Suitability of the fluorescent techniques for the enumeration of probiotic bacteria in commercial non-dairy drinks and in pharmaceutical products

Suitability of the fluorescent techniques for the enumeration of probiotic bacteria in commercial non-dairy drinks and in pharmaceutical products

Food Research International 39 (2006) 22–32 www.elsevier.com/locate/foodres Suitability of the Xuorescent techniques for the enumeration of probiotic...

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Food Research International 39 (2006) 22–32 www.elsevier.com/locate/foodres

Suitability of the Xuorescent techniques for the enumeration of probiotic bacteria in commercial non-dairy drinks and in pharmaceutical products Johanna Maukonen ¤, Hanna-Leena Alakomi, Liisa Nohynek, Katri Hallamaa, Sanna Leppämäki, Jaana Mättö, Maria Saarela VTT Biotechnology, P.O. Box 1500 (Tietotie 2), FIN-02044 VTT, Finland Received 27 April 2005; accepted 24 May 2005

Abstract The suitability of the Xuorescent techniques for the microbiological quality assessment of probiotic non-dairy drinks and probiotic pharmaceutical products was studied. The method optimization was Wrst performed with pure cultures of probiotic strains detected in commercial products (representing Lactobacillus and BiWdobacterium species). Altogether seven diVerent Xuorescent viability stains combined with epiXuorescence microscopy and Xow cytometry were tested. The most applicable stains ChemChrome and LIVE/DEAD BacLight Viability Kit were further used for the viability studies of the commercial probiotic products. The studied products proved to be of good quality. All products contained the probiotic bacterium indicated on the label and the levels of living bacteria were reasonable per dose (108–1010 cells for suggested daily dose of pharmaceutical products and 108–109 cells per 100 ml of drink). The results obtained with Xuorescent stains were mostly in agreement with results obtained with culture, rendering Xuorescent techniques applicable alternatives for rapid viability assessment of probiotic products. Especially the newly developed Xuorometry assay with BacLight proved to be fast and accurate.  2005 Elsevier Ltd. All rights reserved. Keywords: Probiotic product; Lactobacillus; BiWdobacterium; EpiXuorescence microscopy; Flow cytometry; Fluorometry assay

1. Introduction The past two decades have seen a marked increase in the inclusion of probiotic bacteria in various types of food products, especially in fermented milks (Daly & Davis, 1998). During recent years probiotics have been increasingly incorporated also into non-dairy foods such as fruit and berry juices. For the purposes of human nutrition it is suggested that a probiotic is best deWned as ‘a live microbial food ingredient that is beneWcial to health’ (Salminen et al., 1998). Most currently used pro* Corresponding author. Tel.: +358 20 722 7183; fax: +358 20 722 7071. E-mail address: [email protected] (J. Maukonen).

0963-9969/$ - see front matter  2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.05.006

biotics belong to genera BiWdobacterium and Lactobacillus (Kopp-Hoolihan, 2001). Good viability is generally considered a prerequisite for optimal probiotic functionality (Saarela, Mogensen, Fonden, Mättö, & Mattila-Sandholm, 2000). Therefore, the quality assurance of probiotic products is currently based on techniques detecting viable cells. Several factors such as strain characteristics, matrix, temperature (e.g. during storage), pH, oxygen, and accompanying microbes aVect the viability of a probiotic strain (Gomes & Malcatam, 1999; Saarela et al., 2000). Plate count culture technique, which is based on reproduction of bacterial cells on agar plates, is the traditional method used for quality assurance of probiotic products. For the enumeration of probiotic Lactobacillus

