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Commercial Probiotic Products: A Call for Improved Quality Control. A Position Paper by the ESPGHAN Working Group for Probiotics and Prebiotics

Kolaček, Sanja*; Hojsak, Iva*; Berni Canani, Roberto; Guarino, Alfredo; Indrio, Flavia§; Orel, Rok||; Pot, Bruno; Shamir, Raanan#; Szajewska, Hania**; Vandenplas, Yvan††; van Goudoever, Johannes‡‡; Weizman, Zvi§§ESPGHAN Working Group for Probiotics and Prebiotics

Journal of Pediatric Gastroenterology and Nutrition: July 2017 - Volume 65 - Issue 1 - p 117–124
doi: 10.1097/MPG.0000000000001603
Society Papers

ABSTRACT Probiotics have been proposed for a number of indications ranging from the hypothetical long-term immunomodulatory effects to proven benefits in the management of different clinical conditions.

An increasing number of commercial products containing probiotics are available. In those products, irrespective if it is food, food supplement, medical food, or drug, the probiotic microorganisms have to be present in a sufficient number by the end of the shelf-life, to pass through the gastrointestinal tract resisting acid and bile, to colonize the gut, and to retain functional properties required to obtain the suggested beneficial effect. Finally, it should be contamination-free.

Studies organized worldwide and summarized in this article have shown that inconsistencies and deviations from the information provided on the product label are surprisingly common. Frequently strains are misidentified and misclassified, products are occasionally contaminated, sometimes with even facultative or obligatory pathogens, strains are not viable, the labeled number of colonies cannot be verified, or the functional properties are diminished to the extent that preclude the proposed health benefit. As the probiotic preparations are commonly used for a wide range of conditions, the aim of the Working Group was to summarize results of the studies looking into the quality of the probiotic products and to raise the awareness of the important issue of their quality control.

Based on the results obtained, we strongly suggest a more stringent quality control process. This process should ensure that the probiotic content as mentioned on the label meets the actual content throughout the shelf life of the product, while no contamination is present.

*Children's Hospital Zagreb, University of Zagreb School of Medicine, Zagreb, Croatia

Department of Translational Medical Sciences, Paediatric Section, and CEINGE Advanced Biotechnology, and European Laboratory for the Investigation of Food Induced Diseases (ELFID), University of Naples Federico II

Department of Translational Medical Sciences, Paediatric Section, University Federico II, Naples

§Department of Paediatric Gastroenterology Division, Ospedale Pediatrico Giovanni XXIII University of Bari, Bari, Italy

||Department of Gastroenterology, Hepatology and Nutrition, University Medical Centre Ljubljana, University Children's Hospital Ljubljana, Ljubljana, Slovenia

Department IMDO, Vrije Universiteit Brussel, Brussels, Belgium

#Institute for Gastroenterology, Nutrition and Liver Diseases, Schneider Children's Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

**The Medical University of Warsaw, Department of Paediatrics, Warsaw, Poland

††UZ Brussel, Vrije Universiteit Brussel, Brussels, Belgium

‡‡Emma Children's Hospital—AMC and VU University Medical Center, Amsterdam, The Netherlands

§§Ben-Gurion University, Faculty of Health Sciences, Beer-Sheva, Israel.

Address correspondence and reprint requests to Sanja Kolaček, MD, PhD, Referral Center for Pediatric Gatroenterology and Nutrition, Children's Hospital Zagreb, Klaićeva 16, 10000 Zagreb, Croatia (e-mail: sanja.kolacek@gmail.com).

