1. Charakterystyka żywności / składnika

Introduction on the process used for characterisation of food constituents that are microorganisms: Microorganisms or microbes (e.g. bacteria) are living organisms, and can change over time depending on culture conditions. Correct identification of the microorganism‟s species and strain is of critical importance, as the observed effects are species and strain specific. The appropriate classification, identification and nomenclature of microorganisms constitute the starting point for the assessment of microbial properties. Classification assigns an organism to a known taxonomic group according to its similarity to that group. This allows the prediction of the properties of the microorganism on the basis of what is already known about the taxa. A reliable identification confirms the identity of the strain(s) used in a given process and requires the use of appropriate methods. Traditional phenotypic identification of bacteria is not always reliable since certain species cannot be distinguished by these methods. Molecular techniques have emerged in recent years as a replacement or complement to traditional phenotypic tests. DNA-DNA hybridization has become the generally accepted standard for determination of bacterial species identification. However this technique is difficult to perform and requires an expertise not normally present in the food industry. For these reasons phylogenetically based approaches such as sequence analysis of the 16S rRNA gene has proven to be a useful tool for bacterial identification. The EU-funded PROSAFE project concluded that biochemical tests should not be used as a stand-alone approach for identification of bacterial cultures (Vankerckhoven et al., 2008). The use of 16S rRNA gene sequence analysis was considered the best tool for routine species identification. Moreover, the use of sequence-based methods, such as 16S rRNA gene sequencing, was encouraged given their high reproducibility and data exchangeability (Vankerckhoven et al., 2008). The FAO/WHO expert group (FAO, 2006) recommends that phenotypic tests should be done first, followed by genetic identification, using methods such as DNA-DNA hybridization or 16S rRNA sequence analysis. Nevertheless, it is important to underline that in some cases 16S rRNA sequencing has a limited resolution and it may not be enough for discrimination of closely related species (Felis and Dellaglio, 2007; Vankerckhoven et al., 2008) being necessary to use other methods.
For the strain identification (characterisation of the strain by genetic typing), the FAO/WHO working group also recommended that strain typing has to be performed with a reproducible genetic method or using a unique phenotypic trait (FAO, 2006). DNA macrorestriction followed by Pulsed Field Gel Electrophoresis (PFGE) is considered as the generally accepted standard (FAO, 2006) and it has been extensively used for differentiating commercial microorganism strains. Other discriminatory molecular methods, such as Randomly Amplified Polymorphic DNA (RAPD) or Amplified rDNA restriction analysis (ARDRA) among others, are also available for strain characterisation. Hence, species identification and sufficient characterisation (genetic typing) at strain level by using internationally accepted molecular methods is needed. In addition, strains should be named according to the International Code of Nomenclature. Although there is no direct requirement on deposition of the particular strain in an internationally recognised culture collection, the FAO/WHO (FAO, 2006) recommends that strains should also be deposited in an internationally recognised culture collection (with access number). These will assure the tracking and access of scientists and regulatory authorities to the strain and related information in case it is needed. In the context of the Regulation (EC) nº 1924/2006, the purposes of characterisation are to confirm the identity of the food/constituent that is the subject of the health claim, and to establish that the studies provided for substantiation of the health claim were performed with the food/constituent in respect of which the health claim is made. Although not required for substantiation of a claim, characterisation should also be sufficient to allow control authorities to verify that the food/constituent which bears a health claim is the same one that was the subject of a community authorisation. The Panel has decided to use the following criteria for characterisation of food constituents that are microorganisms, which are the subject of health claims:
 Species identification by DNA-DNA hybridization or 16S rRNA sequence analysis.
 Strain identification by DNA macrorestriction followed by PFGE, RAPD, ARDRA or other internationally accepted genetic typing molecular methods.
Only when these two criteria were fulfilled, the microorganism was considered to be sufficiently characterised. In case of combination of several microorganisms, the Panel considers that if one microorganism used in the combination is not sufficiently characterised, the combination proposed is not sufficiently characterised. The characterisation of food constituents that are microorganisms, which are the subject of health claims pursuant to Article 13 of the Regulation (EC) nº 1924/2006, is based on evaluation of available references up to 31 December 2008, including the following:
 The information provided by the Member States in the consolidated list of Article 13 health claims and references that EFSA has received from Member States or directly from stakeholders;
 Generally available data obtained by searching PubMed and Web of Science databases by using the strain name as search term.

