Ministero della Salute

GUIDELINES FOR PROBIOTICS AND PREBIOTICS

FIELD OF APPLICATION
The present guidelines can be applied to products marketed in Italy as food (1) or nutritional
supplements, which are aimed to improve consumers health through several effects, in addiction to their
usual nutritional functions. These additional effects can be achieved through the action of selected
bacteria (probiotics) and/or substances able to promote the growth of specific bacterial strains
(prebiotics).

1. See Reg. 178/2002 of January 28, 2002. For the pharmacological definition mentioned in this last
regulation, please refer to Directive 2004/27/CE, which has modified the previous Directive 2001/83/CE.

DEFINITIONS:

PROBIOTIC
Live and vital microrganisms able to benefit human health when consumed, in adequate amount (2), as
part of a food (3) or a nutritional supplement. Probiotics identification should occur according to the
recommendations listed on the Section Probiotics species and strains identification of this
document.

2. This definition has been adopted by the FAO/WHO Expert Consultation, 2001. Although the above
mentioned document considers this definition for its own purpose, we thought appropriate to adopt it for
the present guidelines.
3. Water included.

FOOD/SUPPLEMENT WITH PROBIOTIC

Food/supplement which contains, in adequately high amount, probiotic microorganisms able to reach the
intestine, multiply and produce a beneficial effect for the health/wellbeing of the host.
The definition of food/supplement with probiotics should be applied to the products which follow the
evaluation process shown in the attached file n1 of these guidelines.
The definition of food (supplement) with probiotics includes as well the interactions between the probiotic
microorganism and the food matrix or the excipients or the other ingredients. The beneficial effect should
be specifically evaluated by using the product in its ready to be consumed form (see file attached n1).

PREBIOTIC
A prebiotic is a non digestible substance of food origin able to be beneficial to the consumer thanks to the
selective promotion of the growth and/or the activity of one or more bacteria already living in the
gastrointestinal tract or taken together with the prebiotic.

FOOD/SUPPLEMENT WITH PREBIOTIC

Food/supplement with prebiotics is a food which contains, in adequate amount, prebiotic molecules able
to promote the development of bacterial strains beneficial to humans.
The definition of food (supplement, etc.) with prebiotics can be applied to the products which follow the
evaluation procedure shown in the file attached n1 of these guidelines.

FOOD/SUPPLEMENT WITH SYMBIOTIC

A food/supplement with symbiotics is made by the association of a food with probiotics with a food with
prebiotics. The definition of symbiotic food or supplement can be applied to the products which follow the
procedure shown in the file attached n1 of these guidelines.

PROBIOTIC FOOD AND MICROORGANISMS

Scientific literature is listing a growing number of probiotic microorganisms. While it is possible to list
bacterial strains now under evaluation and those used as probiotics, it is not easy to make a complete
record of all potentially probiotic species. In addition, it is important to remember that most bacterial
strains which are safe for use and able to produce a beneficial probiotic action are part of Lactobacillus
and Bifidobacterium genus.
As shown by scientific publications, strain specificity is a typical probiotic property.
Although strain specificity has not being strongly confirmed yet (AFFSA, 2005), in almost the entire
totality of published works, FAO/WHO documents included (2001, 2002), probiotic microorganisms
selection at strain level is of primary importance. Choice of strains should take place through a selective
procedure aimed to verify taxonomic identity, main phenotypic properties, safety and efficacy (see file
Genetic characterization
It can be obtained through the application of several techniques; in particular, for species identification:

nucleic acid hybridization

REA (Restriction Endonuclease Analysis
ARDRA (Amplified Ribosomal DNA Restriction Analysis)
Coding DNA sequencing for 16S rRNA
Manual or automated ribotyping

For the bacterial strain typization:

PFGE (Pulse Field Gel Electrophoresis)

RAPD (Random Amplified Polymorphic DNA)-PCR
ERIC (Enterobacterial Ripetitive Intergenic Consensus)
AFLP (Amplified Fragment Length Polymorphism)

Due to rapid evolution of molecular procedures, the above listed methods should not be considered
exaustive, but subject to modifications and integrations.
In addition, FAO/WHO documents (2001, 2002) include the following recommendations:

Application of taxonomic nomenclature recognized by the International Union of Microbiological

Societies;
Deposit of the strains on International Collections under IDA (4) status (bacterial strains
international collections), aimed to allow their availability to the scientific community.