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and BiWdobacterium strains in the products, MRS (de Man Rogosa Sharpe; for biWdobacteria various supplements, e.g. cysteine or antibiotics, can be used) is the favored growth medium (Canganella et al., 1997). In products containing high populations of accompanying microbes and/or a mixture of several probiotic strains, culture on selective media (often including antibiotics) followed by identiWcation of each strain by accurate molecular methods is needed to assess viability of the probiotic strain(s) (Björneholm, Eklöw, Saarela, & Mättö, 2002). Beside culture techniques, Xuorescent stains have commonly been applied in studies on bacterial viability. Fluorescent stains can be applied in the detection of viable, damaged, and dead bacterial cells in a sample, which are detected with Xuorescence microscopy, Xuorometer, or Xow cytometer. Typically, a dual approach includes staining of viable cells with one dye followed by counterstaining of dead or all cells with another stain in order to obtain the total cell number (Breeuwer & Abee, 2000). Fluorescent probes used in viability assessment of lactic acid bacteria include nucleic acid probes such as propidium iodide (PI), TOTO-1 (are excluded from intact cells), SYTO9, DAPI (stain both viable and non-viable cells, are used together with other probes), and physiological indicators such as 2⬘7⬘-bis-(2 carboxyethyl)-5(and-6) carboxyXuorescein (BCECF), carboxyXuorescein diacetate (CFDA), N-(Xuorescein thio-ureanyl)-glutamate (FTUG), and bisoxonol (BOX) (Auty et al., 2001; Bunthof, Bloemen, Breeuwer, Rombouts, & Abee, 2001a; Bunthof & Abee, 2002; Glaasker, Konings, & Poolman, 1996; Karwoski, Venelampi, Linko, & Mattila-Sandholm, 1995; Ueckert et al., 1995; Ueckert, Nebe-von Caron, Bos, & ter Steeg, 1997). Fluorescent probes detect diVerences in, e.g. membrane permeability or enzyme activity of cells thus rendering Xuorescent techniques basically diVerent from culture which relies on microbial reproduction. However, both techniques can be used to measure viability which as a term can comprise all these aspects. In the present study we assessed the probiotic viability of commercial products using Xuorescent techniques and culture. Our aim was to investigate the applicability of Xuorescent techniques on rapid viability studies of commercial probiotic capsules and non-dairy drinks.

2. Materials and methods

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(Oxoid, Basington, Hampshire, UK) or on MRS agar (Oxoid). Cysteine (0.05%, w/v, Merck, Darmstadt, Germany) was added to MRS media for BiWdobacterium animalis subsp. lactis. Lactobacillus plantarum was grown at 30 °C and the other strains at 37 °C for 2–3 days. The culture experiments were repeated three times. 2.2. Probiotic products Pharmaceutical probiotic products and probiotic drinks used are listed in Table 2. These products were purchased at local markets and pharmacies, stored as recommended by the manufacturer, and examined before expiration date. 2.3. Fluorescent detection 2.3.1. Pretreatment of pure cultures Bacterial cultures were grown in MRS broth for 20–24 h (stationary phase) and cells were harvested by centrifugation (1500g, 5 min). Prior to staining, cell suspensions were sonicated (2 £ 10 s on ice, 25 kHz, Amdent 830 Piezo, Amlab, Sweden) to dissociate the aggregates or chains of bacteria. In order to study the stainability of dead cells, bacterial broth cultures were heat-treated in 1 ml aliquots at 85 °C for 10 min. 2.3.2. Pretreatment of drinks Five milliliters of each probiotic drink was suspended in 45 ml of sterile Wltered (0.2 m) phosphate-buVered saline (PBS; pH 7.2) and homogenized for 30 s by a stomacher blender (Colworth Stomacher 400, A.J. Seward UAC House, London, UK). Thereafter the samples were Wltered through 41 and 20 m Wlters to remove large solid particles from the samples. After Wltration, the samples were pelleted by centrifugation (10 min, 1000g) and resuspended in PBS. 2.3.3. Pretreatment of pharmaceutical probiotics Hundred milligrams of each pharmaceutical probiotic product was suspended in 10 ml of sterile Wltered (0.2 m) Ringer solution (Merck), and homogenized for 30 s by stomacher blender (Colworth Stomacher 400). Hundred microliters of cell suspension was washed once with 900 l staining buVer of Xuorochrome to be used, centrifuged (4 min, 12000 rpm, Biofuge 13, Heraeus Instruments, Hanau, Germany), resuspended in 1 ml, and diluted 1:100 in the same buVer for Xuorescence staining.