Received 23 March, 2017

Accepted 29 March, 2017

Conflicts of Interest/Funding: S.K. has participated as a clinical investigator, and/or speaker for Abbott, Arla, Biogaia, Chr. Hansen, Danone, Dukat, Nestle, Nutricia, and MSD. I.H. has participated as a clinical investigator for Biogaia and Chr Hansen and speaker for Biogaia and Medisadria. R.B.C. participated as a clinical investigator, and/or speaker for Dicofarm, Heinz,Mead Johnson Nutrition, Menarini, Nutricia, and Wyeth. A.G. has participated in basic research supported by Biocodex, Mead Johnson, and Dicofarm and in clinical trials and/or advisory boards and/or conference grant by Menarini and Biocodex. F.I., has participated as a clinical investigator and /or consultant and/or speaker for Arla Food, Biogaia, Noos, Nestle, and Nestle Nutrition Institute, Wyeth. R.O. has participated as a clinical investigator or speaker for Medis, Nutricia, Ewopharma, Biogaia, United Pharmaceuticals, Danone, Abbvie, and MSD. R.S. has participated as a clinical investigator, and/or advisory board member, and/or consultant, and/or speaker for Abbott, Danone Institute International, Enzymotec, Nestle Nutrition Institute, and Nutricia. H.S. has participated as a clinical investigator, and/or advisory board member, and/or consultant, and/or speaker for Arla, Biogaia, Biocodex, Danone, Dicofarm, Hipp, Nestle, Nestle Nutrition Institute, Nutricia, Mead Johnson, Merck, and Sequoia. Y.V. has participated as a clinical investigator, and/or advisory board member, and/or consultant, and/or speaker for Abbott Nutrition, Aspen, Biogaia, Biocodex, Danone, Hero, Kabrita, Nestle Nutrition Institute, Nutricia, Mead Johnson Nutrition, Merck, Olygose, Orafti, Phacobel, Rontis, Sari Husada, United Pharmaceuticals, Wyeth and Yakult. J.V.B. has participated as a clinical investigator, and/or advisory board member, and/or consultant, and/or speaker for Danone, Nestle Nutrition Institute, Nutricia, Mead Johnson Nutrition, United Pharmaceuticals, Wyeth, Nutrinia and Enzymotec. He serves on the National Institute of Health, Breast Feeding Council and is founder and director of the national donor human milk bank. Z.W. has participated as a clinical investigator, and/or consultant and/or speaker for BioCodex, BioGaia, Hipp, Materna, Mead Johnson, Nestle, and Sensus.

Disclaimer: ESPGHAN is not responsible for the practices of physicians and provides guidelines and position papers as indicators of best practice only. Diagnosis and treatment is at the discretion of physicians.

The remaining authors report no conflicts of interest.

What Is Known

  • The effects of probiotics seem to be strain-specific and dose-dependent.
  • Manufacturing of probiotic products can affect microbial survival, growth, and viability.
  • Probiotic products are mostly categorized as food or dietary supplements, which, unlike drugs, have to comply with significantly less stringent regulatory criteria.

What Is New

  • Our review provides evidence on the inadequate quality of commercial probiotic products, with regard to microorganism specification, their numbers, functional properties, and the presence of contaminating microorganisms.
  • More stringent quality control procedures are suggested, which should be mandatory for products prescribed for specific clinical situations, and for use in vulnerable populations such as infants and children.

Interest for beneficial microorganisms, whose activities within fermented dairy products were recognized for centuries, resurrected in the present days and resulted in an exponential growth of probiotic preparations on the global market. Owing to increasing interest of the consumers, products containing mono- or mixed cultures of live microorganisms became an important commercial good, arriving on the markets in different forms either within the food, or in pills, sprays, liquids, suspensions, capsules, powder sachets, granulates, chewable bars, and so on. Microorganisms claimed as probiotics are being used in everyday diet for the purpose of “improving health or “to keep a healthy gut” in otherwise fit and healthy population. In addition, probiotics are used to cure or prevent diseases in chronically ill or highly vulnerable populations like preterm infants (1–5). Consequently, a respectable number of studies was undertaken worldwide to provide valid answers. Unfortunately, most studies showing a benefit are not repeated and new studies examine new products. Therefore, the question on whether the quality of the preparations followed “hand in hand” the popularity of the marketed products is posed commonly in the scientific community and by health authorities.

