Microbiological and molecular characterization of commercially available probiotics containing Bacillus clausii from India and Pakistan

    Microbiological and molecular characterization of commercially available probiotics containing Bacillus clausii from India and Pakistan Vania Patrone, Paola Molinari, Lorenzo Morelli PII: DOI: Reference:

S0168-1605(16)30415-9 doi: 10.1016/j.ijfoodmicro.2016.08.012 FOOD 7337

To appear in:

International Journal of Food Microbiology

Received date: Revised date: Accepted date:

24 March 2016 18 July 2016 10 August 2016

Please cite this article as: Patrone, Vania, Molinari, Paola, Morelli, Lorenzo, Microbiological and molecular characterization of commercially available probiotics containing Bacillus clausii from India and Pakistan, International Journal of Food Microbiology (2016), doi: 10.1016/j.ijfoodmicro.2016.08.012

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ACCEPTED MANUSCRIPT Microbiological and molecular characterization of commercially available probiotics

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containing Bacillus clausii from India and Pakistan

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Vania Patrone, Paola Molinari, Lorenzo Morelli*

Istituto di Microbiologia, Università Cattolica del Sacro Cuore, via Emilia Parmense 84,

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29122 Piacenza (Italy)

*Corresponding author:

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[email protected]

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Abstract

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Probiotics are actively used for treatment of diarrhoea, respiratory infections, and prevention of infectious gastrointestinal diseases. The efficacy of probiotics is due to strain-specific

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features and the number of viable cells; however, several reports of deviations from the label in the actual content of strains in probiotic products are a matter of concern. Most of the

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available data on quality focuses on probiotic products containing lactobacilli and/or bifidobacteria, while very few data are available on spore-forming probiotics. The present

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study evaluates the label claims for spore count and species identification in five commercial

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probiotic products marketed in India and Pakistan that claim to contain Bacillus clausii: Tufpro, Ecogro, Enterogermina, Entromax, and Ospor. Bacterial enumeration from three

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batches was done by microbiological plating methods by two independent operators. Species identification was done using PCR amplification and sequence analysis of the 16S rRNA

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gene, and determination of the total amount of species present in the products was done using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by DNA sequencing of the excised bands. Plate count methods demonstrated poor correlations between quantitative label indications and bacteria recovered from plates for Tufpro, Ecogro, and Ospor. The 16S rRNA analysis performed on bacteria isolated from plate counts showed that only Enterogermina and Ospor contained homogenous B. clausii. PCR-DGGE analysis revealed that only Enterogermina had a homogenous B. clausii population while other products had mixed bacterial populations. In conclusion, the current analysis clearly demonstrates that of the five analysed commercial probiotics, only Enterogermina followed the label claims. 2

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Key words: Probiotics, Bacillus clausii, 16S rRNA, Denaturing gradient gel electrophoresis,

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Repetitive sequence-based PCR.

1. Introduction

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The term probiotic is derived from ancient Greek and Latin and means ‘for life’ (Sanders, 2008). The definition of probiotics provided by the Food and Agriculture Organization (FAO)

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of the United Nations and the World Health Organization (WHO) in 2001 has been reworded by the expert panel of the International Scientific Association for Probiotics and Prebiotics which define probiotics as ‘live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (Hill et al., 2014). Species of not only the non-sporing genera Lactobacillus spp. and Bifidobacterium spp., but also of the spore-forming Bacillus spp. are used as probiotics in foods, nutritional supplements, and pharmaceutical products (Saxelin, 2008). The safety of probiotic products containing lactobacilli and bifidobacteria consumed by the general population is supported not only by the so-called “long history of safe use”, but also by a recent literature review (van den Nieuwboer et al., 2014, 2015).

