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.

1.3. Lactobacillus gasseri BCCM/LMG P-18137 (ID 2957, 2958)

The food constituent that is the subject of the proposed health claims is Lactobacillus gasseri BCCM/LMG P-18137.
For L. gasseri BCCM/LMG P-18137, a culture collection number from the BCCM/LMG, P-18137, was provided. Data on the identification and characterisation of L. gasseri BCCM/LMG P-18137 at species and strain level, by different phenotypic (carbohydrate fermentation profiles) and genotypic (16S rRNA gene sequence analyses, ARDRA, Rep-PCR, PFGE) methods, were provided in the applications for further assessment and in the accompanying references (AAT, 2011d, unpublished; BCCM/LMG, 1998, unpublished; Dondi and Morelli, 1999, 2002).
The Panel considers that the food constituent that is the subject of the proposed health claims, L. gasseri BCCM/LMG P-18137, is sufficiently characterised.

1.100. Characterisation of “Lactobacillus gasseri P 18137” (ID 2957, 2958)

The food constituent that is the subject of the health claim is Lactobacillus gasseri P 18137. Reference to phenotypic identification of the strain was found (Dondi and Morelli, 1999; BCCM/LMG Report 04/09/98). However no other information regarding the strain identification/characterisation was found in the literature. The Panel considers that Lactobacillus gasseri P 18137, which is the subject of the health claims ID 2957, 2958, is not sufficiently characterised. According to the patent application by Dondi and Morelli (1999) the strain is deposited at the LMG under nº LMG P-18137. In the LMG, which is a non-public International Depositary Authority under the Budapest Treaty, cultures can be deposited in restricted-access collection as Patent deposits.

2.5. Ochrona przed patogenami pochwy (ID 2950, 2957, 2967)

The claimed effect which is proposed for further assessment is: “Helps in maintaining balanced vaginal flora and pH; Contributes to a healthy colonization of the lactobacilli on the vagina; Helps to redress the healthy balanced vaginal microflora during and after the treatment of vaginal disorders”. The proposed target population is the female population.
The Panel notes that the claimed effect refers to defence against vaginal pathogens by increasing the number of lactobacilli and/or decreasing potentially pathogenic bacteria.
Unlike any other anatomical site of the body, most vaginal vaults are dominated by one or more species of Lactobacillus. In over 70 % of women, the vaginal microbiota is dominated by lactobacilli (> 50 %) (Ling et al., 2010; Ravel et al., 2011; Yamamoto et al., 2009). This microbiota is different from the more complex gut microbiota, where lactobacilli represent less than 3 % of the bacterial population (Franks et al., 1998; Lay et al., 2005; Sghir et al., 2000). The diagnosis of bacterial vaginosis (BV) can be based for example on the Nugent score (microscopic examination of Gram stained smear or vaginal discharge for bacteria and „clue‟ cells). The Panel notes that appropriate outcome measures of the claimed effect include assessment of changes in the Nugent scores. Nugent scores are estimated by measuring the relative amounts of lactobacilli and bacterial pathogens present in the vagina. A Nugent score of 0-3 is classified as normal (lactobacilli are present, but not Gardnerella/Bacteroides or curved Gram-negative bacilli), a score of 4-6 as intermediate (colonisation by Gardnerella/Bacteroides and curved Gram-variable rods [Mobiluncus]), and a score of 7-10 is indicative f BV (with domination of Gardnerella/Bacteroides or curved Gram-negative bacilli and absence of lactobacilli).
The Panel considers that defence against vaginal pathogens is a beneficial physiological effect.

3.4. Ochrona przed patogenami pochwy (ID 2950, 2957, 2967)

Most of the references provided in relation to these claims were on methodologies for bacterial strain identification (Jensen et al., 1993; Ventura et al., 2000); an abstract from a congress on the isolation of specific bacterial strains (Pietronave et al., 2003); patents on methods for selection of Lactobacillus strains (e.g. L. plantarum LMG P-17630) with high ability to adhere to human mucosa (Dondi, 2000), in vitro studies which reported on properties of the strains (e.g. adherence to vaginal epithelial cells (Bonetti et al., 2003; Culici et al., 2004; Escorsell, 2007), or on viability at different pH (Morelli, 2000)). The Panel notes that these references did not address outcome measures related to the claimed effect.
Patents on methods for the selection of Lactobacillus strains (e.g. L. crispatus P-17631, L. gasseri P-17632 and L. gasseri P-18137) with antimicrobial activity, in which the in vitro inhibitory activity of the strain that is the subject of the claim against Candida albicans and Streptococcus β- haemolyticus was mentioned (Dondi and Morelli, 1999, 2002) were provided. Patents for the use of two bacterial strains (i.e. L. gasseri P-17632 and L. salivarius CNCM I-1794) against Candida albicans (Dondi, 2004, 2007), were also provided. The Panel notes that no primary data which could be used for the scientific substantiation of the claim were provided in these patents. The Panel considers that no conclusions can be drawn from these references for the scientific substantiation of the claim
A number of human intervention studies investigated the effects of the specific strain which is the subject of the claim when the strain was administrated intravaginally (Carriero et al., 2007; Escorsell, 2007; Gianella, 2003; Nava et al., 2002). The Panel considers that studies made on substances which are not administered orally cannot be used to substantiate a claim on a food constituent.
The Panel notes that no human studies were provided from which conclusions could be drawn for the scientific substantiation of the claims evaluated in this section.
In relation to ID 2967, in vitro studies which addressed the effects of the strain that is the subject of the claim on the adhesion properties of Candida albicans to vaginal epithelia cells (Culici et al., 2004; Escorsell, 2007) were provided. The Panel considers that in the absence of evidence for an effect on defence against vaginal pathogens in humans, evidence provided in in vitro studies cannot be used alone for the scientific substantiation of a claim on defence against vaginal pathogens.
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 claims evaluated in this section and defence against vaginal pathogens.

Oświadczenia zdrowotne