ID 3016 -
Symbiotyk: Lactobacillus helveticus CNCM I-1722, Bifidobacterium bifidum CNCM I-3426, Bifidobacterium infantis CNCM I-3424, fruktoligosacharydys z sacharozy
PL: Symbiotyk: Lactobacillus helveticus CNCM I-1722, Bifidobacterium bifidum CNCM I-3426, Bifidobacterium infantis CNCM I-3424, fruktoligosacharydys z sacharozy
EN: Symbiotic: Probiotics (Lactobacillus helveticus CNCM I-1722, Bifidobacterium bifidum CNCM I-3426, Bifidobacterium infantis CNCM I-3424 ) and fructoligosaccharides from sucrose
Pdf: a combination of Lactobacillus helveticus CNCM I-1722, Bifidobacterium longum subsp. infantis CNCM I-3424, Bifidobacterium bifidum CNCM I-3426
1. Charakterystyka żywności / składnika
Introduction on the process used for characterisation of food constituents that are bacteria and yeasts:
Correct identification of the bacterium's and yeast's species and strain is of critical importance, as the observed effects in the host 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 a microorganism to a known taxonomic group according to its similarity to that group. This classification allows the prediction of the general properties of the microorganism on the basis of what is already known about the taxon. A reliable identification confirms the identity of the strain(s) used in a given process and requires the use of appropriate methods.
Bacteria - Traditional phenotypic identification of bacteria is not always reliable as certain species cannot be distinguished by these methods. Molecular techniques have emerged as a replacement or complement to traditional phenotypic tests in recent years. DNA-DNA hybridisation has become the generally accepted standard for determination of bacterial species identification. Phylogenetic approaches based on sequence analysis of the 16S rRNA gene have also 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., 2008b). 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., 2008a). The FAO/WHO expert group (FAO/WHO, 2006) recommends that phenotypic tests should be done first, followed by genetic identification, using methods such as DNA-DNA hybridisation or 16S rRNA gene sequence analysis. Nevertheless, it is important to underline that in some cases 16S rRNA gene sequencing has a limited resolution and it may not be enough for discriminating closely related species (Felis and Dellaglio, 2007; Vankerckhoven et al., 2008a) making it 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. DNA macrorestriction followed by Pulsed Field Gel Electrophoresis (PFGE) is considered as the generally accepted standard (FAO/WHO, 2006) and it has been extensively used for differentiating
4 Regulation (EC) No 1924/2006 of the European Parliament and of the Council of 20 December 2006 on nutrition and health claims made on foods. OJ L 404, 30.12.2006, p. 9–25.
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commercial microorganism strains. Other discriminatory molecular methods, such as Randomly Amplified Polymorphic DNA (RAPD) among others, are also available for strain characterisation.
Yeasts - Traditional phenotypic characterisation of yeasts is not always reliable for species and strain identification. Currently, the available molecular techniques complement and replace traditional phenotypic tests for this purpose, because they have higher discrimination power and are independent of growth conditions. Strains of the species Saccharomyces cerevisiae are widely distributed in nature and used for different food fermentation processes (wine, beer, cider, bread, etc.) (Moreno-Arribas et al., 2005; Coton et al., 2006; Legras et al., 2007). This species can also be a saprophytic coloniser of human mucosal surfaces and has been isolated from the digestive tract, vagina, skin and oropharynx of healthy individuals (Aucott et al., 1990). Although the taxonomic designation of Saccharomyces cerevisiae var boulardii to the species Saccharomyces cerevisiae is controversial, currently the former is not accepted as a distinct species (Mitterdorfer et al., 2002).