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ACCEPTED MANUSCRIPT Although probiotics have been advocated for preventing and treating a diverse range of diseases (Boyle et al., 2006), the risk of sepsis is an important area of concern (Ishibashi and

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Yamazaki, 2001). However, a recent study on the safety of probiotics and synbiotics in

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children aged under 18 years, reported no major safety concerns with the probiotic products

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(van den Nieuwboer et al., 2015). Hence, the selection of an appropriate (single or multistrain) probiotic is crucial for their therapeutic efficacy (Sanders et al., 2013). When administered orally, Bacillus probiotics induce the cellular and humoral immune

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systems, resulting in health improvement during intestinal infections (Hao et al., 2015).

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Bacillus spp. (B. cereus, B. clausii, B. pumilus) are characterized as having potential probiotic effects (Duc et al., 2004) including potential colonization, immune-stimulation, and antimicrobial activity. B. clausii is effective in alleviating the symptoms of diarrhoea without

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causing any adverse effects (Sudha et al., 2013) and is used widely in commercial probiotics. Regulations on labelling of commercial probiotic products were initiated by the Joint

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FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics (FAO, 2001). Identification of the genus and species of the probiotic strain using a

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combination of phenotypic and genotypic tests is recommended since the clinical evidence suggests that the health benefits of probiotics are strain-specific (Venugopalan et al., 2010). Subsequently, various health and food draft regulations such as the European Health and Nutrition Policy (Huys et al., 2013; Miquel et al., 2015) and Indian Council of Medical Research and Department of Biotechnology (ICMR-DBT) guidelines (ICMR, 2011) specify indicating the viable bacterial count per gram (or mL) of the product at release and end of the stipulated shelf-life, as well as the full scientific name of the microbial species and strains on the product label. In recent years, the probiotic industry has been experiencing rapid growth in India and Pakistan and new probiotic supplements are becoming readily available in these countries.

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ACCEPTED MANUSCRIPT Previous studies have raised relevant concerns regarding the conformity of probiotics in developing markets with international guidelines; as an example, a number of probiotic

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products commercialized in South Africa do not comply with the content claim stated on their

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labels (Brink et al., 2005; Elliot and Teversham, 2003). To the best of our knowledge, no

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accurate evaluation of the quality and safety standards of the probiotic products sold in India and Pakistan has been performed so far. Until a decade ago, conventional microbiological methods for quantitative (bacterial/spore counts) and genetic tests for qualitative analysis

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were used to evaluate bacterial species and strains in probiotics licensed for medicinal

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purposes (Hanna et al., 2004). In the past 15 years, molecular biological methods are being increasingly used to detect microbes and identify strains in commercial food and drug products. The terminal restriction fragment length polymorphism analysis is employed to

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determine the bacterial composition of probiotic products (Marcobal et al., 2008). Additionally, DNA extraction and polymerase chain reaction (PCR) methods help identify

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discrepancies in label claims (Drisko et al., 2005). Furthermore, sensitive techniques such as repetitive sequence-based PCR (Rep-PCR) are used for rapid identification of Lactobacillus

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spp. and Bifidobacterium spp. (Sul et al., 2007). The FAO/WHO guidelines (FAO, 2001) as well as the European Food Safety Authority (EFSA) (EFSA, 2011) also recommend using Rep-PCR in combination with denaturing gradient gel electrophoresis (DGGE) to evaluate the composition and contamination of marketed probiotic strains. Worldwide studies (De Vecchi et al., 2008; Senesi et al., 2001) demonstrated mismatches between the label information for commercial probiotic products and the results of laboratory assessments that are done (Elliott and Teversham et al., 2004; Fasoli et al., 2003; Theunissen et al., 2005) using either conventional microbiological plating or molecular methods. However, combining the two approaches is hypothesized to improve the sensitivity of evaluation in commercial probiotic products. In the present study, probiotic supplements

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ACCEPTED MANUSCRIPT claiming to contain B clausii spores, marketed in India and Pakistan were evaluated using both conventional and modern methods to identify spore count, species, and strain

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contamination, in order to draw a comprehensive picture of the microbiological quality and

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labelling of spore-forming probiotics in these countries

2. Materials and methods 2.1.

Probiotic products

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A total of five different commercial probiotic supplements marketed in India and Pakistan

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claiming to contain B. clausii spores with a declared dose were collected from local retailers (Table 1). Samples of Tufpro, Ecogro, Enterogermina, Entromax, and Ospor with specific label indication were used. Two of the five products were powder formulation (sachets) and

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three were suspensions (bottles). All batches of each product were analysed separately using

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microbiological plating and molecular techniques prior to their expiration date.