Members of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) Working Group for Pro- and Prebiotics, who have released a number of guidelines or position paper for the clinical use of probiotics in children (6–8), are aware that they are not qualified or authorized to prepare algorithms for the manufacturing practice, or to establish and implement regulatory control mechanisms over commercially available probiotic products. The problem, however, of quality, safety, and validity of the commercial probiotic products, which are used in children, including preterm infants, prompted the ESPGHAN Working Group for Pro- and Prebiotics to perform a literature search and based on the available evidence to raise the awareness of this important issue and to provide recommendations for further actions.

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METHODS

The PubMed and Cochrane Library databases were search up to June 2016. The following key terms were used: (“quality” OR “control” OR “quality control”) AND (“probiotics” OR “probiotic”) AND (“product” OR “products” OR “commercial”). The searches were limited to human studies and to studies published in English language. Only published data were considered. The reference lists of identified studies and key review articles, including previously published reviews, were also searched. A flow diagram documenting the identification process for each research question is presented in Figure 1.

FIGURE 1

FIGURE 1

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ISSUES AFFECTING QUALITY OF THE COMMERCIAL PROBIOTIC PRODUCTS

To fulfill the definition issued by the International Scientific Association for Probiotics and Prebiotics of being “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host(9), probiotics have to be present in a sufficient number within the product by the end of shelf-life, to pass through the gastrointestinal tract resisting acid and alkaline milieu, and to colonize the gut in a sufficient number required for exerting a measurable beneficial effect. Therefore, the quality of the final product depends strongly on the manufacturing processes whereby the procedures such as fermentation, matrix composition, cell harvesting, spray-drying, freeze-drying, and storage conditions like temperature, humidity, and pH are just several of a wider array of manufacturing determinants that can affect microbial survival, growth, viability, and ultimately the study results and/or clinical outcomes (10–15).

Although there are important documents conveying an opinion on a core health benefit of probiotics as a general class (9), also aligning with regulatory approaches in some countries such as Italy (16) and Canada (17), a great majority of recognized effects are strain-dependent. Numerous are the examples of indications such as prevention of nosocomial infections or antibiotic-associated diarrhea, whereby one of the well-known probiotic strains has a scientifically proven efficacy, whereas the others failed in achieving a positive result in the same setting and identical study design (6,8,18,19). Moreover, it has been described that specific properties influencing important determinants of probiotic activity such as mucosal adherence and gut colonization were restricted to the subspecies level, for example for Bifidobacterium longum subsp. infantis in comparison to Bifidobacterium longum subsp. longum (20). Therefore, not only the presence of a sufficient amount of live bacteria at the end of shelf life, but also the confirmed identity of the microorganism at the strain level are prerequisite requirements to ensure that a commercial product will deliver the claimed beneficial health effect.

Another emerging issue related to the product quality is the problem of substrate contamination. Contaminating microorganisms can invalidate and skew the study results making them unrepeatable in future investigations with the same probiotic strain. Much worse could be the clinical outcomes if the contaminants are facultative or obligate pathogens. A recently published case report on fatal gastrointestinal mucormycosis in a premature infant associated with a contaminated commercial dietary supplement is just one of the examples (21).

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ISSUES RELATED TO TAXONOMY, NOMENCLATURE, AND CLASSIFICATION OF STRAINS

As defined in another document, the effectiveness of a probiotic product is the sum of its microbial quality and its functional properties (22). Precise identification and documentation of both are required not only for delineation of a new “potentially useful” microorganism, but also for accurate identification and labeling of the already established strains in the marketed products. When creating and/or manufacturing a probiotic product, precise methodology is mandatory for each of the required steps, from typing (defined as characterization at the individual strain level), over testing of functional capacities such as resistance to acid and bile, mucosal adherence, and adhesion stability, and finally to document viability throughout the storage period. The simplest way to detect and quantify viable microbes is to look for growth of colonies on various nutrient agars, which is routinely used in all microbial laboratories. Different problems, however, could arise during cultivation such as failing to discriminate bacteria at the species and subspecies level, and in particular to distinguish viable cultivable from viable noncultivable microbes (22–24). The advantage of cultivation methods is that they will not pick up dead cells; sometimes however, especially for bifidobacteria, special growth media supplements need to be used for the correct detection and enumeration of strains. Lack of these additions may result in a falsely negative cultivation reaction (25).

