Scientific Opinion on the substantiation of health claims related to  
non-characterised micro-organisms (ID 2936, 2937, 2938, 2941, 2944, 2965,  
2968, 2969, 3035, 3047, 3056, 3059, further assessment) pursuant to Article  
13(1) of Regulation (EC) No 1924/2006[sup]1[/sup]  
EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA)2, 3  
European Food Safety Authority (EFSA), Parma, Italy  
ABSTRACT  
Following a request from the European Commission, the Panel on Dietetic Products, Nutrition and Allergies  
(NDA) was asked to provide a scientific opinion on health claims pursuant to Article 13 of Regulation (EC) No  
1924/2006 in the framework of further assessment. This opinion addresses the scientific substantiation of health  
claims related to microorganisms for which the Panel considered in previous opinions that the data provided were  
not sufficient to characterise the microorganisms in question. The criteria used by the Panel for the  
characterisation of food constituents that are bacteria and combinations thereof, which are the subject of health  
claims, are: species identification by DNA-DNA hybridisation or 16S rRNA gene sequence analysis and/or  
sequence analysis of other relevant genetic markers; strain identification by DNA macrorestriction followed by  
pulsed-field gel electrophoresis, randomly amplified polymorphic DNA analysis, or other internationally  
accepted genetic typing molecular methods. Only when these two criteria are fulfilled is the bacterium considered  
to be sufficiently characterised. In the case of combinations of several bacteria, the Panel considers that if one  
microorganism used in the combination is not sufficiently characterised, the combination proposed is not  
sufficiently characterised. The Panel considers that the food constituents which are the subject of this opinion are  
not sufficiently characterised. On the basis of the data presented, the Panel concludes that a cause and effect  
relationship cannot be established between the consumption of the food constituents which are the subject of this  
opinion and the claimed effects. © European Food Safety Authority, 2012  
Słowa kluczowe:
Microorganism
 
health claims
 
non-characterised
 
	
	1. Charakterystyka żywności / składnika
	
        
		The criteria used by the Panel for the characterisation of food constituents that are bacteria and  combinations thereof, which are the subject of health claims, are specified in previous opinions  (EFSA Panel on Dietetic Products Nutrition and Allergies (NDA), 2009, 2010, 2011) and are:  
 Species identification by DNA-DNA hybridisation or 16S rRNA gene sequence analysis and/or  sequence analysis of other relevant genetic markers.  
 Strain identification by DNA macrorestriction followed by pulsed-field gel electrophoresis  (PFGE), randomly amplified polymorphic DNA analysis (RAPD), or other internationally  accepted genetic typing molecular methods.  
Only when these two criteria are fulfilled is the bacterium considered to be sufficiently characterised.  In the case of combinations of several bacteria, the Panel considers that if one microorganism used in  the combination is not sufficiently characterised, the combination proposed is not sufficiently  characterised.  
		
	
	
    
	
	
		
			
1.1. Bifidobacterium breve BR03 (ID 2936)  
	
	
			The food constituent that is the subject of the health claim is Bifidobacterium breve BR03 related to  the following claimed effect: “intestinal mobility”.  
For B. breve BR03, a culture collection number from the German Collection of Microorganisms and  Cell Cultures (DSMZ), DSM 16604, was provided. The DSMZ is an internationally recognised  culture collection which has the status of an International Depositary Authority under the Budapest  Treaty. In the DSMZ, cultures can be deposited in a restricted-access collection as patent deposits.  Data on the identification and characterisation of B. breve BR03 at species and strain level, by using  both phenotypic (enzymatic activity pattern, carbohydrate fermentation profile, PAGE, antibiotic  resistance profiles) and genotypic (species-specific PCR, ERIC-PCR, PFGE) methods, were provided  in the application for further assessment and in the accompanying references (Del Piano et al., 2010).  The Panel notes that the use of species-specific PCR as unique molecular technique is insufficient to  ensure the correct assignation of the species identity.   
The Panel considers that the food constituent, B. breve BR03, which is the subject of the claim, is not  sufficiently characterised.   
The Panel concludes that a cause and effect relationship cannot be established between the  consumption of B. breve BR03 and the claimed effect considered in this section.  
	