Table 1. Commercially available probiotic supplements analysed and their label indications Products

Batch No.

Declared dose

Virchow Biotech Pvt. Ltd.,

10003014

2×109 spores/ 5 mL

India

10002314

2×109 spores/ 5 mL

10006514

2×109 spores/ 5 mL

Akum Drugs & Pharmaceuticals

XDCW09

2×109 spore in 1 g

Ltd., India

XDCW14

2×109 spores in 1 g

XDCW17

2×109 spores in 1 g

10773

2×109 spores/ 5 mL

10774

2×109 spores/ 5 mL

30094

2×109 spores/ 5 mL

L6AON002

2×109 spores in 1 g

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Tufpro

Manufacturer/Supplier

Bacillus clausii

Spore suspension

Ranbaxy Laboratories Ltd.,

(bottles of 5 mL)

India

Ecogro Bacillus clausii spores (sachet 1 g)

Akumentis Healthcare Ltd., India

Enterogermina Bacillus clausii Spore suspension

Laboratoire Unither, France Sanofi India Ltd., India

(bottles of 5 mL) Entromax

Akum Drugs & Pharmaceuticals

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ACCEPTED MANUSCRIPT Bacillus clausii spores (sachets 1 g)

Ltd., India

L6AON003

2×109 spores in 1 g

Mankind Pharma Ltd., India

L6AON004

2×109 spores in 1 g

16052014

2×109 CFU/ 5 mL

ANA BIO Research &

Ospor (bottles of 5 mL)

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Development JSC., Vietnam

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Matrix Pharma, Pakistan

Quantitation of viable bacteria: bacterial isolation and plate count

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Enumeration of bacteria was performed by means of plate count method, which was done independently by two operators. Brain Heart Infusion (BHI, Oxoid, England) agar was used

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for the isolation and cultivation of bacteria. Except for Ospor, for which only one lot was available, three different batches of each product were analysed separately. For each batch,

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the contents of three separate sachets or three bottles were pooled and 1 g (powder) or 1 mL

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(liquid) was diluted in 9 ml of maximum recovery diluent (MRD, Oxoid, England). One hundred microliters of a tenfold dilution series of each product were plated in triplicate and

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incubated aerobically for 48 hours. Before counting, spores from different samples were heat inactivated at 85ºC for 10 mins to kill any residual vegetative cells or germinated spores.

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After incubation, visible colonies were counted and expressed as colony-forming units per gram (CFU/g) or mL (CFU/mL), representing the number of viable bacteria present in each product. This methodology is based on the manual issued by the National Health Institute (Istituto Superiore di Sanità –ISS) (Aureli et al., 2008).

2.3. Colony fingerprinting using Rep-PCR A total of 75 selected colony isolates showing different morphologies on the agar plates were collected for further characterization. Crude DNA was extracted from bacterial colonies using the micro LYSIS kit (Microzone, Italy) according to the manufacturer’s instructions. RepPCR fingerprinting using the (GTG)5 primer was applied as the typing method for 7

ACCEPTED MANUSCRIPT discriminating colony isolates (Versalovic et al., 1994). Reaction mixture (25 µL) was prepared using 1 µL of template DNA, 2 µM of primer, and 23.5 µL Megamix (Microzone,

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Italy). PCR amplification was performed using a BioRad T100 Thermal cycler (BIORAD,

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USA) with an initial denaturation step (95 °C, 7 min) followed by 30 cycles of denaturation

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(90 °C for 30 s), annealing (40°C for 1 min) and extension (65 °C for 8 min), and a single final extension step (65 °C for 16 min). The PCR products were separated on 2.5% (w/v)

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agarose gels and the resulting fingerprints were compared directly by visual examination.

