Journal of Chemotherapy Volume 16, number 5 October 2004

MICROBIOLOGICAL EVALUATION OF COMMERCIAL PROBIOTIC PRODUCTS AVAILABLE IN ITALY

L. DRAGO -E, DE VECCHI -L. NICOLA -A. COLOMBO -M.R. GISMONDO

Laboratory of Clinical Microbiology, Dept of Clinical Sciences "L. Sacco",

L. Sacco Teaching Hospital, University of Milan, Italy.

Correspondence: Lorenzo Drago, Lab of Clinical Microbiology, Dept Clinical Sciences L. Sacco, Via GB. Grassi 74,

20157 Milano, Italy. Tel: +390239042469; Fax: +390250319651. E-mail: lorenzo.drago@unimi.it

Summary

Scientific evidence of the prevention and therapy of some intestinal diseases is accumulating in regard to probiotic products. However, sufficient information on the use of probiotics in specific therapies is not yet available and, above all, there is no clear legislation about these products in Europe.

In this study, we evaluated five different probiotic products commercially avail- able in Italy for their qualitative and quantitative microbial content after about 12 and 22 months of storage. We also evaluated the stability of lactobacilli to 0.3% bile salts and to pH of 3.58 and 7.98. There were discrepancies between the declared content and our results found after storage for 4 of the tested products. Bile salts and basic pH did not affect the growth of the lactobacilli tested, while for 2 tested products 6 hours at acid pH produced a complete inhibition of bacterial growth.

Our results suggest the need for clear legislation and adequate control of the manufacturing of probiotic products.

Key words: Probiotics, lactobacilli, product stability.

INTRODUCTION

The term probiotic was initially introduced by Fuller in 1989 to indicate "live microbial food supplement which beneficially affects the host by improving the intestinal microbial balance" 1. More recently, an Expert Committee has redefined probiotics as "living micro-organisms which upon ingestion in certain numbers exert health benefits beyond inherent general nutrition" 2.3. Several bacteria have been proposed for use as probiotics, mainly lactobacilli, bifidobacteria, other lactic acid bacteria (LAB) such as enterococci, and other microorganisms comprising Bacillus subtilis, Bacillus cereus, Saccharomyces boulardii and Saccharomyces cerevisiae 4.

Most of these commercial products are used for the treatment of a wide range of conditions such as antibiotic-associated diarrhoea, alteration of intesti- nal microflora balance, food and lactose intolerance and intestinal bowel diseases 5.6.

However, to exert their healthy action in the intestinal lumen, all these microorganisms should be able to reach the gut as viable organisms and in suf- ficient number 7.8.

The aim of the present study was to evaluate the stability over time of the microbial content of some commercial probiotic-based products present in the Italian market, by considering qualitative and quantitative features. Moreover, the lactobacilli found in the tested products were also tested for their ability to survive for a prolonged time in contact with acid and basic pH and in the presence of high concentrations of bile salts.

MATERIAL AND METHODS

Commercial probiotic products

Five different products defined as "probiotics" by

the manufacturer were included in the study. Of these, 2 contained only one species (Lactobacillus spp.) and the other 3 had a polymicrobial content (Lactobacillus spp. with other microorganisms). The tested products were named A (Bifilact capsules - Fidia Nutraceutical, Abano Terme, Italy), B (Yovis sachets to be stored at 4°C -Sigma Tau, Rome, Italy), C (Dicoflor sachets -Dicofarm, Rome, Italy), D (Neolactoflorene capsules -Newpharma, Milan, Italy) and E (Enterolactis sachets -Sofar, Trezzano Rosa, Italy). Product A was stored both at room temperature, as stated by the manufacturer, and at 4 oc to evaluate differences between storage temper- atures. Two different batches of each product were evaluated.

Stability of microbial load over time

Since we were unable to find recently manufactured products, the microbial count of each commer- cial preparation was performed at the half-life and near the expiry date stated on the label, correspond- ing to 12 and 22 months after production, respectively.

For microbial counts, one gram of each products was suspended in 10 mL of sterile saline (0.9% NaCI) and since Iyophilised products were not easily dissolved, solutions were allowed to stabilize for 15 min and vortexed at regular intervals of 5 minutes. Differential counts of microorganisms contained in the probiotic products were carried out by spreading 10 and lOO pL from opportune tenfold diluted solu- tions on 5% sheep blood agar plates (to evaluate streptococci), on de Man-Rogosa-Sharpe (MRS) agar (selective medium for lactobacilli) and on tryptone soy agar (medium used to evaluate growth of a wide range of microorganisms). Plates were incubated at 37°C in 10% CO2 enriched atmosphere for 48 h. Counts of bifidobacteria were performed by plating 10 and 100 pL from tenfold dilutions on blood agar plates, and incubating at 37°C for 48 h in Gas-Pak system for anaerobiosis.

Colonies were counted and preliminarily identi- fied according to their morphology (shape, appear- ance and size) and Gram stain was performed. Final identification was carried out by biochemical tests with API system (bioMerieux, Rome, Italy) by using API20A for anaerobes, API Coryne for Gram-posi- tive bacilli and API20 Strep for streptococci. Lactobacilli were identified by means of API 50 CH cards

Stability of lactobacilli at acid and basic pH

Lactobacilli belonging to the species present at the highest load in each product were further evalu- ated for their ability to survive at acid and basic pH for a period of 48ho In particular, we tested Lo bul- garicus, Lo acidophilus, Lactobacillus GG and Lo casei contained in products A, B, C and E, respec- tively, while Lo acidophilus (grown at 5 x 102 CFU/cps) was chosen for product Do MRS broth was acidified with 6N HCI to reach a final pH of 3.58, while a basic pH (7.98) was obtained by addi- .

tion of 1M KOH to the broth. After an overnight growth on MRS agar, lactobacilli were suspended in normal sterile saline and added to acidified and basi- fied MR5 broths in order to obtain inocula of about 1~ 8 x 105 CFU/mL. Bacterial counts were per- formed immediately after addition of the inoculum and after 6, 24 and 48 h of incubation at 37°C in 10% CO2 enriched atmosphere, by plating 10 and 100pl from tenfold dilutions onto MRS agar plates. After 48 h at 37°C in 10% CO2 enriched atmos- phere, colonies were counted and the mean value from the two aliquots was calculated. At the same intervals, broth pH values were measured.