Description of presently established methods, particularly those on a molecular level, is not within the scope of this document, particularly as there are many other articles covering the topic (22–24,26–32). It is important, however, to acknowledge that inappropriate identification methods are the major cause for incorrect species designations and mislabeling of probiotic products (22,33).

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REGULATORY ISSUES

Regulation over probiotic products varies in respect to legal or statutory position. Although there are probiotic preparations licensed as medicinal products (pharmaceuticals/drugs), most of them are categorized as food or dietary supplements (United States, Europe), as natural health products (Canada), or as food for specific health uses (Japan) (17,34,35). In contrast to drugs that are rigorously regulated in respect to premarketing and post market safety control, including obligation for continuous monitoring, dietary supplements have to comply with significantly less stringent regulatory criteria in most parts of the world. And yet probiotics are the only group of preparations that contain live micro-organisms requiring specific manufacturing conditions to allow viable and active delivery into the correct part of the gastrointestinal tract, while retaining all the beneficial properties throughout the shelf life. Moreover, unlike other food products and drugs, there are specific safety concerns such as systemic infection, metabolic production of harmful substances, gene transfer including those responsible for antibiotic resistance, and immunomodulation, all of which are extensively covered elsewhere (22).

In the year 2006, Food and Agriculture Organization/World Health Organization has issued recommendations on the information that should be present on the probiotic product label: genus, species, and strain designation; minimum viable number of each probiotic strain at the end of the shelf life; the suggested serving size that must deliver the effective dose of probiotics related to the health claim; health claim; proper storage conditions; corporate contact details for consumer information (36). Despite the clear recommendations, however, a wide “gray zone” is handled by the authorities responsible for controlling the product quality, including the periodical screening of the market, and the validation of the information on the labels.

In Europe, probiotic-containing foods and food supplements are subjected to European Union (EU) regulation covered by the Food Products Directive and Regulation (37). In 2006, a novel regulation regarding all nutritional and health claims, related to all types of food, was published by the European Parliament (38). The European Food Safety Authority (EFSA) is the responsible agency in the EU for foods, food supplements, and therefore for the majority of probiotic products evaluation. More precisely, EFSA as the EU risk assessor is responsible for providing scientific advice regarding food and feed safety to support a decision-making process or setting legislation by the EU risk managers (ie, the European Commission, the Member States, and the European Parliament). It has developed a list of safe microbial cultures defined as QPS-list (Qualified Presumption of Safety) (http://www.efsa.europa.eu/en/topics/topic/qps) designated for premarket safety assessment of the biological agents. Furthermore, EFSA is responsible for the assessment of health claims made on foods (including food supplements and probiotics) that are submitted by food manufacturers and member states. A huge number of health claims were assessed (>3000), and among them were many claims on different probiotic strains, either in more general terms such as “boosts immune system/promotes gut health,” or more specific regarding preventive or therapeutic efficacy in defined clinical conditions. As of October 2016, all of the claims related to probiotics were rejected (39) except for a generic claim on better lactose digestion promoted by yogurt cultures of Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus. Despite such rigorous and scientifically based evaluation in relation to health claims, there is insufficient control during the manufacturing process and virtually no follow-up once the probiotic product is on the market.