2.1. Bacterial strains and culture conditions The bacterial strains used in the study are listed in Table 1. Strains were obtained from VTT culture collection (Espoo, Finland) and chosen on the basis that they represent diVerent probiotic species and products. The strains were cultured anaerobically in MRS broth

2.3.4. Fluorescent staining methods The used stains are listed in Table 1. Since BacLight and ChemChrome were found to be the most promising alternatives with pure cultures, they were used for staining of bacteria in probiotic drinks and pharmaceutical products. The staining procedure was the same for

¡ +/¡ ¡ +/¡ ¡ ¡ ¡ ¡ ¡ ¡ +/¡ ¡ +/¡ +/¡ ¡ +/¡ +/¡ +/¡

Table 1 The applicability of Xuorescent probes to probiotic pure cultures Table 1 Strain probes The applicability of Xuorescent probes toFluorescent probiotic pure cultures LIVE/DEAD ChemChromeb ChemChromeb CFDA CAM Strain Fluorescent probes BacLight & PBS & ChemSol (5,6-carboxyXuorescein (calcein acetoxymethyl b b CAM LIVE/DEAD Viability Kita ChemChrome ChemChrome CFDA diacetate)c ester)a & PBS & ChemSol (5,6-carboxyXuorescein (calcein acetoxymethyl BacLight Lactobacillus rhamnosus VTT E-96666 Viability ++ ++ + ¡ c diacetate) ester)a Kita + (ATCC 53103) Lactobacillus E-96666 +++ ++ + ++ + ¡ Lactobacillus rhamnosus reuteri VTTVTT E-97849 +++ +++ +++ ++ (ATCC 53103) Lactobacillus paracasei VTT E-94510 ++ + ++ +/¡ ¡ Lactobacillus plantarum reuteri VTT E-97849 +++ +++ +++ ++ Lactobacillus VTT E-981065 +++ +++ ++ ++ + ¡ Lactobacillus paracasei E-94510 ++ + ++ +/¡ ¡ BiWdobacterium animalisVTT subsp. lactis +++ ++ ++ ++ + Lactobacillus plantarum VTT E-981065 +++ ++ ++ + ¡ VTT E-94508 BiWdobacterium animalis subsp. lactis +++ ++ ++ ++ + +++, stained very well; ++, stained well; +, stained weakly; +/¡, stained very weakly, cells uncountable; ¡, did not stain. VTT E-94508 a Manufacturer: Molecular Probes, Eugene, OR, USA. b +++, stained very well; ++, stained well; +, stained Manufacturer: Chemunex, Ivry-sur-Seine Cedex,weakly; France.+/¡, stained very weakly, cells uncountable; ¡, did not stain. ac Manufacturer: Probes,MO, Eugene, Manufacturer: Molecular Sigma, St. Louis, USA.OR, USA. b Manufacturer: Chemunex, Ivry-sur-Seine Cedex, France. c Manufacturer: Sigma, St. Louis, MO, USA.

SFDA (5-sulfo-Xuorescein SFDA diacetate, sodium salt)a (5-sulfo-Xuorescein ¡ diacetate, sodium salt)a

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CFDA-AM (5-carboxyXuorescein CFDA-AM diacetate acetoxy-methyl ester)a (5-carboxyXuorescein ¡ diacetate acetoxy-methyl ester)a

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samples examined with epiXuorescent microscope, Xow cytometer, and Xuorometer. Nucleic acid probes SYTO9 and PI of LIVE/DEAD BacLight Bacterial Viability Kit were used as described by the manufacturer. The viability Xuorochromes based on intracellular enzymatic reaction and intact cell membrane were ChemChrome, CFDA and CFDA-AM, SFDA, and CAM. Various staining buVers were tested to Wnd the optimal one for each stain. BrieXy, 1 ml of sample was prepared in staining buVer, 10 l of Xuorogenic esterase substrate was added, and the sample was incubated at 38 °C in the dark for 10 min for ChemChrome and 15–60 min for the other esterase stains. To visualize also damaged and dead cells, selected samples were stained also with 1.5 l of PI (from BacLight) according to manufacturer’s instructions. The stained samples were analyzed with epiXuorescent microscope (Olympus BX60, Olympus Optical Ltd., Tokyo, Japan) and/or Xow cytometer (Partec PAS, Partec GmbH, Müster, Germany). In addition, the drink samples were examined with microtiter plate Xuorometer (Fluoroscan Ascent FL, LabSystems, Helsinki, Finland). 2.3.5. EpiXuorescence microscopy The stainability and viability of the bacterial cells in pure cultures and probiotic products were evaluated by epiXuorescent microscope through a 40 £ 0.75 objective, whereas the probiotic drinks were examined through a 100 £ 0.75 objective. The samples were observed using multiband Wlter set (double Wlter for PI and Texas Red; Chroma Technology Corp., Brattleboro, VT, USA). The enumeration of Xuorescent cells in each sample was performed using Thoma-chamber cell counter (Knittel Gläser, Germany). 2.3.6. Flow cytometric analysis Particle analyzing system (PAS) Xow cytometer was used for the analysis of several pure cultures and probiotic drink samples stained with Xuorescent probes. Analysis of the samples was performed with Partek FloMax Operation and analysis software (version 2.4d). One milliliter pretreated sample was stained as described above and 25 l of Xuorescent Flow-CountTM Fluorospheres (Beckman Coulter, Allendale, NJ, USA) was added in each sample as internal standard. The running parameters were optimized for each sample. Nearly all samples were analyzed in triplicate. However, a few Xow cytometric experiments with pharmaceutical probiotic products were performed once with two replicates. 2.3.7. Microtiter plate Xuorochrome assay Microtiter plate Xuorochrome assay with LIVE/ DEAD BacLight Viability Kit was performed as previously described by Alakomi, Mättö, Virkajärvi, and Saarela (2005). Each sample was examined in triplicate.