4. IDA=International Depositary Authority, bacterial strains collections recognized at international level.

Considering the strain specificity of probiotic action, it is also recommended to keep stable products
composition at strain level. It is also suggested that at the moment of notification of food and
supplements mentioned in this document, analytical method utilized for the probiotic microorganisms
numbering should be specified. The specification should also include the genetic characterization
procedure.

PROBIOTIC ACTIVITY AND PROBIOTIC LACTIC ACID BACTERIA AMOUNT NECESSARY TO GET A
PROBIOTIC ACTION
As shown by the growing number of reviews published on Medline type databases, evolution of
knowledge regarding probiotic activities is extremely rapid.
Some of the properties of several probiotic bacterial strains have been evaluated by some meta-analysis
(Elmer et al, 1996, Van Niel et al, 2002, Huang et al, 2002, Cremonini et al, 2002, D'Souza et al, 2002,
Szajewska e Mrukowiz, 2003, Szajewska e Mrukowiz, 2005), two FAO/WHO documents (2001, 2002),
one publication of the European Society of Pediatric Gastroenterology, Epatology and Nutrition
(ESPGHAN) (Agostoni et al, 2004) and one AFSSA dossier (2005).
In all of the seven above mentioned meta-analysis, probiotics have shown a positive action in reducing
recovery times of diarrea induced by antibiotic therapy.
Several of the above listed documents suggest the relevance of the dose administered with the aim of
getting a positive effect; also, the definition of probiotic itself implies the importance of this factor.
It is not possible at the moment to give precise indications on this matter, as after ingestion probiotic
strains activity is influenced by several factors. Among them:

Probiotic species
Probiotic properties of the selected strain
Strain vitality at the moment of utilization

From a general point of view, based on recent publications, 109 live cells per day per adult is considered
to be a sufficient number to get a temporary colonization. A similar cell number should be present inside
the portion/daily dose of the food/supplement. Different daily doses could be accepted, if proved by
specific studies.
The number of live microbic cells present inside the product at the end of the shelf life should be at least
equal to the amount utilized for the efficacy trials. Indications on label should refer to the minimum dose
guaranteed at the end of shelf life, under the suggested storing conditions; this amount should not be
significantly different from the quantity used for the efficacy tests. Analytic evaluation of live bacterial
cells number can require specific methods, according to the species unter testing.
 PROBIOTIC BACTERIA SAFETY

As defined on EFSA documents preceding the introduction of QPS status for some bacterial groups inside
the European Union, characterization at every single strain level of safety of strains belonging to species
sufficiently known is not required. The only exception concerns antibiotic-resistance profile determination,
which should be tested on every single strain under trial, with the aim of excluding acquired or potentially
transmitted antibiotic-resistances. For safety evaluation, taxonomic identification at genetic level at least
as species is also necessary, besides the need to preserve the stability of strains inside the products.
Introduction of new microbial strains as part of a product, even if they are belonging to the same strain,
should require a new safety and efficacy evaluation.