In the United States, probiotic products mostly fall within the Food and Drug Administration (FDA) category of dietary supplements with the granted (GRAS) status (generally recognized as safe), and as such are not subjected to close monitoring. This issue is extensively discussed elsewhere (34,40). Since 2007, a standardized manufacturing process is required for dietary supplements that comply with Good Manufacturing Practice guidelines issued by the FDA. These rules, however, do not address control or verification of products’ quality and efficacy (41). In the United States, claims that address normal functioning known as (nonspecific) “structure/functioning claims” do not require governmental approval, and therefore are frequently used with probiotic products.

In summary, regulatory status of probiotic products is not established on an international basis, there is no label control, and there are no periodic screenings of the products’ quality and safety.

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SUMMARY OF THE RESULTS ON QUALITY ASSESSMENT OF THE COMMERCIAL PROBIOTIC PRODUCTS

Quality assessment studies are carried out worldwide with the aim to evaluate the quality of the commercial probiotic products with most coming from Europe, United States, Asia, South Africa, and Australia. Results of these studies are presented in Table 1 . The major findings are summarized as follows:

TABLE 1

TABLE 1

TABLE 1

TABLE 1

TABLE 1

TABLE 1

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Misidentification at the Genus/Species/Strain Level and Therefore Mislabeling With Regard to Incorporated Probiotic Strains (20,29,32,42–50)

Products were found to contain nonclaimed species/strains, mostly because inappropriate identification methods used. This was a common finding, in particular among the products with multiple strains, whereby some of the strains were correctly labeled, whereas the others were incorrectly designated. In one of the latest studies, aimed to determine how well label claims describe the species of detectable bifidobacteria in the product, only 1 in 16 commercial probiotic products perfectly matched its bifidobacterial label claims in all samples tested (20). There are many examples documenting that instead of claimed microorganisms with well known Generally Recognized As Safe/Qualified Presumption of Safety (GRAS/QPS) status, products were composed of potentially pathogenic genera such as Micromonas, Staphylococcus, Enterococcus, Bacillus, and so on (46,48,51).

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Incongruent Numbers of Viable Cells Per Dose (29,32,43,44,46–48,52)

Many tested products contained significantly lower number of viable bacteria as compared to the numbers on the labels. A respectable number of products (up to 23%–33%) contained too few viable cells precluding the possibility of any claimed health effect (43). The viability decreased significantly over time, although still being within the declared shelf life. The quality varied between different lots, but also among pills originating from the same lot (20).

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Contamination (43,45–48,53)

This is a common and particularly worrisome finding with potentially severe consequences (21).

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Decreased Functional Properties (11,54,55)

Decreased acid or bile tolerance, impaired abilities to colonize and to adhere to intestinal cells, and inability to inhibit or exclude a pathogen were all found within the same species and it was influenced with the manufacturing processes and the food matrix used (11).

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Conclusion

In summary, few studies yielded satisfactory results; the majority reported on >1 labeling inconsistency in most of the tested products. This finding applies for single and multistrain products, irrespective of the country of origin. Moreover, probiotic preparations licensed as medicinal products were also affected, although not to the same extent (49,51).

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CONCLUDING REMARKS AND RECOMMENDATIONS

Subjects across all pediatric age groups, from birth up to transition to adult health care, are using probiotic products with increasing frequency, and are also commonly involved in the clinical studies. Furthermore, the pediatric age is particularly vulnerable with respect to safety issues, with the special emphasis on the long term outcomes. Therefore, the Working Group members address the problem and agree to provide initiatives as follows.

  1. Probiotics may profoundly differ in their effects on health. Hence, precise identification of microorganisms to the strain level is required to reproduce documented effect on health.
  2. Irrespective of the field addressed (research, manufacturing, quality control, and surveillance of the final product), it would be useful that probiotic products intended to improve otherwise normal diet in the healthy population are differentiated from drug-like probiotic preparations prescribed for specific clinical situations/indications. The later need to be subjected to rigorous clinical trials required for the respective application envisaged.
  3. Probiotic products should be submitted to systematic quality control procedures by the respective authorities to confirm the viability and strain-level identification of the active ingredient (strain or strains). Results of these evaluations should be made public.
  4. In view of the rapidly developing technology, the quality control should be performed in certified laboratories using validated and standardized methodology. Standardization and validation control should be carried out by the reference laboratories under the auspices of the respective regulatory agencies.
  5. Recommendations 3 and 4 should ideally apply to all probiotic-containing products, but are mandatory for products intended for use in vulnerable populations such as neonates (preterm and term), infants, and children, or in defined clinical conditions or if marketed as pharmaceutical products.
  6. Adverse events, potentially related to probiotic products, should be reported and a register of those events should be maintained by health authorities.
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REFERENCES