    
			
	
		
			
1.2. Bifidobacterium longum BL03 (ID 2937)  
	
	
			The food constituent that is the subject of the health claim is Bifidobacterium longum BL03 related to  the following claimed effect: “intestinal mobility”.   
For B. longum BL03, a culture collection number from the DSMZ, DSM 16603, was provided. Data  on the identification and characterisation of B. longum BL03 at species and strain level, by using both  phenotypic (enzymatic activity pattern, carbohydrate fermentation profile, PAGE, antibiotic resistance  profiles) and genotypic (species-specific PCR, PFGE) methods, were provided in the application for 
further assessment. The Panel notes that the use of species-specific PCR as unique molecular  technique is insufficient to ensure the correct assignation of the species identity.  
The Panel considers that the food constituent, B. longum BL03, which is the subject of the claim, is  not sufficiently characterised.  
The Panel concludes that a cause and effect relationship cannot be established between the  consumption of B. longum BL03 and the claimed effect considered in this section.  
	
    
			
	
		
			
1.3. A combination of Bifidobacterium breve BR03 and Lactobacillus plantarum LP01  (ID 2938)  
	
	
			The food constituent that is the subject of the health claim is a combination of Bifidobacterium breve  BR03 and Lactobacillus plantarum LP01 related to the following claimed effect: “reducing gastro- intestinal discomfort associated with increased transit time”.  
For B. breve BR03, a culture collection number from the DSMZ, DSM 16604, was provided. Data on  the identification and characterisation of B. breve BR03 at species and strain level, by using both  phenotypic (enzymatic activity pattern, carbohydrate fermentation profile, PAGE, antibiotic resistance  profiles) and genotypic (ERIC-PCR, species-specific PCR, PFGE) methods, were provided in the  application for further assessment and in the accompanying references (Del Piano et al., 2010). The  Panel notes that the use of species-specific PCR as unique molecular technique is insufficient to  ensure the correct assignation of the species identity, and considers that the strain B. breve BR03 is  not sufficiently characterised.  
For L. plantarum LP01, a culture collection number from the Belgian Co-ordinated Collections of  Microorganisms (BCCM/LMG), LMG P-21021, 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. plantarum LP01 at  species and strain level, by using both phenotypic (enzymatic activity pattern, carbohydrate  fermentation profile, PAGE, antibiotic resistance profiles) and genotypic (species-specific PCR,  PFGE) methods, were provided in the application for further assessment and in the accompanying  references (Del Piano et al., 2010). The Panel notes that the use of species-specific PCR as unique  molecular technique is insufficient to ensure the correct assignation of the species identity, and  considers that the strain L. plantarum LP01 is not sufficiently characterised.  
The Panel considers that the food constituent, a combination of B. breve BR03 and L. plantarum  LP01, which is the subject of the health claim, is not sufficiently characterised.  
The Panel concludes that a cause and effect relationship cannot be established between the  consumption of a combination of B. breve BR03 and L. plantarum LP01 and the claimed effect  considered in this section.  
	
    
			
	
		
			
1.4. A combination of Bifidobacterium animalis subsp. lactis BS01, Lactobacillus rhamnosus  LR04, Lactobacillus plantarum LP02 and short-chain fructo-oligosaccharides (ID 2941)  
	