1.38. Characterisation of a combination of “Lactobacillus helveticus CNCM I-1722 and Bifidobacterium longum CNCM I-3470” (ID 938, 939)

The food constituent that is the subject of the health claim is a combination of “Lactobacillus helveticus CNCM I-1722 and Bifidobacterium longum CNCM I-3470”. Lactobacillus helveticus CNCM I-1722 - The identification/characterisation of the strain Lactobacillus helveticus CNCM I-1722 is not included in the studies used as reference material and no information regarding the strain identification/characterisation was found in the literature. The Panel considers that Lactobacillus helveticus CNCM I-1722, which is the subject of the health claims ID 938, 939, is not sufficiently characterised. A culture collection number from the Collection Nationale de Cultures de Microorganismes (CNCM) was provided, but instead referring to another strain, Lactobacillus acidophilus, under the same collection number (CNCM I-1722) which is included in a patent (Durand and Panes, 2001). This indicates problems or a mistake in identification of the strain. Bifidobacterium longum CNCM I-3470 - The identification/characterisation of the strain Bifidobacterium longum CNCM I-3470 is not included in the studies provided as reference material and no information regarding the strain identification/characterisation was found in the literature. The Panel considers that Bifidobacterium longum CNCM I-3470, which is the subject of the health claims ID 938, 939, is not sufficiently characterised. A culture collection number from the Collection Nationale de Cultures de Microorganismes (CNCM) was provided. The CNCM is a restricted-access non-public collection which has the status of International Depositary Authority under the Budapest Treaty. The Panel considers that the combination of “Lactobacillus helveticus CNCM I-1722 and Bifidobacterium longum CNCM I-3470”, which is the subject of the health claims ID 938, 939, is not sufficiently characterised.

2. Znaczenie oświadczenia dla zdrowia człowieka

The claimed effects which are proposed for further assessment are “maintenance of the defence against pathogenic gastro-intestinal (GI) microorganisms” and “decreasing potentially pathogenic intestinal microorganisms”. The proposed target population is the general population.
The presence of pathogenic microorganisms in the GI tract (e.g. viruses and bacteria) may lead to the development of GI infections. Defence against GI pathogenic microorganisms may protect against the development of GI infections.
The Panel considers that defence against pathogenic gastro-intestinal microorganisms is a beneficial physiological effect.