1. Embleton ND, Zalewski S, Berrington JE. Probiotics for prevention of necrotizing enterocolitis and sepsis in preterm infants. Curr Opin Infect Dis 2016; 29:256–261.
2. Fiocchi A, Pecora V, Dahdah L. Probiotics, prebiotics& food allergy prevention: clinical data in children. J Pediatr Gastroenterol Nutr 2016; 63 (suppl 1):S14–S17.
3. Rodenas CL, Lepage M, Ngom-Bru C, et al. Effect of formula containing Lactobacillus Reuteri DSM 17938 on fecal microbiota of infants born by cesarean-section. J Pediatr Gastroenterol Nutr 2016; 63:681–687.
4. Lien TH, Bu LN, Wu JF, et al. Use of Lactobacillus casei rhamnosus to prevent cholangitis in biliary atresia after kasai operation. J Pediatr Gastroenterol Nutr 2015; 60:654–658.
5. Sanders ME, Merenstein DJ, Ouwehand AC, et al. Probiotic use in at-risk populations. J Am Pharm Assoc (2003) 2016; 56:680–686.
6. Szajewska H, Canani RB, Guarino A, et al. Probiotics for the prevention of antibiotic-associated diarrhea in children. J Pediatr Gastroenterol Nutr 2016; 62:495–506.
7. Szajewska H, Guarino A, Hojsak I, et al. Authors’ response. J Pediatr Gastroenterol Nutr 2016; 62:e12–e13.
8. Szajewska H, Guarino A, Hojsak I, et al. Use of probiotics for management of acute gastroenteritis: a position paper by the ESPGHAN Working Group for Probiotics and Prebiotics. J Pediatr Gastroenterol Nutr 2014; 58:531–539.
9. Hill C, Guarner F, Reid G, et al. Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 2014; 11:506–514.
10. Auclair J, Frappier M, Millette M. Lactobacillus acidophilus CL1285, Lactobacillus casei LBC80R, and Lactobacillus rhamnosus CLR2 (Bio-K+): characterization, manufacture, mechanisms of action, and quality control of a specific probiotic combination for primary prevention of Clostridium difficile infection. Clin Infect Dis 2015; 60 (suppl 2):S135–S143.
11. Grzeskowiak L, Isolauri E, Salminen S, et al. Manufacturing process influences properties of probiotic bacteria. Br J Nutr 2011; 105:887–894.
12. Nivoliez A, Camares O, Paquet-Gachinat M, et al. Influence of manufacturing processes on in vitro properties of the probiotic strain Lactobacillus rhamnosus Lcr35(R). J Biotechnol 2012; 160:236–241.
13. Broeckx G, Vandenheuvel D, Claes IJ, et al. Drying techniques of probiotic bacteria as an important step towards the development of novel pharmabiotics. Int J Pharm 2016; 505:303–318.
14. Alvarez-Calatayud G, Margolles A. Dual-coated lactic acid bacteria: an emerging innovative technology in the field of probiotics. Future Microbiol 2016; 11:467–475.
15. Whorwell PJ, Altringer L, Morel J, et al. Efficacy of an encapsulated probiotic Bifidobacterium infantis 35624 in women with irritable bowel syndrome. Am J Gastroenterol 2006; 101:1581–1590.
16. Ministero della Salute, Commissione unica per la nutrizione e la dietetica. Guidelienes on probiotics and prebiotics. Ministero della salute (online), http://www.salute.gov.it/alimentiParticolariIntegratori/newsAlimentiParticolariIntegratori.jsp?id=1729&menu=inevidenza&lingua=italiano.
17. Health Canada. Accepted claims about nature of probiotic microorganisms in food. Health Canada, http://www.inspection.gc.ca/food/labelling/food-labelling-for-industry/health-claims/eng/1392834838383/1392834887794?chap=9.