	
			The food constituent that is the subject of the health claim is a combination of Bifidobacterium lactis  BS01, Lactobacillus rhamnosus LR04, Lactobacillus plantarum LP02 and short-chain fructo- oligosaccharides related to the following claimed effect: “defence against upper respiratory tract  infections”.  
For B. lactis BS01, hereafter B. animalis subsp. lactis BS01, since the species B. lactis has been  reclassified as B. animalis subsp. lactis (Masco et al., 2004), a culture collection number from the  BCCM/LMG, LMG P-21384, was provided. Data on the identification and characterisation of  B. lactis BS01 at species and strain level, by using both phenotypic (enzymatic activity pattern,  carbohydrate fermentation profile, PAGE, antibiotic resistance profiles) and genotypic (species- specific PCR, Rep-PCR, MLST, and genome sequencing [publicly available at genbank, Project ID  59607]) methods, were provided in the application for further assessment and in the accompanying  references (Del Piano et al., 2010). The Panel considers that the strain B. animalis subsp. lactis BS01  is sufficiently characterised.  
For L. rhamnosus LR04 a culture collection number from the DSMZ, DSM 16605, was provided.  Data on the identification and characterisation of L. rhamnosus LR04 at species and strain level, by  using both phenotypic (carbohydrate fermentation pattern, enzymatic activity pattern, PAGE,  antibiotic resistance profile) and genotypic (species-specific PCR, PFGE) methods, were provided in  the application for further assessment. The Panel notes that the use of species-specific PCR as unique  molecular technique is insufficient to ensure the correct assignation of the species identity, and  considers that the strain L. rhamnosus LR04 is not sufficiently characterised.   
For L. plantarum LP02 a culture collection number from the BCCM/LMG, LMG P-21020, was  provided. Data on the identification and characterisation of L. plantarum LP02 at species and strain  level, by using both phenotypic (carbohydrate fermentation pattern, enzymatic activity pattern, PAGE,  antibiotic resistance profile) and genotypic (species-specific PCR, PFGE) methods, were provided in  the application for further assessment and in the accompanying references (Del Piano et al., 2010).  The Panel notes that the use of species-specific PCR as unique molecular technique is insufficient to  ensure the correct assignation of the species identity, and considers that the strain L. plantarum LP02  is not sufficiently characterised.   
From the references provided, the Panel assumes that the fructo-oligosaccharides (FOS) are obtained  from sucrose. They are prepared by enzymatic elongation of sucrose, and consist of a mixture of  kestose (glucose-fructose-fructose, GF2), nystose (GF3) and fructosylnystose (GF4), with an average  degree of polymerisation (DPav) of 3.6, and are sometimes referred to as short-chain fructo- oligosaccharides. FOS from sucrose differ from natural fructans by degree of polymerisation (DP)  (only 10 % of native chicory inulin have a DP between 2 and 5) (Roberfroid, 2007), and differ from  oligofructoses prepared by inulin hydrolysis (DP from 2 to 7, DPav 4) by the presence of a glucose  moiety.   
The Panel considers that the food constituent, a combination of B. animalis subsp. lactis BS01,  L. rhamnosus LR04, L. plantarum LP02 and short-chain fructo-oligosaccharides, which is the subject  of the health claim, is not sufficiently characterised.  
The Panel concludes that a cause and effect relationship cannot be established between the  consumption of a combination of B. animalis subsp. lactis BS01, L. rhamnosus LR04, L. plantarum  LP02 and short-chain fructo-oligosaccharides and the claimed effect considered in this section.  
	
    
			
	
		
