grown overnight until visible colonies appeared. Then, the upper agar containing nutrient medium with an extract from the brain-heart, and dilution of the isolated pathogenic microorganisms were poured onto plates with agar medium. The plates were incubated at 37 ° C. The next day, the diameters of the zones of inhibition of pathogenic microorganisms were measured.

A score of 1 corresponds to a diameter of 1 to 3 mm, a score of 2 corresponds to a diameter of 4 to 6 mm and a score of 3 corresponds to a diameter of more than 6 mm. The experiments were conducted on three different media: MRS (De Man Rogosa and Sharpe), Elliker and TGV (Tryptone-glucose-meat extract). Each experiment was carried out independently in triplicate. The scores obtained for each medium summarized the identification of a specific scoring for each strain and each pathogenic microorganism. Then, the scores obtained for each pathogenic microorganism were summarized to obtain for each strain an overall assessment of antimicrobial activity. The results are shown in table 1 below.

Table 1 Antimicrobial activity of selected strains of Lactococcus lactis Kinds Link E. coli L.
monocytogenes S.
enteritidis General antimicrobial scoring L. lactis subsp. lactis
DN 030 066 PCT
request
WO 97/16529 3 8 4 fifteen L. lactis subsp. lactis
DSMZ 20729 Harris et al.,
1992 3 7 2 2 L. lactis subsp. cremoris DSMZ 4645 Ryan et al.,
1996 one one 2 14

The above results show that the strain in accordance with the invention DN 030 066 has antimicrobial activity superior to the activity of two strains of Lactococcus lactis, known for their anti-pathogenic activity.

EXAMPLE 2: TEER-EVALUATION OF THE STRAPS L. LACTIS DN 030 066 AND DN 030087

The integrity of the intestinal barrier can be assessed by measuring the potential difference observed between the apical and basal sides of the monolayer of T84 cells. This experimental model is called transepithelial electrical resistance (TEER) (Hirotani et al., 2008).

Suspensions of cultures of various strains of bacteria were washed with PBS. Bacteria (100 cfu / cell) were then added to the apical portion of the monolayer of T84 cells. In the analysis, 96 strains were tested, including 64 Bifidobacteria, 32 Lactobacilli and 2 Lactococcus lactis (DN 030 0664 and DN 030 087). After 4 hours and 6 hours of incubation with various bacteria, the TEER value was determined to evaluate the epithelial barrier function. All experiments were performed three times independently and in triplicate. The value of T84 cells at t = 0 was taken as 100%. The results for 7 strains having the highest TEER value are shown in table 2 below.

table 2 TEER score of selected bacterial strains Strains Link TEER (%) at t = 4 h TEER (%) at t = 4 h L.lactis
DN 030 066 PCT application
WO 97/16529 110.90 113.94 L.lactis
DN 030 087 The present invention 108.54 102.79

L. rhamnosus
LGG ATCC 53103 Patent
US 4839281 96.94 101.43 L.acidophilus LA1 Link-Amster et al., 1994 93.29 100.78 T84 cells (control) one hundred 100.49 Bifidobacterium
longum NCC2705 PCT application
WO 2002/074798 99.83 94.87 Bifidobacterium
Longum w11 Patent
EP 1 609 852 100.81 87.68 Bifidobacterium
Longum bb536 Patent application
JP 2006081429 87.50 77.89

The above results show that the strains in accordance with the invention DN 030 0664 and DN 030 087 improve the integrity of the epithelial barrier compared with known strains (including strains having anti-inflammatory activity).

LINKS

Claims (1)

The use of strain DN 030087 CNCM I-2807 Lactococcus lactis to obtain a fermented dairy product.