Species identification by 16S rRNA analysis

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For the identification of different bacterial species, one representative isolate from each fingerprinting pattern was selected for amplification of the 16S rRNA gene using PCR. DNA

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fragments of approximately 1.5 kb (corresponding to the size of the 16S rRNA gene) were amplified using the primers P0 (5'-GAAGAGTTTGATCCTGGCTCAG-3') and P6

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(5'CTACGGCTACCTTGTTACGA-3') (Dicello et al., 1996). Reaction mixture (25 µL) was prepared using 2 µL template DNA, 1 µM concentration of primers, and 22 µL Megamix

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(Microzone, Italy). PCR amplification was performed in a thermal cycler with an initial denaturation step (94 °C, 5 min), 30 cycles of denaturation (94 °C for 30 s), annealing (58 °C for 30 s), extension (72 °C for 1 min), and a final extension step (72 °C for 7 min). PCR products were analysed on 1.5% (w/v) agarose gels, purified with Nucleo Spin Gel and PCR Clean Up (Macherey-Nagel, Germany), and quantified with the Marker VI (Roche, Germany) molecular weight standard. Samples were then sequenced at the BMR genomics sequencing facility (BMR Genomics, Italy).

2.5.

Identification of viable/nonviable bacteria by PCR-DGGE

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ACCEPTED MANUSCRIPT Total DNA was extracted directly from the probiotic products using the FastDNA® SPIN Kit for Soil (MP Biomedicals, USA) with the FastPrep®-24 instrument following the

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manufacturer’s instructions. The V2-V3 region of the 16S rRNA gene was amplified using

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the primers HDA1-GC and HDA2, and the thermo-cycling program described by Fasoli et al.

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(2003). DGGE analysis was performed using the INGENYphorU System (Ingeny International BV, Netherlands). Amplicons were analysed on 8% (w/v) polyacrylamide (acrylamide:bis, 37.5:1) gels in 1× Tris acetate EDTA (TAE) buffer containing a 40% to 60%

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linear denaturing gradient of 7.0 M urea and formamide (40% w/v). Electrophoresis was

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performed at a constant voltage of 100 V at 60 °C for 18 h. The gel was then stained in 1× TAE buffer with SYBR Green I nucleic acid stain (Roche, Germany) and photographed. DGGE bands were excised from the gel, eluted in 25 µl of sterile distilled water, and re-

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amplified with primers HDA1 (without the GC-clamp) and HDA2. The PCR products were purified from the reaction mixture using the Nucleo Spin Gel and PCR clean-up (Macherey-

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Nagel, Germany). Sequencing of the PCR amplicons was done at the BMR Genomics Sequencing facility (BMR Genomics, Italy). The sequences were taxonomically assigned by

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comparison with classified sequences present in the Ribosomal Database Project (RDP II; http://rdp.cme.msu.edu/) using the Sequence Match tool.

3. Results 3.1.

Plate counts of viable bacteria

The bacterial/spore count analysed using the plate count method showed poor correlation with the label indications for three of the five analysed products (Table 2). Enterogermina and Entromax matched the label claim (2×109 spores/ 5 ml and 2×109 CFU/g, respectively), while spore counts lower than those stated in the label indication were noted for Tufpro and Ospor

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ACCEPTED MANUSCRIPT (4×108 CFU/mL each). Ecogro showed minor deviations in the spore count (for one batch and

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not confirmed by one of the operators) from the label indication (2×109 CFU/g).

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Table 2. Bacterial spore counts of the five probiotic products before and after heat treatment to kill vegetative cells

Condition↓ Batch No.

Operator 1

Operator 2

Operator 1

10006514

10006514

10002314

Operator 1

Operator 2

10002314

10003014

10003014

Operator 2

RI

(spore count)

PT

Operator

Product

→

(CFU/mL)

Before

2.55×106

1.26×106

1.30×107

2.17×107

1.20×107

1.81×106

After

1.30×106

3.97×105

6.00×105

2.58×107

3.00×105

1.25×106

Batch No.