			
1.5. A combination of Lactobacillus acidophilus LA02 and Lactobacillus plantarum LP01  (ID 2944)  
	
	
			The food constituent that is the subject of the health claim is a combination of Lactobacillus  acidophilus LA02 and Lactobacillus plantarum LP01 related to the following claimed effect: “relief  of abdominal discomfort and pain”.   
For L. acidophilus LA02 a culture collection number from the DSMZ, DSM 21717, was provided.  Data on the identification and characterisation of L. acidophilus LA02 at species and strain level, by 
using both phenotypic (enzymatic activity pattern, carbohydrate fermentation profile, PAGE,  antibiotic resistance profiles) and genotypic (species-specific PCR, PFGE) methods, were provided in  the application for further assessment. The Panel notes that the use of species-specific PCR as unique  molecular technique is insufficient to ensure the correct assignation of the species identity, and  considers that the strain L. acidophilus LA02 is not sufficiently characterised.   
For L. plantarum LP01 a culture collection number from the BCCM/LMG, LMG P-21021, was  provided. Data on the identification and characterisation of L. plantarum LP01 at species and strain  level, by using both phenotypic (enzymatic activity pattern, carbohydrate fermentation profile, PAGE,  antibiotic resistance profiles) and genotypic (species-specific PCR, PFGE) methods, were provided in  the application for further assessment and in the accompanying references (Del Piano et al., 2010).  The Panel notes that the use of species-specific PCR as unique molecular technique is insufficient to  ensure the correct assignation of the species identity, and considers that the strain L. plantarum LP01  is not sufficiently characterised.   
The Panel considers that the food constituent, a combination of L. acidophilus LA02 and  L. plantarum LP01, which is the subject of the health claim, is not sufficiently characterised.  
The Panel concludes that a cause and effect relationship cannot be established between the  consumption of a combination of L. acidophilus LA02 and L. plantarum LP01 and the claimed effect  considered in this section.  
	
    
			
	
		
			
1.6. Lactobacillus plantarum LP01 (ID 2965)  
	
	
			The food constituent that is the subject of the health claim is Lactobacillus plantarum LP01 related to  the following claimed effect: “intestinal mobility”.   
A culture collection number from the BCCM/LMG, LMG P-21021, was provided. Data on the  identification and characterisation of L. plantarum LP01 at species and strain level, by using both  phenotypic (enzymatic activity pattern, carbohydrate fermentation profile, PAGE, antibiotic resistance  profiles) and genotypic (species-specific PCR, PFGE) methods, were provided in the application for  further assessment and in the accompanying references (Del Piano et al., 2010). The Panel notes that  the use of species-specific PCR as unique molecular technique is insufficient to ensure the correct  assignation of the species identity.  
The Panel considers that the food constituent, L. plantarum LP01, which is the subject of the health  claim, is not sufficiently characterised.  
The Panel concludes that a cause and effect relationship cannot be established between the  consumption of L. plantarum LP01 and the claimed effect considered in this section.  
	
    
			
	
		
			
1.7. Lactobacillus rhamnosus LR04 (ID 2968, 2969)  
	
	
			The food constituent that is the subject of the health claims is Lactobacillus rhamnosus LR04 related  to the following claimed effect: “balancing intestinal flora, improves skin, scalp and hair health” (ID  2968) and “reduce the daily number of bowel movements” (ID 2969).   
A culture collection number from the DSMZ, DSM 16605, was provided. Data on the identification  and characterisation of L. rhamnosus LR04 at species and strain level, by using both phenotypic  (carbohydrate fermentation pattern, enzymatic activity pattern, PAGE, antibiotic resistance profile)  and genotypic (species-specific PCR, PFGE) methods, were provided in the application for further  assessment and in the accompanying annexes. The Panel notes that the use of species-specific PCR as  unique molecular technique is insufficient to ensure the correct assignation of the species identity. 
The Panel considers that the food constituent, L. rhamnosus LR04, which is the subject of the health  claims, is not sufficiently characterised.  
The Panel concludes that a cause and effect relationship cannot be established between the  consumption of L. rhamnosus LR04 and the claimed effects considered in this section.  
	