3. Naukowe uzasadnienia wpływu na zdrowie człowieka - Obrona przed patogenami przewodu pokarmowego

Of the references provided in relation to the claim, nine were human studies (five studies were on antibiotic-associated diarrhoea, two related to Helicobacter pylori (hereafter H. pylori) eradication therapy, and two on the treatment of diarrhoea), eight were animal studies, and six were in vitro studies. Five papers were published in Ukrainian and one in Czech, and their full English translations were given.
All the studies were carried out with the combination of microorganisms that is the subject of the health claim, consisting of L. rhamnosus CNCM I-1720 and L. helveticus CNCM I-1722 in a 95:5 ratio.
Studies on antibiotic-associated diarrhoea (AAD)
One study in adults and four studies in children which investigated the effect of the combination of L. rhamnosus CNCM I-1720 and L. helveticus CNCM I-1722 on AAD were provided.
Two studies were conducted in Korean hospitalised adult patients and in Ukrainian children with cystic fibrosis, respectively, under antibiotic treatment for respiratory tract infections (Aryayev and Kononenko, 2009; Song et al., 2010). The Panel notes that the infectious nature of the diarrhoeal episodes was not clearly established by the diagnostic criteria used, that causes of diarrhoea other than GI infections were not systematically excluded, that microbiological analyses were not performed, and that antibiotic treatment may induce diarrhoea by mechanisms unrelated to GI infections. The Panel considers that no conclusions can be drawn from these studies for the scientific substantiation of a claim related to defence against pathogenic gastro-intestinal microorganisms.
In a randomised, open-label study, Marushko and Shef (2007) evaluated the effect of the combination of L. rhamnosus CNCM I-1720 and L. helveticus CNCM I-1722 added to antibiotic therapy vs. antibiotic therapy alone (control) in a group of 34 hospitalised children with pneumonia or bronchitis aged from 10 months to 3 years. The presence and duration of diarrhoea, abdominal pain, abdominal bloating and vomiting were measured, and microbiological analyses of stools before and after treatment were performed. The Panel notes that the methodology used for the microbiological analyses was not well described, that the bacterial groups analysed were not sufficiently characterised as pathogens, and that the evidence provided, therefore, did not establish the infectious aetiology of the episodes of diarrhoea. The Panel considers that no conclusions can be drawn from this study for the scientific substantiation of a claim related to defence against pathogenic gastro-intestinal microorganisms.
Maydannik et al. (2010), in a multicentre, randomised, open-label study, assessed the effect of the combination of L. rhamnosus CNCM I-1720 and L. helveticus CNCM I-1722 together with antibiotic therapy vs. antibiotic therapy alone (control) on the incidence and duration of diarrhoea, and on the presence of C. difficile toxins A and B in 244 children with infections of the respiratory, urinary or digestive tracts, and who received antibiotic therapy for not less than seven days. The Panel notes that
no information was provided about the baseline characteristics of the two study groups (e.g. age and carrier status for С. difficile toxins), that precise information on type and duration of the antibiotic treatment in the two study groups was not provided even though development of AAD was reported to be influenced by the type of antibiotic used, and that the statistical tests used in the data analyses were not described. The Panel considers that no conclusions can be drawn from this study for the scientific substantiation of the claim.
In an open-label study, Gnaytenco et al. (2009) compared the incidence of AAD related to standard triple H. pylori eradication therapy (control; omeprazole, amoxicillin and clarithromycin for seven days) with the same therapeutic regimen taken together with the combination of L. rhamnosus CNCM I-1720 and L. helveticus CNCM I-1722 in 45 subjects. The incidence of diarrhoea was measured and the assessment for C. difficile toxins A and B was performed at the beginning and at the end of the intervention. The Panel notes that it is unclear whether the study was randomised, that no information was provided on the baseline characteristics of the two study groups (e.g. clinical features, age and carrier status for С. difficile toxins), and that the statistical tests used in the data analyses were not described. The Panel considers that no conclusions can be drawn from this study for the scientific substantiation of the claim.
Studies on H. pylori eradication
Two open-label trials conducted in subjects with H. pylori infection undergoing treatment with antibiotics and proton pump inhibitor therapy for H. pylori eradication assessed the effect of the combination of L. rhamnosus CNCM I-1720 and L. helveticus CNCM I-1722 as co-adjuvant therapy for H. pylori eradication (Vdovychenkon et al., 2008; Ziemniak, 2006). The Panel notes that no evidence was provided that results obtained in patients with H. pylori infection under antibiotics with respect to treatment of the disease can be extrapolated to healthy subjects with respect to the development of H. pylori infection. The Panel considers that no conclusions can be drawn from these studies for the scientific substantiation of a claim on defence against pathogenic gastro-intestinal microorganisms targeted to the general population (i.e. subjects without infections).
Treatment of diarrhoea
Two studies addressed the use of the combination of L. rhamnosus CNCM I-1720 and L. helveticus CNCM I-1722 for the treatment of acute and chronic diarrhoea of various origins, including GI infections, in hospitalised children (Tlaskal et al., 1995; 2005). The Panel notes that the status of the GI tract in subjects with diarrhoea due to a GI infection may not be comparable to the status of the GI tract in subjects without GI infection. The Panel considers that no conclusions can be drawn from these studies for the scientific substantiation of the claim.
Animal and in vitro studies
The animal studies provided evaluated the effect of the combination of L. rhamnosus CNCM I-1720 and L. helveticus CNCM I-1722 on the treatment of experimental Citrobacter rodentrium infection in adult mice (Johnson-Henry et al., 2005) and neonatal mice (Gareau et al., 2010), on bacterial translocation from the intestine to the lymph nodes and on the intestinal barrier function in rats experiencing chronic psychological stress (Zareie et al., 2006), on mucin expression in a rat model (Dykstra et al., 2011), on colonic macromolecular permeability and corticosterone level in an animal model of stress induced by neonatal maternal separation (Gareau et al., 2007), on the rate of H. pylori eradication in Mongolian gerbils (Brzozowski et al., 2006), on H. pylori colonisation in mice (Johnson-Henry et al., 2004), and on GI functions and immune markers (e.g. gastric emptying, intestinal permeability and gastric CD3+ cell counts) after chronic H. pylori infection in mice (Verdu et al., 2008).
The in vitro studies submitted investigated the effects of a combination of L. rhamnosus CNCM I-1720 and L. helveticus CNCM I-1722 on the interaction of Camplylobacter jejuni with a cell line or
invasion of T48 epithelial cell line (Alemka et al., 2010; Wine et al., 2009), on cytokine secretion by a human intestinal epithelial cell line (Wallace et al., 2003), on epithelial injury (paracellular permeability) following exposure to Escherichia coli O157:H7 (Sherman et al., 2005), on adhesion of Escherichia coli O157:H7 to epithelial cells (Johnson-Henry et al., 2007), and on the immune effects of Escherichia coli O157:H7 infection in epithelial cells (Jandu et al., 2009).
The Panel notes that no human intervention studies were provided from which conclusions could be drawn for the scientific substantiation of the claim. The Panel also notes that animal and in vitro studies cannot predict the occurrence of an effect of a combination of L. rhamnosus CNCM I-1720 and L. helveticus CNCM I-1722 on defence against pathogenic gastro-intestinal microorganisms in humans.
The Panel concludes that a cause and effect relationship has not been established between the consumption of a combination of L. rhamnosus CNCM I-1720 and L. helveticus CNCM I-1722 and defence against pathogenic gastro-intestinal microorganisms.

Oświadczenia zdrowotne

ID 939 - Lactobacillus rhamnosus CNCM I-1720, Lactobacillus helveticus CNCM I-1722

1. Charakterystyka żywności / składnika

1.38. Characterisation of a combination of “Lactobacillus helveticus CNCM I-1722 and Bifidobacterium longum CNCM I-3470” (ID 938, 939)

2. Znaczenie oświadczenia dla zdrowia człowieka

3. Naukowe uzasadnienia wpływu na zdrowie człowieka - Obrona przed patogenami przewodu pokarmowego

Warunki i możliwe ograniczenia stosowania oświadczenia