Product name Composition of the product

Bacterial strain announced by the manufacturer

Number of probiotics Stainability with LIVE/DEAD (cfu/g) announced by BacLight Viability Kit the manufacturer Cells Background

Probiotic drinks GeWlus-1 Fruit juice with 5 diVerent fruitsa

L. rhamnosus GG

No information

GeWlus-2

Apple-grape (dark grapes) juice

L. rhamnosus GG

No information

Proviva-3

Blueberry soup with oat

L. plantarum 299v

No information

Proviva-4

Raspberry soup with oat

L. plantarum 299v

No information

Rela-5

L. reuteri Fruit juice with fruits and carrotb, supplemented with calcium and Wber L. reuteri Fruit juice with fruit and berriesc, supplemented with calcium and Wber

Rela-6

Pharmaceutical probiotics GeWlus Capsules with lyophilized cells Lactophilus Lyophilized cells a b c

No information No information

L. rhamnosus GG 1.9 £ 1010 L. casei variety rhamnosus 1 £ 108

Contains orange, grape (green grapes), peach, mangos, and passion fruit juices. Contains orange, grape (green grapes), mango, and carrot juices. Contains apple, grape (dark grapes), raspberry, and elderberry juices.

Brightly Xuorescing

Fluorescing particles (both red and green) Brightly Xuorescing, Contains no extra cells clumps Xuorescing particles Brightly Xuorescing Fluorescing particles (both red and green) Brightly Xuorescing Fluorescing particles (mainly red) Brightly Xuorescing Semi-brightly Xuorescing particles (mainly green) Brightly Xuorescing Fluorescing particles (mainly red)

Stainability with ChemChrome Cells

Background

Brightly Xuorescing, Weak Xuorescence but only a few cells Weak Xuorescence Brightly Xuorescing

Weak Xuorescence

Brightly Xuorescing

Weak Xuorescence

Brightly Xuorescing

weak Xuorescence

Brightly Xuorescing

Weak Xuorescence

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Table 2 The commercial probiotic products studied and their stainability as observed by microscopic analysis

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Intensities of green Xuorescence was used to estimate the amount of colony forming units in the samples by using standard curves previously determined for Lactobacillus sp. (Alakomi et al., 2005). 2.4. Plate count The culturable numbers of probiotic bacteria in pharmaceutical probiotic products and drinks were determined by the spread plate count method. The pretreated samples were diluted in peptone saline (PS, Maximum Recovery Diluent, Lab103, Lab M, Amersham, UK), and plated in duplicate on the media indicated for pure cultures. Plates were incubated for 2–3 days at 30 or 37 °C in anaerobic jars (H2/CO2/N2; 10:5:85, Anoxomat WS8000, Mart® Microbiology, Lichtenvoorde, Holland) and thereafter the colonies were enumerated. The culture experiments were performed in triplicate. The culturable numbers of probiotic bacteria were also determined for the original probiotic drinks and samples after homogenization but before Wltration to ensure that the used pretreatments did not have an eVect on the viability of the studied bacteria. The bacteria were identiWed with the RiboPrinter® Microbial Characterization System (Qualicon™, Wilmington, USA). 2.5. Statistical analysis Mean and standard deviation were calculated for each experiment. Student’s t test was used for the statistical analysis after log conversion of the results.