XDCW09

XDCW09

XDCW17

XDCW17

XDCW14

XDCW14

Before

1.70×109

1.90×109

1.50×109

1.94×109

3.10×109

2.43×109

After

1.00×109

1.55×109

2.00×109

5.45×108

2.00×109

1.98×109

30094

30094

10774

10774

10773

10773

2.73×108

3.20×108

3.20×108

3.30×108

3.30×108

1.84×108

3.40×108

3.40×108

3.30×108

3.30×108

L6AON002

L6AON002

L6AON003

L6AON003

L6AON004

L6AON004

Before

1.10×109

1.86×109

2.43×109

1.30×109

1.70×109

1.71×109

After

2.00×109

1.55×109

2.30×109

1.00×109

2.00×109

1.31×109

Batch No.

16052014

16052014

-

-

-

-

Before

1.50×108

2.41×108

-

-

-

-

After

5.10×107

5.60×107

-

-

-

-

Enterogermina (CFU/mL)

Before

3.00×108

After

2.25×108

Batch No. Entromax (CFU/g)

Ospor (CFU/mL)

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Batch No.

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(CFU/g)

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Ecogro

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Tufpro

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ACCEPTED MANUSCRIPT 3.2.

Species identification using Rep-PCR and 16S rRNA gene sequence analysis

A total of 75 isolated colonies that were representative of all morphologies from the five

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products were processed for Rep-PCR analysis. Thirty-two colonies showed heterogeneous

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rep-PCR profiles indicating distinct genetic diversity among them (Figure 1).

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Sequence analysis of the 16S rRNA gene amplicons obtained from the 32 genotypically diverse isolates revealed that the 2 colonies from Enterogermina and the 3 colonies from Ospor showed 100% sequence identity with B. clausii (Table 3). Among the 11 colonies

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sequenced from Tufpro, 10 colonies showed high sequence similarity with B. cereus, whereas

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only 4 of 12 colonies sequenced from Ecogro showed sequence similarity with B. clausii. All 4 colonies sequenced from Entromax showed sequence similarity with B. subtilis (Table 3).

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Table 3. Sequence analysis of the 16S rRNA gene amplicons of 32 genotypically diverse

Sequenced

Species with

Positive

colonies, no.

S_ab score ≥ 0.98

colonies, no.

Bacillus cereus

10

Alcaligenes faecalis

1

Bacillus subtilis

6

Bacillus clausii

4

Bacillus amyloliquefaciens

2

Product

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Tufpro

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bacterial colonies obtained from the five probiotic samples

Ecogro

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12

Enterogermina

2

Bacillus clausii

2

Entromax

4

Bacillus subtilis

4

Ospor

3

Bacillus clausii

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Identification of viable/nonviable bacteria

PCR-DGGE analysis has a much higher sensitivity than culture-dependent analysis for detecting bacterial strains in probiotics, although it does not differentiate between 12

ACCEPTED MANUSCRIPT viable/nonviable bacteria. Results from the PCR-DGGE analysis of probiotic samples suggested that all products contained B. clausii spores, including those that demonstrated low

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bacterial counts with the plate count method (Table 4).

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Table 4. Identification of DGGE bands of the five probiotic samples after sequence analysis; the number of sequenced bands are as indicated in Figure 2 Band no.

DGGE results

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Product

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0.96

Alcaligenes faecalis

0.73

Staphylococcus/Lysinibacillus

0.93

Xanthomonas/Pseudomonas

0.94

Bacillus clausii

0.89

Bacillus amyloliquefaciens

≥0.87

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Lysinibacillus

0.92

2, 9, 10, 12, 14, 18, 20,

Bacillus subtilis/Bacillus

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Tufpro

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2, 22

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4

Entromax

Ospor

≥0.91

21

amyloliquefaciens

1, 4

Bacillus clausii

6

Bacillus licheniformis

15

Acinetobacter subsp.

13

Paenibacillus subsp.