Some of the most useful molecular methods to identify yeasts are based on the spacer regions between the highly conserved rRNA genes (rDNA). The spacer regions between the rDNA subunits (internal transcribed spacer; ITS) and between the repeats (non-transcribed spacer; NTS) are highly variable between and within species, which allows their use for taxonomy purposes with different discrimination power (Baleiras Couto et al., 1996). One of the widely accepted techniques used for the adscription of yeasts to the species Saccharomyces cerevisiae is the restriction fragment length polymorphisms (RFLP) analysis of the PCR products of the 5.8S-ITS region (Mitterdorfer et al., 2002; De Llanos et al., 2004, 2006). Other alternative molecular techniques used for identification of yeast species is the sequence analysis of the D1 and D2 domains of 26S rDNA (Kurtzman and Robnett, 1998; Coton et al., 2006) and rDNA spacer regions (Posteraro et al., 2005).
For identification of strains, diverse molecular typing techniques are available, including chromosome length polymorphism analysis by PFGE, RAPD, and microsatellite DNA polymorphism analysis (Mitterdorfer et al., 2002; van der Aa Kühle et al., 2005; Posteraro et al., 2005).
Hence, species identification and sufficient characterisation (genetic typing) at strain level of the bacteria/yeasts 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/WHO, 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 bacteria, which are the subject of health claims:
Species identification by DNA-DNA hybridisation or 16S rRNA gene sequence analysis.
Strain identification by DNA macrorestriction followed by PFGE, RAPD, or other internationally accepted genetic typing molecular methods.
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Only when these two criteria were fulfilled, the bacterium was considered to be sufficiently characterised.
The Panel has decided to use the following criteria for characterisation of food constituents that are yeasts, which are the subject of health claims:
Species identification of by restriction fragment length polymorphism analysis (RFLP) (e.g. RFLP of PCR products the 5.8S rDNA internal transcribe spacer [ITS] region) or by sequencing analysis of DNA taxonomic markers (e.g. the D1 and D2 domains of 26S rDNA or ITS regions).
Strains identification by chromosome length polymorphism (PFGE), RAPDs, microsatellite DNA polymorphism analysis or other internationally accepted genetic typing molecular techniques.
Only when these two criteria were fulfilled, the yeast was considered to be sufficiently characterised.
In the case of combination of several bacteria and/or yeasts, 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 bacteria and yeasts, 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.
1.32. Characterisation of a combination of “Lactobacillus helveticus CNCM I-1722, Bifidobacterium bifidum CNCM I-3426 and Bifidobacterium infantis CNCM I-3424” (ID 3016)
The food constituent that is the subject of the health claim is a combination of “Lactobacillus helveticus CNCM I-1722, Bifidobacterium bifidum CNCM I-3426 and Bifidobacterium infantis CNCM I-3424”. For Lactobacillus helveticus CNCM I-1722, a culture collection number from the Collection Nationale de Cultures de Microorganismes (CNCM) is erroneously provided, as it refers to another strain, i.e. Lactobacillus acidophilus, under the same collection number (CNCM I-1722) included in a patent (Durand and Panes, 2001). For Bifidobacterium bifidum CNCM I-3426, a culture collection number from the Collection Nationale de Cultures de Microorganismes (CNCM) is
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provided. The CNCM is a restricted-access non-public collection which has the status of International Depositary Authority under the Budapest Treaty. For Bifidobacterium infantis CNCM I-3424, a culture collection number from the Collection Nationale de Cultures de Microorganismes (CNCM) is provided. The CNCM is a restricted-access non-public collection which has the status of International Depositary Authority under the Budapest Treaty.
Lactobacillus helveticus CNCM I-1722 - The identification/characterisation of the strain Lactobacillus helveticus CNCM I-1722 is not included or cited in the studies provided 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 is not sufficiently characterised.
Bifidobacterium bifidum CNCM I-3426 - The identification/characterisation of the strain Bifidobacterium bifidum CNCM I-3426 is not included or cited in the studies provided as reference material and no information regarding strain Bifidobacterium bifidum CNCM I-3426 identification/characterisation was found in the literature. The Panel considers that Bifidobacterium bifidum CNCM I-3426 is not sufficiently characterised.