    
			
	
		
			
1.8. Bifidobacterium adolescentis BA02 (ID 3035)  
	
	
			The food constituent that is the subject of the health claim is Bifidobacterium adolescentis BA02  related to the following claimed effect: “intestinal motility”.  
A culture collection number from the DSMZ, DSM 17103, was provided. Data on the identification  and characterisation of B. adolescentis BA02 at species and strain level, by using both phenotypic  (carbohydrate fermentation pattern, enzymatic activity pattern, PAGE, antibiotic resistance profile)  and genotypic (species-specific PCR, PFGE) methods, were provided in the application for further  assessment and in the accompanying annexes. The Panel notes that the use of species-specific PCR as  unique molecular technique is insufficient to ensure the correct assignation of the species identity.   
The Panel considers that the food constituent, B. adolescentis BA02, which is the subject of the health  claim, is not sufficiently characterised.  
The Panel concludes that a cause and effect relationship cannot be established between the  consumption of B. adolescentis BA02 and the claimed effect considered in this section.  
	
    
			
	
		
			
1.9. A combination of Bifidobacterium animalis subsp. lactis BS01, Lactobacillus rhamnosus  LR04, Lactobacillus rhamnosus LR05, Lactobacillus plantarum LP01 and Lactobacillus  
	
	
			plantarum LP02 and short-chain fructo-oligosaccharides or galacto-oligosaccharides  
(ID 3047)  
The food constituent that is the subject of the health claim is a combination of Bifidobacterium lactis  BS01, Lactobacillus rhamnosus LR04, Lactobacillus rhamnosus LR05, Lactobacillus plantarum  LP01, Lactobacillus plantarum LP02 and short-chain fructo-oligosaccharides or galacto- oligosaccharides related to the following claimed effect: “defence against upper respiratory tract  infections”.   
For B. lactis BS01, hereafter B. animalis subsp. lactis BS01, since the species B. lactis has been  reclassified as B. animalis subsp. lactis (Masco et al., 2004), a culture collection number from the  BCCM/LMG, LMG P-21384, was provided. Data on the identification and characterisation of  B. animalis subsp. lactis BS01 at species and strain level, by using both phenotypic (enzymatic  activity pattern, carbohydrate fermentation profile, PAGE, antibiotic resistance profiles) and  genotypic (species-specific PCR, Rep-PCR, MLST and genome sequencing [publicly available at  genbank, Project ID 59607]) methods, were provided in the application for further assessment and in  the accompanying references (Del Piano et al., 2010). The Panel considers that the strain  Bifidobacterium animalis subsp. lactis BS01 is sufficiently characterised.  
For L. rhamnosus LR04 a culture collection number from the DSMZ, DSM 16605, was provided.  Data on the identification and characterisation of L. rhamnosus LR04 at species and strain level, by  using both phenotypic (carbohydrate fermentation pattern, enzymatic activity pattern, PAGE,  antibiotic resistance profile) and genotypic (species-specific PCR, PFGE) methods, were provided in  the application for further assessment. The Panel notes that the use of species-specific PCR as unique  molecular technique is insufficient to ensure the correct assignation of the species identity, and  considers that the strain L. rhamnosus LR04 is not sufficiently characterised. 
For L. rhamnosus LR05 a culture collection number from the DSMZ, DSM 19739, was provided.  Data on the identification and characterisation of L. rhamnosus LR05 at species and strain level, by  using both phenotypic (carbohydrate fermentation pattern, enzymatic activity profile, PAGE,  antibiotic resistance profile) and genotypic (species-specific PCR, PFGE) methods, were provided in  the application for further assessment. The Panel notes that the use of species-specific PCR as unique  molecular technique is insufficient to ensure the correct assignation of the species identity, and  considers that the strain L. rhamnosus LR05 is not sufficiently characterised.   