Bacterial growths at acid and basic pH were compared with growth of the same strains in opti- mal conditions (MRS broth).

Stability of lactobacilli in presence of high concentrations of bile salts

The ability of lactobacilli belonging to the species present at the highest loads (see above), isolated from probiotic commercial products, to survive in the presence of elevated concentrations of bile salts was evaluated by adding a mixture of bile salts (Oxgall, Sigma, St. Louis, MO) to MRS broth in order to obtain a final concentrations of 0.3%. Broths supplemented with bile salts were inoculated with lactobacilli suspension to give a final inoculum of 1-8 x 105 CFU/mL.

Bacterial counts were performed after 0, 6, 24 and 48 h of incubation at 37°C in 10% CO2 enriched atmosphere: aliquots of 10 and 100 J.lL from opportune tenfold dilutions were spread onto MRS agar plates and incubated for 48 h at 37°C in 10% CO2 enriched atmosphere. MRS broth inocu- lated with the same bacterial suspension was used as growth control.

RESULTS

Stability over time

The qualitative and quantitative compositions of each commercial product at 12 and 22 months after production are summarized in Table 1. Near the expiry date, only Lactobacillus sporogenes was found in product A, while in product B lactobacilli and streptococci were found in lower amounts than stated on the label. As declared, product C con- tained only Lactobacillus casei subsp. rhamonosus GG, but only about one-half of the original content was found at the expiry date. The label on product D reported only the qualitative composition, while quantitative data for each microbial species were lacking. However, there were some discrepancies between the species declared on the label and the microorganisms found. For product E, a 1 loglo decrease in CFU/g was observed after storage.

Stability of lactobacilli in modified conditions

Tables 2-4 show the growth of lactobacilli in the presence of altered conditions, resembling what may happen in vivo, in the gut with acid pH and high concentrations of bile salts.

The growth of lactobacilli was not affected by

bile salts (Table 2) or basic pH (Table 3), while low pH values influenced the growth of some strains. In particular, acid pH inhibited the growth of lactobacil­li isolated from products A and D after 6 h, while strains contained in B and E well tolerated such con­ditions (Table 4).

link Tabelle

DISCUSSION

A correct taxonomic identification of microor-ganisms used as probiotics for commercial use as well as their quali-quantitative characteristics are fun- damental when a new product is launched onto the market. Evaluations of some probiotics in the USA and Europe have often shown poor correlation between label claims and actual contents. For instance, in an American study, the bacterial compo- sition of only 7 out of 30 products corresponded well with what was claimed on the label 9. In the same way, in the United Kingdom 63% of the tested products were below the requested standards 10. Recently, an Italian study analyzed the content of yoghurt and lyophilized products available in the Italian market, reporting some discrepancies between label claims and the actual content, including inappropriate identifications, the presence of undeclared microorganisms, and bacterial loads lower than declared 11. However, none of these study evaluated the stability of the commercial prod- ucts until the expiration date or the capabilities of lactobacilli contained in these products to grow at acid and basic pH and in the presence of high concentrations of bile salts.

According to some authors, a daily intake of at least 109 viable cells/day is necessary to obtain a transient colonization of the gut 12. Therefore, the amount of viable microorganisms contained in commercial products plays an important role in deter- mining its clinical efficacy. Nonetheless, intestinal colonization also depends on the strain used and on its intrinsic probiotic properties, such as adhesion, hydrophobicity and interaction with other intestinal microorganisms.

In some of the products tested in the present study, Lactobacillus sporogenes (in products A and D) and Streptococcus thermophilus (in products A, B and D), might have been used to increase and/or maintain the declared microbial load, since these bacteria are known for their stability during storage. However, L. sporogenes does not belong to the Lactobacillus genus, since it has been reclassified as Bacillus coagulans 13.

A decrease in microbial load was observed after 12 and 22 months of storage; since we were unable to evaluate the content of the tested products immediately after production, it might be hypothesized that this decrement could occur during the lyophilization process. In fact, the stress induced by some industrial large-scale procedures, such as freeze-drying or spray-drying techniques, may heaviIy condition the viability of some microorganisms, such as anaerobes. Differences in survival of the industrial process were also found among the different species of lactobacilli.

Our results, however, may have been influenced by the method used: plate count may be affected by the medium used and by interactions between bacteria, and needs to be improved 3. This issue once again indicates the lack of current regulatory requirements and, therefore, the need for clear legislation in Italy and in the European Union about the pro- duction and quality control of the probiotic industry to define qualitative and quantitative characteristics of a probiotic as well as to periodically screen the manufacturing by competent organizations 7.14.

The term "probiotic" is often improperly used and information on specific probiotic properties of the strains contained in the products are not exhaustive.

In conclusion, the findings of the present study, although do not deny the beneficial effects and the potential utility of probiotics, raises some concerns about the quality of some commercial products, emphasizing the need for clear legislation on this matter .

ACKNOWLEDGEMENT: This work was partially supported by a grant from the Associazione Malattie Infiammatorie Croniche Intestinali (A.M.I.C.I.) Lombardia.

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