18. Hojsak I, Abdovic S, Szajewska H, et al. Lactobacillus GG in the prevention of nosocomial gastrointestinal and respiratory tract infections. Pediatrics 2010; 125:e1171–e1177.
19. Hojsak I, Tokic Pivac V, Mocic Pavic A, et al. Bifidobacterium animalis subsp. lactis fails to prevent common infections in hospitalized children: a randomized, double-blind, placebo-controlled study. Am J Clin Nutr 2015; 101:680–684.
20. Lewis ZT, Shani G, Masarweh CF, et al. Validating bifidobacterial species and subspecies identity in commercial probiotic products. Pediatr Res 2016; 79:445–452.
21. Vallabhaneni S, Walker TA, Lockhart SR, et al. Notes from the field: fatal gastrointestinal mucormycosis in a premature infant associated with a contaminated dietary supplement–Connecticut, 2014. MMWR Morb Mortal Wkly Rep 2015; 64:155–156.
22. Huys G, Botteldoorn N, Delvigne F, et al. Microbial characterization of probiotics--advisory report of the Working Group “8651 Probiotics” of the Belgian Superior Health Council (SHC). Mol Nutr Food Res 2013; 57:1479–1504.
23. Sohier D, Pavan S, Riou A, et al. Evolution of microbiological analytical methods for dairy industry needs. Front Microbiol 2014; 5:16.
24. Ercolini D. PCR-DGGE fingerprinting: novel strategies for detection of microbes in food. J Microbiol Methods 2004; 56:297–314.
25. Sule J, Korosi T, Hucker A, et al. Evaluation of culture media for selective enumeration of bifidobacteria and lactic acid bacteria. Braz J Microbiol 2014; 45:1023–1030.
26. Tuomola E, Crittenden R, Playne M, et al. Quality assurance criteria for probiotic bacteria. Am J Clin Nutr 2001; 73:393S–403S.
27. Davis C. Enumeration of probiotic strains: review of culture-dependent and alternative techniques to quantify viable bacteria. J Microbiol Methods 2014; 103:9–17.
28. Markiewicz L, Biedrzycka E. Identification of Lactobacillus and Bifidobacterium species with PCR applied to quality control of fermented dairy beverages. Pol J Food Nutr Sci 2005; 14/55:359–365.
29. Masco L, Huys G, De Brandt E, et al. Culture-dependent and culture-independent qualitative analysis of probiotic products claimed to contain bifidobacteria. Int J Food Microbiol 2005; 102:221–230.
30. Huys G, Vancanneyt M, D’haene K, et al. Accuracy of species identity of commercial bacterial cultures intended for probiotic or nutritional use. Res Microbiol 2006; 157:803–810.
31. Duc Le H, Hong HA, Barbosa TM, et al. Characterization of Bacillus probiotics available for human use. Appl Environ Microbiol 2004; 70:2161–2171.
32. Temmerman R, Scheirlinck I, Huys G, et al. Culture-independent analysis of probiotic products by denaturing gradient gel electrophoresis. Appl Environ Microbiol 2003; 69:220–226.
33. Sanders ME. Probiotics: definition, sources, selection, and uses. Clin Infect Dis 2008; 46 (suppl 2):S58–S61.
34. Federal Regulation of Probiotics: An Analysis of the. Existing Regulatory Framework and Recommendations for. Alternative Frameworks. NIH Grant Number:5R01HG005171-02; 2016. http://www.law.umaryland.edu/ProbioticsWhitePaper.
35. Health Food Regulatory System in Japan. Available at: https://food.chemlinked.com/chempedia/health-food-regulatory-system-japan.
36. FAO/WHO. Food and Agriculture Organization - World Health Organization. Report of a Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food. 202.