For L. plantarum LP01 a culture collection number from the BCCM/LMG, LMG P-21021, was  provided. Data on the identification and characterisation of L. plantarum LP01 at species and strain  level, by using both phenotypic (enzymatic activity pattern, carbohydrate fermentation profile, PAGE,  antibiotic resistance profiles) and genotypic (species-specific PCR, PFGE) methods, were provided in  the application for further assessment and in the accompanying references (Del Piano et al., 2010).  The Panel notes that the use of species-specific PCR as unique molecular technique is insufficient to  ensure the correct assignation of the species identity, and considers that the strain L. plantarum LP01  is not sufficiently characterised.   
For L. plantarum LP02 a culture collection number from the BCCM/LMG, LMG P-21020, was  provided. Data on the identification and characterisation of L. plantarum LP02 at species and strain  level, by using both phenotypic (carbohydrate fermentation pattern, enzymatic activity pattern, PAGE,  antibiotic resistance profile) and genotypic (species-specific PCR, PFGE) methods, were provided in  the application for further assessment and in the accompanying references (Del Piano et al., 2010).  The Panel notes that the use of species-specific PCR as unique molecular technique is insufficient to  ensure the correct assignation of the species identity, and considers that the strain L. plantarum LP02  is not sufficiently characterised.  
From the references provided, the Panel assumes that the fructo-oligosaccharides (FOS) are obtained  from sucrose. They are prepared by enzymatic elongation of sucrose, and consist of a mixture of  kestose (glucose-fructose-fructose, GF2), nystose (GF3) and fructosylnystose (GF4), with an average  degree of polymerisation (DPav) of 3.6, and are sometimes referred to as short-chain fructo- oligosaccharides. FOS from sucrose differ from natural fructans by degree of polymerisation (DP)  (only 10 % of native chicory inulin have a DP between 2 and 5) (Roberfroid, 2007), and differ from  oligofructoses prepared by inulin hydrolysis (DP from 2 to 7, DPav 4) by the presence of a glucose  moiety.   
Galacto-oligosaccharides (GOS) are formed by enzymatic treatment of lactose with β-galactosidases  with transgalactosylation activities to produce several oligomers of different chain lengths. In the  reaction 4’- or 6’-galactosyl-lactose, longer oligosaccharides, transgalactosylated disaccharides and  non-reducing oligosaccharides are formed. The microbial source of β-galactosidase affects the  utilisation of the substrate by gut bacteria (Depeint et al., 2008). The Panel notes that there are  different GOS with different chain lengths.   
The Panel considers that the food constituent, a combination of B. animalis subsp. lactis BS01, L.  rhamnosus LR04, L. rhamnosus LR05, L. plantarum LP01, L. plantarum LP02, and short-chain  fructo-oligosaccharides or galacto-oligosaccharides, which is the subject of the health claim, is not  sufficiently characterised.  
The Panel concludes that a cause and effect relationship cannot be established between the  consumption of a combination of B. animalis subsp. lactis BS01, L. rhamnosus LR04, L. rhamnosus  LR05, L. plantarum LP01, L. plantarum LP02 and short-chain fructo-oligosaccharides or galacto- oligosaccharides and the claimed effect considered in this section. 
	