3. Results 3.1. Staining of probiotic pure cultures with Xuorescent stains and evaluation of the viability by Xuorescence microscopy and Xow cytometry The stainability of the selected probiotic strains was Wrst assessed with epiXuorescence microscopy. The detailed results are shown in Table 1. All the studied probiotic strains were stained well with the BacLight kit (viable:green, dead:red), whereas there were great diVerences between the stainability of the selected bacteria when esterase stains were used (Table 1). Altogether CAM, CFDA-AM, and SFDA did not stain the used bacteria properly. ChemChrome stained all and CFDA most of the studied bacteria (viable:green, dead: does not stain at all). BacLight, ChemChrome, and CFDA were used in further studies where the numbers of microscopically detected viable cells were compared to the numbers of viable cells obtained with Xow cytometry and colony forming units obtained by culture technique (Fig. 1(a)). The enumeration results are presented only for those

species – namely Lactobacillus rhamnosus, Lactobacillus plantarum, and Lactobacillus reuteri – which were found in the studied probiotic drinks and pharmaceutical products. For Xow cytometric analysis, L. rhamnosus and L. plantarum were stained with ChemChrome and L. reuteri with CFDA. The viable cells stained with esterase dyes were separated in Xow cytometry by size and green Xuorescence. The viable cell groups were easily distinguished, except in case of L. plantarum, where the cell groups were more widely spread. The numbers of viable cells, as assessed by epiXuorescence microscopy and Xow cytometry, were similar to the numbers obtained by culture technique (p > 0.05). When living and heat-treated cells were mixed 1:1 and stained with ChemChrome (L. rhamnosus and L. plantarum) or CFDA (L. reuteri) together with PI or with BacLight kit (Fig. 1(b)), the number of live and dead cells enumerated in epiXuorescence microscopy was the same (50% + 50% within one stain combination; p > 0.05). However, esterase stains together with PI on L. rhamnosus and L. reuteri staining gave slightly higher cell numbers than the BacLight kit (dead cells: p < 0.05). Furthermore, L. reuteri stained with CFDA + PI gave higher results than plate count (p < 0.05). When the living and dead cells in the mixtures were separated in Xow cytometry by green and red Xuorescence signals, respectively, or by the particle size and red or green Xuorescence, the groups in dot plots were not as clearly distinguishable as compared to the samples containing only living or heat-treated bacteria. Regardless, living and dead cells were enumerable in the samples of L. rhamnosus and L. reuteri and numbers of viable cells correlated with the results obtained with plate count (Fig. 1(b); p > 0.05). However, the number of dead cells detected Xow cytometrically from L. rhamnosus samples was smaller when compared to the number of Xow cytometrically detected viable cells (p D 0.01). The groups of living and heat-treated cells in L. plantarum Xow cytometric analysis were not distinguishable. However, an estimate of viable cells was obtained based on size and green Xuorescence intensities of the cells. When comparing the ChemChrome/CFDA + PI results obtained with microscopy to the results of the same stain combination obtained with Xow cytometry, all the corresponding enumeration results obtained with microscopy were signiWcantly higher (p < 0.05). When heat-treated cells alone were stained with either BacLight or ChemChrome, only dead cells were observed with the microscope (detection limit »106; data not shown). Heat-treated pure cultures stained with PI were separated in Xow cytometry by their size and red Xuorescent signal. The analysis of L. rhamnosus cells was easy due to strong Xuorescent signal, whereas the analysis of L. reuteri and L. plantarum cells was diYcult, since the cells did not create strong Xuorescent signals. Altogether, the number of dead cells detected with

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Fig. 1. (a) The numbers of viable cells in 20–24 h old (stationary phase) pure cultures as assessed by plate count method, epiXuorescence microscopy, and Xow cytometry. Lactobacillus rhamnosus and Lactobacillus plantarum were stained with ChemChrome and Lactobacillus reuteri with CFDA (5,6-carboxyXuorescein diacetate) for the Xow cytometric analysis, whereas all the strains were stained with LIVE/DEAD BacLight Viability kit, CFDA, and ChemChrome for microscopical analysis. Mean and standard deviation were calculated for each experiment (two repeated experiments with very similar results were performed for those Xow cytometric analysis where standard deviation is not visible). (b) The numbers of dead and living cells in mixtures (containing equal numbers of living and heat-treated pure cultures) as assessed by plate count method, epiXuorescence microscopy, and Xow cytometry. Lactobacillus rhamnosus and Lactobacillus plantarum were stained with ChemChrome and Lactobacillus reuteri with CFDA (5,6-carboxyXuorescein diacetate) for the Xow cytometric and microscopic analyses. The groups of living and heat-treated cells in L. plantarum Xow cytometric analysis were not distinguishable. However, an estimate of viable cells was obtained based on size and green Xuorescence of the cells ( denotes dead cells, which were not computable). Mean and standard deviation were calculated for each experiment (two repeated experiments with very similar results were performed for those Xow cytometric analysis where standard deviation is not visible). Statistically signiWcant diVerences (p < 0.05) when compared to the culture results are represented by the symbol (¤).