11

Geobacillus

5

Xanthomonas/Pseudomonas

6

Bacillus cereus/licheniformis

8

Staphilococcus/Lysinibacillus

1

Bacillus clausii

0.96

9, 10, 16, 17, 19

Bacillus subtilis

≥0.91

2, 18

Bacillus amyloliquefaciens

≥0.90

1, 4

Bacillus clausii

0.89

1

Bacillus clausii

0.96

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Enterogermina

score

Bacillus cereus/licheniformis

3

Ecogro

S_ab

0.92 0.91 0.76 0.83 0.76 0.94 0.96 0.93

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ACCEPTED MANUSCRIPT The sequences of the bands obtained from the Enterogermina and Ospor showed similarity only with B. clausii. Furthermore, among these two only Enterogermina showed a single band

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of denatured DNA (B. clausii) on the gel (Figure 2).

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Additional bands were observed in the DGGE profiles of the other products suggesting a

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mixed bacterial population. Among these, other species belonging to the genus Bacillus were found including B. cereus from Tufpro, B. subtilis from Ecogro, and B. amyloliquefaciens

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from Entromax (Figure 2).

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4. Discussion

Validation of the probiotic strains and species in commercial samples is an important safety issue in the use of medicinal products. This study evaluated the qualitative and quantitative

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aspects of label claims from five commercial probiotic products from India and Pakistan containing B. clausii. Overall, using conventional and modern methods of analysis, a poor

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correlation between label indications and probiotics recovered was demonstrated in most products. Only Enterogermina and Ospor contained B. clausii spores indicated by the label

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whereas the other products exhibited mixed bacterial populations.

Probiotics are of great interest to the medical world for their potential therapeutic and preventive health benefits. B. clausii together with other spore-forming Bacillus spp. is an important human probiotic (Sanders et al., 2003). It has the ability to germinate after an acid challenge and grow as vegetative cells, both in the presence of bile and under limited oxygen availability (Cenci et al., 2006). It contributes similarly for its spore formation and probiotic action if administered orally, either as a lyophilized or liquid formulation (Ghelardi et al., 2015). Moreover, the four isogenic B. clausii strains known to have differential proteome expression can cooperate as probiotics, with complementary contributions to overall probiotic

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ACCEPTED MANUSCRIPT activity (Lippolis et al., 2013). However, concerns about the validity of unsubstantiated claims of strain composition and spore count often pose questions about probiotic quality.

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Hence, it becomes increasingly important to test the label indications using independent

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laboratory analysis.

In the present study, Tufpro and Ospor among the five probiotics analysed showed poor correlations between the spore count indicated by the label and laboratory analysis, whereas

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Ecogro showed a minor deviation. A similar mismatch between label and laboratory assay for

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the B. clausii spore count was reported for six different commercial probiotic products manufactured and marketed in Italy (De Vecchi et al., 2008).

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A report on the bifidobacterium claims for 58 probiotic products obtained worldwide showed a relatively high degree of genomic homogeneity among the various strains used in the

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industry (Masco et al., 2005). Genotypic characterization of the probiotic products at strain level using culture-dependent and independent approaches followed by pulsed-field gel

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electrophoresis (PFGE) revealed the genomic homogeneity of bifidobacterium strains. In contrast, species identified by bacterial culture and Rep-PCR fingerprinting in the current study revealed approximately 40% (32/75 isolates) diverse genotypes among the five probiotic products. Analysis of the 16S rRNA gene confirmed that only Enterogermina and Ospor contained B. clausii, whereas the remaining samples were confirmed for undeclared species. In addition to the conventional methods, molecular methods such as RAPD analysis have been used for species identification in probiotic samples (Senesi et al., 2001). One such study confirmed low intra-specific genome diversity among four B. clausii strains constituting Enterogermina from samples that were decades old, and indicated that each strain had remained the same for the past 25 years (Senesi et al., 2001). On the contrary, analysis of 58

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ACCEPTED MANUSCRIPT commercial probiotic products by 16S rDNA sequencing in an exploratory study revealed intra-specific discrepancies among 26 of 58 probiotic strains, including two ATCC

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Lactobacillus strains, and complete mismatch of labelled species for six products (Yeung et

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al., 2002). Interestingly, the maximum homogeneity observed with a B. cereus strain among

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the majority of colony isolates (10 out of 11) of the Tufpro sample in our study raises medical concerns as it has not received Qualified Presumption of Safety (QPS) status by the EFSA in the European Union (EFSA, 2014). Also, the presence of B. licheniformis in the Tufpro and

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animal studies (Sorokulova et al., 2008).