37. Food Products Directive and Regulation. Regulation 178/2002/EC; directive 2000/13/EU.
38. EU - European Union. Regulation (EC) No. 1924/2006 of the European Parliament ad of the Council of 20 December 2006 on nutritional and health claims made on foods. OJ L 404, 30.12.2006, 9–25.
39. FAQ on Nutrition and Health Claims, EFSA, Http://www.efsa.europa.eu/en/fasq/faqnutrition.htm.
40. Donovan SM, Schneeman B, Gibson GR, et al. Establishing and evaluating health claims for probiotics. Adv Nutr 2012; 3:723–725.
41. US Food and Drug Administration. Current Good Manufacturing Practice in Manufacturing, Packaging, Labelling, or Holding Operation for Dietary Supplements, 2007.
42. Canganella F, Paganini S, Ovidi M, et al. A microbiology investigation on probiotic pharmaceutical products used for human health. Microbiol Res 1997; 152:171–179.
43. Drago L, Rodighiero V, Celeste T, et al. Microbiological evaluation of commercial probiotic products available in the USA in 2009. J Chemother 2010; 22:373–377.
44. Fasoli S, Marzotto M, Rizzotti L, et al. Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis. Int J Food Microbiol 2003; 82:59–70.
45. Theunissen J, Britz TJ, Torriani S, et al. Identification of probiotic microorganisms in South African products using PCR-based DGGE analysis. Int J Food Microbiol 2005; 98:11–21.
46. Aureli P, Fiore A, Scalfaro C, et al. National survey outcomes on commercial probiotic food supplements in Italy. Int J Food Microbiol 2010; 137:265–273.
47. Hamilton-Miller JM, Shah S, Smith CT. “Probiotic” remedies are not what they seem. BMJ 1996; 312:55–56.
48. Marcobal A, Underwood MA, Mills DA. Rapid determination of the bacterial composition of commercial probiotic products by terminal restriction fragment length polymorphism analysis. J Pediatr Gastroenterol Nutr 2008; 46:608–611.
49. Szajewska H, Fordymacka A, Bardowski J, et al. Microbiological and genetic analysis of probiotic products licensed for medicinal purposes. Med Sci Monit 2004; 10:BR346–BR350.
50. Morovic W, Hibberd AA, Zabel B, et al. Genotyping by PCR and high-throughput sequencing of commercial probiotic products reveals composition biases. Front Microbiol 2016; 7:1747.
51. Hamilton-Miller JM, Shah S, Winkler JT. Public health issues arising from microbiological and labelling quality of foods and supplements containing probiotic microorganisms. Public Health Nutr 1999; 2:223–229.
52. Allgeyer LC, Miller MJ, Lee SY. Sensory and microbiological quality of yogurt drinks with prebiotics and probiotics. J Dairy Sci 2010; 93:4471–4479.
53. Temmerman R, Pot B, Huys G, et al. Identification and antibiotic susceptibility of bacterial isolates from probiotic products. Int J Food Microbiol 2003; 81:1–10.
54. De Vecchi E, Nicola L, Zanini S, et al. In vitro screening of probiotic characteristics of some italian products. J Chemother 2008; 20:341–347.
55. Vanhee LM, Goeme F, Nelis HJ, et al. Quality control of fifteen probiotic products containing Saccharomyces boulardii. J Appl Microbiol 2010; 109:1745–1752.
56. Goldstein EJ, Citron DM, Claros MC, et al. Bacterial counts from five over-the-counter probiotics: are you getting what you paid for? Anaerobe 2014; 25:1–4.
Keywords:

children; commercial products; food supplement; probiotics; quality control; regulation

© 2017 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,