microscopy and Xow cytometry for every pure culture studied (data not shown) was similar (p > 0.05). 3.2. Staining of probiotic cultures in drinks with Xuorescent stains and evaluation of the viability by epiXuorescence microscopy, Xow cytometry, and Xuorometry BacLight and ChemChrome were used for all drinks studied. The studied drinks are listed in Table 2. The

numbers of viable bacteria obtained with plate count, epiXuorescence microscopy, Xow cytometry, and Xuorometry for GeWlus, Proviva, and Rela drinks are presented in Fig. 2(a), (b), and (c), respectively. When examined with epiXuorescence microscope, BacLight was suitable for staining the probiotic bacteria in all the samples and ChemChrome for all the other samples except the GeWlus-1 drink (Table 2). In all drinks BacLight also stained other material besides cells, but due to brighter Xuorescence of the cells, the cells were

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Fig. 2. (a) The number of viable probiotic bacteria detected in GeWlus probiotic drinks by plate count, epiXuorescent microscopy, Xow cytometry, and Xuorometry. ChemChrome and LIVE/DEAD BacLight Viability Kit were used for microscopical and Xow cytometric analysis and LIVE/DEAD BacLight Viability Kit for Xuorometric analysis. Mean and standard deviation were calculated for each experiment (three experiments in duplicate with very similar results were performed for those Xuorometric analyses where standard deviation is not visible). Statistically signiWcant diVerences (p < 0.05) when compared to the culture results are represented by the symbol (¤). denotes result below detection limit (105 cells/ml). (b) The number of viable probiotic bacteria detected in Proviva probiotic drinks by plate count, epiXuorescent microscopy, Xow cytometry, and Xuorometry. ChemChrome and LIVE/DEADBacLight Viability Kit were used for microscopical and Xow cytometric analysis and LIVE/DEAD BacLight Viability Kit for Xuorometric analysis. Mean and standard deviation were calculated for each experiment (three experiments in duplicate with very similar results were performed for those Xuorometric analyses where standard deviation is not visible). Statistically signiWcant diVerences (p < 0.05) when compared to the culture results are represented by the symbol (¤). (c) The number of viable probiotic bacteria detected in Rela probiotic drinks by plate count, epiXuorescent microscopy, Xow cytometry, and Xuorometry. ChemChrome and LIVE/DEAD BacLight Viability Kit were used for microscopical and Xow cytometric analysis and LIVE/DEAD BacLight Viability Kit for Xuorometric analysis. Mean and standard deviation were calculated for each experiment (three experiments in duplicate with very similar results were performed for those Xuorometric analyses where standard deviation is not visible). Statistically signiWcant diVerences (p < 0.05) when compared to the culture results are represented by the symbol (¤).

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Fig. 3. The number of cells in pharmaceutical probiotic products determined by plate count method and epiXuorescence microscopy. Those columns where standard deviation is not visible, experiments were not repeated, but two replicates had very similar results (except the number of living cells announced by the manufacturer, which was a single number).

enumerable. However, only viable cells (stained with SYTO9) in drinks were detectable in Xow cytometry, because PI also stained other particles in the drinks, thus preventing reliable distinction of cells from other particles in the drink. In both GeWlus drinks, the number of cells observed microscopically was higher than with culture (Fig. 2(a); p < 0.05 for GeWlus 2). The bacteria in GeWlus-1 drink did not stain properly with ChemChrome, even though the staining procedure was repeated Wve times with two diVerent drink-batches. The poor stainability with ChemChrome was also seen in Xow cytometric results. However, ChemChrome was able to stain the same bacteria in GeWlus-2 drink. In both Proviva drinks, the results obtained with culture were higher than the results obtained with Xuorescent techniques (Fig. 2(b); p < 0.05 for all experiments in Proviva-3 when compared to culture). However, all the results obtained with Xuorescent techniques were in good agreement, the only exception being that in Proviva-3 drink microscopic results of a given stain were slightly higher than the ones observed with Xow cytometry. In both Rela-drinks the number of cells observed with Xuorescent techniques were higher than with culture (p < 0.05 for all experiments as compared to culture in Rela-6 drink, in addition to microscopical and Xow cytometric (BacLight) experiments as compared to culture in Rela-5 drink). In both samples the number of viable cells observed with ChemChrome was higher in microscopic enumeration than in the Xow cytometric enumeration (p < 0.05). The bacteria from probiotic drinks were identiWed with the RiboPrinter? to be the same strains that the manufacturers have claimed on the product label (data not shown).

