ID 934 - Lactobacillus fermentum 57A, Lactobacillus plantarum 57B, Lactobacillus gasseri 57C

PL: Lactobacillus fermentum 57A, Lactobacillus plantarum 57B, Lactobacillus gasseri 57C
EN: A combination of three probiotic strains:-Lactobacillus gasseri 57C -Lactobacillus fermentum 57A -Lactobacillus plantarum 57BTrade name of the food supplement: prOVag
Pdf: a combination of Lactobacillus fermentum 57A, Lactobacillus plantarum 57B

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.

1.35. Characterisation of a combination of “Lactobacillus gasseri 57C, Lactobacillus fermentum 57A, Lactobacillus plantarum 57B” (ID 934)

The food constituent that is the subject of the health claim is a combination of “Lactobacillus gasseri 57C, Lactobacillus fermentum 57A, Lactobacillus plantarum 57B”. Lactobacillus gasseri 57C - The only reference to strain Lactobacillus gasseri 57C species identification/characterisation found in the studies provided refers to the use of phenotypic tests and species identification by 16S-23S rDNA sequence analysis (IBSS Biomed Report). No reference regarding the strain characterisation was found in the studies provided or in the literature. The Panel considers that Lactobacillus gasseri 57C, which is the subject of the health claim ID 934, is not sufficiently characterised. No indication of the deposit of the strain in an internationally recognised culture collection was found in the information provided or the literature. Lactobacillus fermentum 57A - The only reference to strain Lactobacillus fermentum 57A species identification/characterisation found in the studies provided refers to the use of phenotypic tests and species identification by 16S-23S rDNA sequence analysis (IBSS Biomed Report). No reference regarding the strain characterisation was found in the studies provided or in the literature. The Panel considers that Lactobacillus fermentum 57A, which is the subject of the health claim ID 934, is not sufficiently characterised. No indication of the deposit of the strain in an internationally recognised culture collection was found in the information provided or the literature. Lactobacillus plantarum 57B - The only reference to strain Lactobacillus plantarum 57B species identification/characterisation found in the studies provided refers to the use of phenotypic tests and species identification by 16S-23S rDNA sequence analysis (IBSS Biomed Report). No reference regarding the strain characterisation was found in the studies provided or in the literature. The Panel considers that Lactobacillus plantarum 57B, which is the subject of the health claim ID 934, is not sufficiently characterised. No indication of the deposit of the strain in an internationally recognised culture collection was found in the information provided or the literature. The Panel considers that the combination of “Lactobacillus gasseri 57C, Lactobacillus fermentum 57A, Lactobacillus plantarum 57B”, which is the subject of the health claim ID 934, is not sufficiently characterised.

2. Znaczenie oświadczenia dla zdrowia człowieka

The claimed effect, which is proposed for further assessment, is “a beneficial effect on vaginal bacterial flora increasing total number of Lactobacillus rods, colonisation with tested strains and decreasing pH value and Nugent score”. The proposed target population is “women with no symptoms of urogenital tract infections but with disturbed or abnormal vaginal flora”.
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, and that the target population is the female population.
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, vaginal microbiota are dominated by lactobacilli (>50 %) (Ling et al., 2010; Ravel et al., 2010; 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 on, for example, 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 the 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 of 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. Naukowe uzasadnienia wpływu na zdrowie człowieka - Ochrona przed patogenami pochwowymi przez zwiększenie ilości bakterii kwasu mlekowego i/lub zmniejszenie ilości potencjalnie patogennych bakterii lub drożdży

The references provided for the scientific substantiation of the claim included textbooks, narrative reviews, industrial certifications and consensus documents that did not provide original data for the scientific substantiation of the claim. The Panel considers that no conclusions can be drawn from these references for the scientific substantiation of the claim.
Among six human studies provided, four did not use the combination of bacterial strains which is the subject of the claim (Caillouette et al., 1997; Priestley et al., 1997; Samet et al., 2003; Vasquez et al., 2002). The Panel considers that no conclusions can be drawn from these references for the scientific substantiation of the claim.
Two references described the same human study (Strus et al., 2008; Strus et al., 2011, unpublished). In an open-label, not randomised, uncontrolled study the combination of the three bacterial strains which is the subject of the claim,was administered orally (1×109 CFU/day of lactic acid bacteria in the ratio: L. gasseri 57C 50 %, L. fermentum 57A 25 %, L. plantarum 57B 25 %) for 60 days to a group of outpatient women with intermediate Nugent score and increased vaginal pH but without clinical symptoms of urogenital infection to evaluate the colonisation of the vaginal epithelium by at least one of the given strains. The secondary endpoints included measurement of numbers of total lactobacilli in the vagina and rectum, vaginal pH, and Nugent score values. The Panel notes that this study was a
preliminary, open-label, uncontrolled study and considers that no conclusions can be drawn from this study for the scientific substantiation of the claim.
The animal and in vitro studies provided evaluated bacterial adhesion properties, bacterial binding to cell surfaces, production of hydrogen peroxide by bacteria, bacterial resistance to gastric acid and bile salts, and bactericidal activity. 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 notes that animal and in vitro studies cannot predict the occurrence of an effect of a combination of Lactobacillus fermentum 57A, Lactobacillus plantarum 57B and Lactobacillus gasseri 57C on defence against vaginal pathogens in vivo in humans.
The Panel concludes that a cause and effect relationship has not been established between the consumption of a combination of Lactobacillus fermentum 57A, Lactobacillus plantarum 57B and Lactobacillus gasseri 57C and defence against vaginal pathogens.

Warunki i możliwe ograniczenia stosowania oświadczenia

One dose must contain 1 billion (at least 108 CFU) lyophilized lactic acid bacteria strains in the following proportion: 50% Lactobacillus gasseri 57C, 25% Lactobacillus fermentum 57A, 25% Lactobacillus plantarum 57B. To also present a statement which conveys that pregnant or lactating women should consult with a physician prior to taking prOVag. The following statements should be included on the product: 1 capsule daily Do not exceed recommended daily dosage Store in refrigerator (2°C-8°C).