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.

1.1. Lactobacillus crispatus BCCM/LMG P-17631 (ID1030, 2950)

The food constituent that is the subject of the proposed health claims is Lactobacillus crispatus BCCM/LMG P-17631.
For L. crispatus BCCM/LMG P-17631, a culture collection number from the Belgian Co-ordinated Collections of Microorganisms (BCCM/LMG P-17631) was provided. The BCCM/LMG is an internationally recognised culture collection which has the status of an International Depositary Authority under the Budapest Treaty. In the LMG, cultures can be deposited in a restricted-access collection for safe deposit or for patent purposes. Data on the identification and characterisation of L. crispatus BCCM/LMG P-17631 at species and strain level, by different phenotypic (carbohydrate fermentation profiles, PAGE) and genotypic (16S rRNA gene sequence analyses, plasmidic profile, ARDRA, Rep-PCR, AFLP) methods, were provided in the applications for further assessment and in the accompanying references (AAT, 2011a, unpublished, 2011b, unpublished; BCCM/LMG, 1997, unpublished; Dondi and Morelli, 1999, 2002; Morelli, 1997, unpublished).
The Panel considers that the food constituent that is the subject of the proposed health claims, L. crispatus BCCM/LMG P-17631, is sufficiently characterised.

1.5. Lactobacillus paracasei CNCM I-1688 (ID 2962, 2963)

The food constituent that is the subject of the proposed health claims is Lactobacillus paracasei CNCM I-1688.
For L. paracasei CNCM I-1688, a culture collection number from the CNCM, I-1688, was provided. Data on the identification and characterisation of L. paracasei CNCM I-1688 at species and strain level, by different phenotypic (carbohydrate fermentation profiles) and genotypic (16S rRNA gene sequence analyses, plasmidic profile, ARDRA, RAPD, Rep-PCR, PFGE) methods, were provided in the applications for further assessment and in the accompanying references (AAT, 2011f, unpublished; Bonetti et al., 2002; Morelli, 1996, unpublished; Morelli, 1997, unpublished).
The Panel considers that the food constituent that is the subject of the proposed health claims, L. paracasei CNCM I-1688, is sufficiently characterised.

1.101. Characterisation of “Lactobacillus paracasei I1688” (ID 2962, 2963)

The food constituent that is the subject of the health claim is Lactobacillus paracasei I1688. Information on the strain identification/characterisation by sugar fermentation and plasmidic profile of the strain was found in the references provided (Pedraglio, 2004; Morelli, 1996). RAPD and ARDRA for this strain were also performed in a study (Bonetti et al., 2002). No other information regarding the identification/characterisation of the strain Lactobacillus paracasei I1688 was found in the literature. The Panel considers that Lactobacillus paracasei I1688, which is the subject of the health claims ID 2962, 2963, is not sufficiently characterised. According to a patent cited in the references provided as reference the strains is deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), as Lactobacillus paracasei CNCM I-1688 (Pedraglio, 2004). The CNCM is a restricted-access non-public collection which has the status of International Depositary Authority under the Budapest Treaty.

2.6. Zwiększenie produkcji interleukiny 10 (IL-10) i/lub zwiększenie aktywności komórek NK (Natural Killers) (ID 2960, 2962, 2971)

The claimed effects which are proposed for further assessment are: “Supports your natural (immune) defence system by increasing the IL-10 production and enhancing NK cell activity in peripheral blood mononuclear cells (PBMC); Necessary to maintain the natural defences/helps to maintain a balanced immune system (increasing the IL-10 production and enhancing NK cell activity)”, and “Supports your natural (immune) defence system by increasing the IL-10 production in peripheral blood mononuclear cells (PBMC); Necessary to maintain the natural defences/helps to maintain a balanced immune system (increasing the IL-10 production)”. The proposed target population is the general population.
The Panel notes that the claimed effect “supports your natural (immune) defence system /necessary to maintain the natural defences /helps to maintain a balanced immune system” is not sufficiently defined, and assumes that the claimed effect relates to increasing IL-10 production by peripheral blood mononuclear cells and/or enhancing the lytic activity of natural killer cells.
The Panel notes that increasing IL-10 production by peripheral blood mononuclear cells and/or enhancing the lytic activity of natural killer cells is not a beneficial physiological effect per se, but needs to be linked to a beneficial physiological or clinical outcome.
Most of the references provided in relation to these claims were on methodologies for bacterial strain identification; in vitro studies on immunomodulatory properties (i.e. lymphocyte proliferation,
phenotype of the lymphocytic subpopulations, cytokine production) (Castellazzi, 2007a, unpublished; Castellazzi et al., 2007b; Castellazzi, 2007c, unpublished); and a patent (Dondi and Malfa, 2007) which reported the results of the in vitro study by Castellazi et al. (2007a, unpublished).
In relation to ID 2962 and 2971, two references reported on the same single-arm (no control group) human intervention study (Castellazzi, 2007a, unpublished; Valsecchi et al., 2008) which investigated the effects of two strains in combination, while the single strains are the subject of the claims, on allergy symptoms (atopic dermatitis, rhino-conjunctivitis, urticaria, contact dermatitis, oral allergy syndrome and food allergies) and immunological parameters (i.e. lymphocyte population, natural killer activity and cytokine production) in 20 children with atopic disorders. The Panel notes that this single-arm study was not controlled and investigated a combination of strains rather than the single strains which are the subject of the claim, and considers that no conclusions can be drawn from this study for the scientific substantiation of the claim.
The Panel considers that the evidence provided does not establish that increasing IL-10 production by peripheral blood mononuclear cells and/or enhancing the lytic activity of natural killer cells is a beneficial physiological effect.
The Panel concludes that a cause and effect relationship has not been established between the consumption of the food constituents which are the subject of the health claims and a beneficial physiological effect related to an increase in IL-10 production and/or an enhancement of the lytic activity of natural killer cells.

Oświadczenia zdrowotne

ID 2962 - Lactobacillus paracasei I1688

1. Charakterystyka żywności / składnika

1.1. Lactobacillus crispatus BCCM/LMG P-17631 (ID1030, 2950)

1.5. Lactobacillus paracasei CNCM I-1688 (ID 2962, 2963)

1.101. Characterisation of “Lactobacillus paracasei I1688” (ID 2962, 2963)

2.6. Zwiększenie produkcji interleukiny 10 (IL-10) i/lub zwiększenie aktywności komórek NK (Natural Killers) (ID 2960, 2962, 2971)

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