PREBIOTICS PROPERTIES

Prebiotics definition is based on non digestability and selected fermentescibility by some bacterial groups,
not taking into consideration chemical nature; nevertheless, most prebiotics are carbohydrates.
At the moment, datas available seem to point out that even among bacteria belonging to the same
biochemical group, polymerization degree and type of bonding between monomers can have a profound
effect upon prebiotic action efficacy (Crittenden & Payne, 1996; Grizard & Barthomeuf, 1999; Frank et
al., 2002); in any case, there are a few exceptions.
Even inside the same biochemical family, prebiotic ingredients are often made of several compounds.
These molecules not only differ for the polymerization degree but also for the production technology
(e.g., fructans can be obtained either by enzimatic hydrolysis or by extraction); according to the method
selected, the two manifacturing processes lead up to different mixtures of final products.
Also, intestinal bacteria metabolize these compounds in a different way (Roberfroid et al., 1998, Perrin et
al., 2002, Kaplan et Hutkins, 2000, 2003). Therefore, product chemical structure (polymerization degree
included) should be clearly and accurately specified on label. In addition, results achieved with one
prebiotic ingredient cannot be extrapolated in any way to another prebiotic, even if belonging to the
same biochemical class.

PREBIOTIC ACTIVITIES AND TOTAL AMOUNT REQUIRED TO GET A PREBIOTIC ACTION

Several studies (see ESPGHAN documents (Aggett et al, 2003, Agostoni et al, 2004) AFSSA dossier
(2005), and ENDO European Union project final report (Van Loo et al, 1999) underline the importance of
daily dose in relation with prebiotic efficacy. Therefore, comparably with probiotic products
recommendations, any reference to prebiotic definition should mention the carried dose, inside a
normal daily consumption, and should be supported by specific studies.

PREBIOTICS SAFETY

While safety studies are available in adequate number for some prebiotics, this does not hold true for all
the ingredients capable of a prebiotic action. The effect of newly introduced prebiotic ingredients should
be verified through proper microbiological studies. Research should be aimed to verify the absence of a
stimulating action on pathogenic and opportunistic bacteria living inside human intestine.

BIBLIOGRAPHY

1. AFFSA. Effets des probiotiques et prbiotiques sur la flore et l'immunit de l'homme adulte. 2005
(www.afssa.fr)

2. Aggett PJ, Agostoni C, Axelsson I, Edwards CA, Goulet O, Hernell O, Koletzko B, Lafeber HN,
Micheli JL, Michaelsen KF, Rigo J, Szajewska H, Weaver LT; ESPGHAN Committee on Nutrition.
Nondigestible carbohydrates in the diets of infants and young children: a commentary by the
ESPGHAN Committee on Nutrition. J Pediatr Gastroenterol Nutr. 2003 Mar;36(3):329-37.

3. Agostoni C, Axelsson I, Braegger C, Goulet O, Koletzko B, Michaelsen KF, Rigo J, Shamir R,

Szajewska H, Turck D, Weaver LT; ESPGHAN Committee on Nutrition. Probiotic bacteria in
dietetic products for infants: a commentary by the ESPGHAN Committee on Nutrition. J Pediatr
Gastroenterol Nutr. 2004 Apr;38(4):365-74
4. Agostoni C, Axelsson I, Goulet O, Koletzko B, Michaelsen KF, Puntis JW, Rigo J, Shamir R,
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products for infants: a commentary by the ESPGHAN Committee on Nutrition. J Pediatr
Gastroenterol Nutr. 2004 Nov;39(5):465-73

5. Cremonini F, Di Caro S, Nista EC, Bartolozzi F, Capelli G, Gasbarrini G, Gasbarrini A. Meta-

analysis: the effect of probiotic administration on antibiotic-associated diarrhoea. Aliment
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6. Crittenden, R. G., and Playne, M. J. Production, properties and applications of food-grade

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including Powder Milk with Live Lactic Acid Bacteria Cordoba (Argentina) 200
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http://www.fao.org/es/ESN/food/foodandfood_probiocons_en.stm)

10. FAO/WHO EXPERT CONSULTATION: Guidelines for the Evaluation of Probiotics in Food London
Ontario (Canada) 2002.
(http://www.who.int/foodsafety/publications/fs_management/probiotics/en/ oppure:
http://www.fao.org/es/ESN/food/foodandfood_probiocons_en.stm )

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1095).Br J Nutr. 1999 Feb;81(2):121-32

15. Kaplan, H., and Hutkins, R. W. Fermentation of fructooligosaccharides by lactic acid bacteria and
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December2005