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Ecogro samples furthers the concern because of the reported risks associated with their use in

Further to the identification of variations in label claims, PCR-DGGE was used to perform a

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more comprehensive analysis of the bacterial composition of the probiotic products, although such methodology does not discriminate viable/non-viable spores. DGGE analysis indicated

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that all samples contained B. clausii, including those that demonstrated low bacterial counts in the plate count method. Subsequent re-amplification of the bands and sequencing identified

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that, among the five samples, only Enterogermina had a single band corresponding to a homogenous B. clausii population, whereas the results from other products suggested mixed populations. Similar mismatch results were reported with probiotic samples from South Africa (Elliott and Teversham, 2004) and Italy (Fasoli et al., 2003) based on culture methods and DGGE analysis, where an independent laboratory confirmed that a majority (six of nine samples from South Africa) of probiotic samples did not correspond to the label claims. Identification of unlabelled bacterial species using PCR-based DGGE analysis was also investigated in another South African probiotic study; only 54.5% of yogurt samples and 33.3% of lyophilised products had label corroboration (Theunissen et al., 2005).

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ACCEPTED MANUSCRIPT 5. Conclusions Quality control of products containing viable bacterial cells or spores is a sensitive matter as

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several papers are available about the poor quality of these products, which was mainly due to

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mislabelling of the species and/or a content of viable cells that was lower than declared.

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Surprisingly, official methods covering this specific analytical need do not exist; the International Organization for Standardization-International Dairy Federation (ISO-IDF)

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methods are available only for dairy-based products.

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In Italy, the National Health Institute has released a set of recommended methods specifically developed for assessing food supplements claiming to contain probiotic bacteria (Aureli et al., 2008). Even if not directly focused on B. clausii, we followed the general procedure provided

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by these guidelines. In the present study, we followed these guidelines whenever applicable. Outcomes of this study strongly suggest that of the five commercially available probiotic

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supplements from India and Pakistan claiming to contain Bacillus clausii spores, only Enterogermina complies with label indications. The plate count method demonstrated

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correlations between the amount of spores specified on the label and laboratory results only for Enterogermina and Entromax, whereas the PCR-DGGE analysis revealed that only Enterogermina had a homogenous B. clausii population.

Results of the present study have analytical significance in terms of bacterial spore counts in the commercial products, and may have regulatory significance because of the presence of species that have not received QPS status. Periodic surveillance of label claims of approved food and therapeutic probiotics is essential to ensure the safety and efficacy of ‘for life’ products.

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ACCEPTED MANUSCRIPT Acknowledgments This work was supported by Sanofi-Aventis, France. The authors acknowledge Sashi Kiran

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Goteti from Jeevan Scientific Technology Limited (Hyderabad, India) and Anahita Gouri of

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Sanofi (India) for providing writing and editing assistance in the development of this

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manuscript.

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Figure captions

Figure 1. Representative Rep-PCR profiles of some probiotic samples. 200bp, molecular weight marker; G, Ecogro; E, Enterogermina; O, Ospor; T, Tufpro. Figure 2. PCR-DGGE analysis of DNA extracted from five probiotic samples. E, Enterogermina; Tuf, Tufpro; ECO, Ecogro; Max, Entromax; OS, Ospor. Numbers indicate different batches.

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Few data are available on quality and reliability of spore-forming probiotics. Most B. clausii probiotics from India and Pakistan do not comply with label claims. Bacteria contained in some products are not univocally ascribed to B. clausii. Some bacteria found have not received Qualified Presumption of Safety status. Surveillance of food and therapeutic probiotics is needed to ensure safety/efficacy.

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