    
			
	
		
			
1.10. Bifidobacterium longum W11 (ID 3056)  
	
	
			The food constituent that is the subject of the health claim is Bifidobacterium longum W11 related to  the following claimed effect: “relief of abdominal discomfort and bloating”.   
A culture collection number from the BCCM/LMG, LMG P-21586, was provided. Data on the  identification and characterisation of B. longum W11 at species and strain level, by using both  phenotypic (enzymatic activity pattern, carbohydrate fermentation profile, PAGE, antibiotic resistance  profile) and genotypic (species-specific PCR, PFGE) methods, were provided in the application for  further assessment and in the accompanying references (Morelli, 2003; No authors listed, 2011). The  Panel notes that the use of species-specific PCR as unique molecular technique is insufficient to  ensure the correct assignation of the species identity.   
The Panel considers that the food constituent, B. longum W11, which is the subject of the health  claim, is not sufficiently characterised.  
The Panel concludes that a cause and effect relationship cannot be established between the  consumption of B. longum W11 and the claimed effect considered in this section.  
	
    
			
	
		
			
1.11. A combination of Bifidobacterium animalis subsp. lactis BS01, Lactobacillus rhamnosus  LR04, Lactobacillus plantarum LP02, lactoferrin and short-chain fructo-oligosaccharides  
	
	
			(ID 3059)  
The food constituent that is the subject of the health claim is a combination of Bifidobacterium lactis  BS01, Lactobacillus rhamnosus LR04, Lactobacillus plantarum LP02, lactoferrin and short-chain  fructo-oligosaccharides related to the following claimed effect: “defence against upper respiratory  tract infections”.   
For B. lactis BS01, hereafter B. animalis subsp. lactis BS01, since the species B. lactis has been  reclassified as B. animalis subsp. lactis (Masco et al., 2004), a culture collection number from the  BCCM/LMG, LMG P-21384, was provided. Data on the identification and characterisation of  B. lactis BS01 at species and strain level, by using both phenotypic (enzymatic activity pattern,  carbohydrate fermentation profile, PAGE, antibiotic resistance profiles) and genotypic (species- specific PCR, Rep-PCR, MLST and genome sequencing [publicly available at genbank, Project ID  59607]) methods, were provided in the application for further assessment and in the accompanying  references (Del Piano et al., 2010). The Panel considers that the strain B. animalis subsp. lactis BS01  is sufficiently characterised.  
For L. rhamnosus LR04 a culture collection number from the DSMZ, DSM 16605, was provided.  Data on the identification and characterisation of L. rhamnosus LR04 at species and strain level, by  using both phenotypic (carbohydrate fermentation pattern, enzymatic activity pattern, PAGE,  antibiotic resistance profile) and genotypic (species-specific PCR, PFGE) methods, were provided in  the application for further assessment. The Panel notes that the use of species-specific PCR as unique  molecular technique is insufficient to ensure the correct assignation of the species identity, and  considers that the strain L. rhamnosus LR04 is not sufficiently characterised.   
For L. plantarum LP02 a culture collection number from the BCCM/LMG, LMG P-21020, was  provided. Data on the identification and characterisation of L. plantarum LP02 at species and strain  level, by using both phenotypic (carbohydrate fermentation pattern, enzymatic activity pattern, PAGE,  antibiotic resistance profile) and genotypic (species-specific PCR, PFGE) methods, were provided in  the application for further assessment and in the accompanying references (Del Piano et al., 2010).  The Panel notes that the use of species-specific PCR as unique molecular technique is insufficient to  ensure the correct assignation of the species identity, and considers that the strain L. plantarum LP02  is not sufficiently characterised. 
Lactoferrin is a globular glycoprotein with a molecular mass of approximately 77 kDa which occurs  naturally in cow’s milk. The tertiary structure of this glycoprotein has two iron-binding sites, which  enables it to bind two Fe3+ ions per molecule of protein.   
From the references provided, the Panel assumes that the fructo-oligosaccharides (FOS) are obtained  from sucrose. They are prepared by enzymatic elongation of sucrose, and consist of a mixture of  kestose (glucose-fructose-fructose, GF2), nystose (GF3) and fructosylnystose (GF4), with an average  degree of polymerisation (DPav) of 3.6, and are sometimes referred to as short-chain fructo- oligosaccharides. FOS from sucrose differ from natural fructans by degree of polymerisation (DP)  (only 10 % of native chicory inulin have a DP between 2 and 5) (Roberfroid, 2007), and differ from  oligofructoses prepared by inulin hydrolysis (DP from 2 to 7, DPav 4) by the presence of a glucose  moiety.   
The Panel considers that the food constituent, a combination of B. animalis subsp. lactis BS01, L.  rhamnosus LR04, L. plantarum LP02, lactoferrin and short-chain fructo-oligosaccharides, which is  the subject of the health claim, is not sufficiently characterised.  
The Panel concludes that a cause and effect relationship cannot be established between the  consumption of B. animalis subsp. lactis BS01, L. rhamnosus LR04, L. plantarum LP02, lactoferrin  and short-chain fructo-oligosaccharides and the claimed effect considered in this section.