Bifidobacterium infantis CNCM I-3424 - The identification/characterisation of the strain Bifidobacterium infantis CNCM I-3424 is not included or cited in the studies provided as reference material and no information regarding the strain identification/characterisation was found in the literature. The species Bifidobacterium infantis has been reclassified as subspecies of Bifidobacterium longum, Bifidobacterium longum ssp. infantis (Sakata et al., 2002). The Panel considers that Bifidobacterium infantis CNCM I-3424 is not sufficiently characterised.
The Panel considers that the combination of “Lactobacillus helveticus CNCM I-1722, Bifidobacterium bifidum CNCM I-3426 and Bifidobacterium infantis CNCM I-3424”, which is the subject of the health claim ID 3016, is not sufficiently characterised.
2. Znaczenie oświadczenia dla zdrowia człowieka
The claimed effect, which is proposed for further assessment, is “helps to stimulate the immune system and thereby decreases the risk of occurrence of common infectious diseases”. The proposed target population is the general population.
The Panel assumes that the claimed effect refers to contribution to immune defence against pathogens. The Panel considers that contribution to immune defence against pathogens is a beneficial physiological effect.
3. Naukowe uzasadnienia wpływu na zdrowie człowieka - Udział w obronie immunologicznej organizmu przeciwko patogenom
The references provided in relation to the claim included one human study, one meta-analysis of human studies, animal studies, in vitro studies and general review articles. Of these references, many were not related to the combination of food constituents which is the subject of the claim, or they addressed characterisation aspects of the microorganisms. The Panel considers that no conclusions can be drawn from these references for the scientific substantiation of the claim.
One human intervention study (Cazzola et al., 2010b) and one animal study (Cazzola et al., 2010a) were carried out with the combination which is the subject of the health claim, consisting of L. helveticus CNCM I-1722, B. infantis CNCM I-3424 and B. bifidum CNCM I-3426 in the ratio of 6:2:2, with a total bacterial count ≥3 x 109 CFU and 750 mg FOS from sucrose.
In a multicentre, randomised, double-blind, placebo-controlled, parallel intervention study, Cazzola et al. (2010b) assessed the effect of consumption of the combination of bacterial strains and fructo-oligosaccharides, which is the subject of the claim, during a winter period (December – March) on the incidence of infections in 135 children (mean age 4.1±1.0 years) who had suffered from at least three episodes of ear, nose and throat infections, other respiratory tract infections or gastro-intestinal infections in the previous winter. The children were randomised in blocks of six to consume either the intervention (n=62) or the placebo (starch, n=73) for three months. All health problems of any type were recorded by parents in a diary. The primary outcome was the percentage of children who suffered from at least one health problem of any nature during the intervention period. Secondary outcomes comprised the percentages of children suffering from at least one health problem characterised by respiratory or gastro-intestinal symptoms, the percentage of children with at least one febrile episode, the number of reported health problems per included child, and the percentage of children with at least one health problem causing one or more days of school loss. The Panel notes that no information was provided on the diagnostic criteria and on the validity of diaries used to assess the incidence of infections, and that the evidence provided did not establish that the outcome measures used in this study were appropriate measures of infections in the study population. The Panel also notes that no information was provided in the publication about the use of rescue medication, which may have confounded the results and that different type of health problems were considered together. The Panel considers that no conclusions can be drawn from this study for the scientific substantiation of the claim.
One animal study (Cazzola et al., 2010a) explored the mechanisms by which the combination of bacterial strains and fructo-oligosaccharides which is the subject of the claim could exert an effect on the response to infections by measuring serum levels of pro- and anti-inflammatory cytokines in a rat model. The Panel considers that in the absence of evidence for an effect on immune defence against pathogens in humans, evidence provided in this animal study cannot be used for the scientific substantiation of a claim on immune defence against pathogens.
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 concludes that a cause and effect relationship has not been established between the consumption of a combination of L. helveticus CNCM I-1722, B. longum subsp. infantis CNCM I-3424, B. bifidum CNCM I-3426 and fructo-oligosaccharides from sucrose and contribution to immune defence against pathogens.
Warunki i możliwe ograniczenia stosowania oświadczenia
3.109 CFU/ day