3.3. Staining of pharmaceutical probiotic products with Xuorescent stains and evaluating the viability by epiXuorescence microscopy The probiotic bacteria in two commercial pharmaceutical products were stained with Xuorescent dyes selected based on the results obtained from studies with corresponding pure cultures. Both ChemChrome and BacLight kit were suitable for pharmaceutical products tested. The numbers of viable cells obtained with epiXuorescence microscopy were somewhat higher than the numbers obtained by culture (Fig. 3), especially when Lactophilus was stained with ChemChrome (about 2.5 log higher). When PI and SYTO9 from LIVE/DEAD BacLight kit were used separately, the obtained cell numbers were higher than when the BacLight kit was used according to the manufacturer’s instructions (data not shown). The other particles in samples were stained brightly with all the other stains except ChemChrome. The bacteria from pharmaceutical products were identiWed with the RiboPrinter® to be the same strains that the manufacturers have announced (data not shown).

4. Discussion In this study, the stainability of several probiotic strains was initially studied with six diVerent viability stains in order to determine the best possible stain–buVer combinations for viability determination of commercial probiotic products. The results showed that pretreatment of the samples and the choice of the Xuorescent probe, staining buVer, incubation time, and temperature were of importance when staining diVerent lactic acid

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bacteria strains, as has previously been reported (Diaper, Tither, & Edwards, 1992). In this study, the nucleic acid stain LIVE/DEAD BacLight Viability Kit stained well all the studied pure cultures and was suitable for evaluating the viability of cells, as has been reported for pure cultures and probiotic products (Auty et al., 2001; Bunthof, van Schalkwijk, Meijer, Abee, & Hugenholtz, 2001b). Of the esterase stains tested in this study, ChemChrome stained all the pure cultures studied and CFDA all the other pure cultures, except L. paracasei. In the literature, CFDA has been reported to be a suitable viability stain for L. plantarum cells (Nebe-von Caron & Badley, 1995; Ueckert et al., 1995). BacLight and ChemChrome were selected in our study for additional experiments with commercial probiotic products (six drinks and two pharmaceutical products). Both stains were suitable for measuring the viability of most of the probiotic drinks, the only exception being ChemChrome with GeWlus-1 drink. In GeWlus-1 drink the number of viable cells in microscopic enumeration with ChemChrome was below the detection limit (105 cells/ml) although the staining procedure was repeated several times with diVerent juice batches. However, the same bacterium stained well in another drink-matrix, so it seems that the speciWc juice rather than the bacterium itself had an eVect on the stainability. Furthermore, the pH could not have aVected the stainability, since both drinks were diluted in PBS and the original pH of the drinks was similar (pH 3.8 for GeWlus1 and pH 3.9 for GeWlus-2). According to the literature, microbes lacking esterase activity completely have not been found (Mulard, 1995). However, there are a number of reasons why living cells do not stain with esterase substrates; the cells may lack the speciWc esterase activity or the esterase substrate may be impermeable to the cell wall. In addition, the level of retention is also dependent on the membrane potential since the dye is retained based on its charge (Nebe-von Caron & Badley, 1995). Moreover, many bacteria, including Lactococcus lactis and L. plantarum, have been shown to leak Xuorescein derivatives from intact cells. It has been suggested that this is due to energy-dependent extrusion mechanism (Glaasker et al., 1996). In both GeWlus and Rela drinks the cfu counts obtained with culture were mostly lower than the viable cell numbers obtained with Xuorescent techniques. Diaper and Edwards (1994) also found that higher number of viable bacteria was estimated by Xow cytometric enumeration of ChemChrome stained cells compared to cfu counts. This may be interpreted as evidence for the adoption of a viable but non-culturable state (Diaper & Edwards, 1994). Furthermore, in both our drink ‘families’ the sample, which contained dark grapes had a lower cfu count than the one that did not contain dark grapes when compared to overall results. It has been shown previously that phenolic compounds of fruits

