ID 938 - Lactobacillus helveticus CNCM I-1722, Bifidobacterium longum CNCM I-3470

PL: Lactobacillus helveticus CNCM I-1722, Bifidobacterium longum CNCM I-3470
EN: Lactobacillus helveticus CNCM I-1722 and Bifidobacterium longum CNCM I-3470
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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.

2.1. Łagodzenie stresu (ID 938)

The claimed effect, which is proposed for further assessment, is “significant improvement of stress- induced psychological and gastrointestinal symptoms like anxiety, anger-hostility, depressive symptoms, nausea and abdominal pain”. The proposed target population is healthy adults experiencing moderate stress or anxiety.
The Panel assumes that the claimed effect refers to alleviation of psychological stress. The Panel considers that alleviation of psychological stress is a beneficial physiological effect.

3.1. Łagodzenie stresu (ID 938)

Among the references provided in relation to the claim were human, animal and in vitro studies which were unrelated to the combination of L. helveticus CNCM I-1722 and B. longum CNCM I-3470 (Estrada et al., 2001; Firmesse et al., 2008; Haskey and Dahl, 2006; Johnson-Henry et al., 2007; Wine et al., 2009). The Panel considers that no conclusions can be drawn from these studies for the scientific substantiation of the claim.
Three human intervention studies on a combination of L. helveticus CNCM I-1722 and B. longum CNCM I-3470 (Diop et al., 2008; Messaoudi et al., 2010; 2011) were provided for the scientific substantiation of the claim.
Diop et al. (2008) in a double-blind, placebo-controlled, randomised, parallel study assessed the effect of a combination of L. helveticus CNCM I-1722 and B. longum CNCM I-3470 on symptoms claimed to be related to stress. Adult volunteers aged 18-60 years with at least two symptoms (anxiety, nervousness, irritability, sleeping problems, gastro-intestinal disturbances) perceived as induced by stress during the preceding month, but not receiving any medical treatment for stress-induced symptoms, were eligible for the study. Subjects randomly received a combination of the two bacterial strains (3 x 109 CFU per sachet, L. helveticus CNCM I-1722 and B. longum CNCM I-3470 in a ratio of 9:1, one sachet daily) or placebo for three weeks. Participants completed a questionnaire on stress- induced symptoms at the beginning and at the end of the intervention. The questionnaire consisted of 62 items related to symptoms in the following areas: gastro-intestinal, cardiovascular, sleeping, locomotor, physical, psychological, intellectual, spiritual and social. Each symptom was evaluated using a 10-cm visual analogue scale (VAS). A global score for each area was determined as the mean of each item. The changes of the score between the baseline and the end of the study were calculated and compared between the two groups using the unpaired Student’s t test. A total of 75 subjects (54 females, aged 38±11 years) were randomised (37 in the study group and 38 in the placebo group) and 64 finished the study (31 in the intervention group and 33 in the placebo group). The reasons for drop- outs were given. The Panel notes that no information was given about the validation of the questionnaire used for subjective measurements, that the process of randomisation was insufficiently described, that inclusion criteria (at least two symptoms perceived as induced by stress) were not sufficiently defined and justified, and that no correction for multiple testing was performed in the statistical analysis of the results. The Panel considers that no conclusions can be drawn from this study for the scientific substantiation of the claim.
Messaoudi et al. (2010) studied the potential anxiolytic effect of a combination of L. helveticus CNCM I-1722 and B. longum CNCM I-3470 in a double-blind, placebo-controlled, randomised, parallel study. The Hospital Anxiety and Depression Scale (HADS) was used for the enrolment of subjects. Their results had to be ≤20 in the HADS total score, ≤12 in HADS-anxiety subscale and ≤12 in HADS-depression subscale. Sixty-six subjects were randomised to take either the combination of bacterial strains which is the subject of the claim (in the form of sticks, 3 x 109 CFU daily, L. helveticus CNCM I-1722 and B. longum CNCM I-3470 content in a ratio of 90:10) or placebo, identical in taste and appearance, for 30 days. Fifty-five subjects finished the study (26 subjects in the intervention group, 29 subjects in the control group, mean age 42.4 years in the intervention group and 43.2 years in the control group). At the beginning and at the end of the intervention the participants completed a set of questionnaires: the Hopkins Symptom Checklist (HSCL-90), the HADS, the Perceived Stress Scale (PSS) and the coping checklist (CCL). Moreover, 24 h urinary free cortisol was measured. In a separate publication (Messaoudi et al., 2011), a secondary sub-group analysis of the results of the above-described study for 25 subjects (10 in the study group and 15 in the control group) with urinary free cortisol concentrations less than 50 ng/ml at baseline was presented. The Panel notes that this study was designed for measuring anxiety and not changes induced by stress. The Panel considers that no conclusions can be drawn from these studies for the scientific substantiation of the claim.
The submitted studies in animals evaluated the results of administration of the combination of bacterial strains which is the subject of the claim on the conditioned defensive burying test (Messaoudi et al., 2010), on depressive behaviour after experimental myocardial infarction (Arseneault-Breard et al., 2011), and on apoptosis in the limbic system after myocardial infarction (Girard et al., 2009). The in vitro studies measured the effect of the combination of bacterial strains which is the subject of the claim on the proliferation rate of splenocytes and on immunoglobuline production (Easo et al., 2002), their survival in simulated gastro-intestinal conditions (Possemiers et al., 2010), the susceptibility of several bacterial strains affected by chemical stress to different antibiotics (Kheadr, 2006; Kheadr et al., 2007), and the effect of various Lactobacillus and Bifidobacterium strains on cytokine production by human intestinal epithelial cells (Wallace et al., 2003). The Panel considers that in the absence of evidence for an effect on the alleviation of
psychological stress in humans, evidence provided in these animal and in vitro studies cannot be used for the scientific substantiation of a claim on alleviation of psychological stress.
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 and B. longum subsp. longum CNCM I-3470 and alleviation of psychological stress.

Warunki i możliwe ograniczenia stosowania oświadczenia

at least 3x109 cfu/ day