have an eVect on the viability of, e.g. Leuconostoc oenos (Vivas, Lonvaud-Funel, & Glories, 1997), and dark grapes are known to contain several phenolic compounds. If this is the reason for our lower cfu counts, the phenolic compounds apparently did not aVect membrane integrity or esterase activity, but only reproducibility. In Proviva drinks the number of cfu/ml was higher than the numbers of viable cells obtained with Xuorescent techniques. However, the standard deviations of the experiments were low, giving statistical signiWcance to small diVerences (below 0.5 log), which are not normally considered as signiWcant due to accuracy limitations of the used methods. In Proviva-3 drink microscopic method with a particular stain gave slightly higher cell numbers than the same stain with Xow cytometric analysis. In this case, overlap between background Xuorescence and labeled cells cannot be discarded, and it may have lead to an underestimation of the true viable count by Xow cytometry (Porter, Diaper, Edwards, & Pickup, 1995). However, the diVerence here was only »0.3 log at the most. The large diVerences seen in GeWlus drinks Xow cytometric analyses were mainly due to the juice-matrices. In GeWlus-1 drink the other particles besides cells were also stained with BacLight red (dead) and green (viable), and some were about the same size as the bacteria, thus resulting in overestimation of the viable bacteria in Xow cytometry. In GeWlus-2 drink the cells were in clumps (even after pretreatment), which resulted in underestimation of cell viability. One clump was counted as one cell in Xow cytometry, whereas the cells in clumps were enumerable in microscopy. The Xuorescent staining methods (1–2 h) are considerably faster than the traditional cultivation methods (2–5 days) (Breeuwer and Abee, 2002). Moreover, the plate counts may underestimate the number of viable cells due to the viable but non-culturable state of bacteria, which is caused by cell stress or injury (Camper & McFeters, 1979). Altogether, our results of the microscopic experiments with two diVerent stains on a given probiotic drink were highly similar, the only exception being the GeWlus-1 drink. Flow cytometry seemed to be more prone to miscalculations, since it counts one cell clump as one cell, and other Xuorescing particles may lead to overestimated cell numbers. With Xow cytometry the choice of Xuorescent stain and the pretreatment of the sample are thus even more important than with microscopic evaluation. However, since Xow cytometry permits simultaneous measurements of multiple cellular parameters and can more readily be automated (Wallner, Erhart, & Amann, 1995), it will be more convenient for large sample numbers. ChemChrome was faster and easier for microscopic analysis than BacLight, since with ChemChrome other particles did not Xuoresce. According to the literature, the staining procedure by ChemChrome’s manufacturer (ChemFlow) has been

J. Maukonen et al. / Food Research International 39 (2006) 22–32

considered a promising tool for the detection of microbes from foodstuVs (Laplace-Builhé, Hahne, Hunger, Tirilly, & Drocourt, 1993). On the other hand, BacLight was more informative in our studies, since it enabled also the enumeration of dead cells and the overall view of the sample. The most convenient method, when all our studied stain–method combinations were included was Xuorometry with BacLight. In most of the samples the number of cells obtained with Xuorometry, when compared to culture results, was within 0.3 log, the only exception being Rela-6 drink (diVerence »0.7 log). The Xuorometry method is truly ‘a rapid method’, since after staining the results are obtained in less than 1 min. In addition, 96 samples can be detected simultaneously and the use of the device itself is straightforward and inexpensive. Both the membrane integrity (BacLight) and esterase (ChemChrome) Xuorescence probes were suitable for measuring the viability of cells in pharmaceutical probiotic products. However, in samples where there were other particles besides bacterial cells, staining with BacLight caused a Xuorescent background, which could prevent the use of Xow cytometry, unless cells cannot be diVerentiated from other particles by size. Fluorogenic esterase substrate ChemChrome was suitable for pharmaceutical samples even when there were other particles than bacterial cells present. Concerns about the quality of probiotic products have been widely expressed. Commercial probiotic products often contain bacterial species not indicated in the label, they contain unacceptably low numbers of the added probiotic, and/or an erroneous name is given for the probiotic bacterium (Canganella et al., 1997; Temmerman, Pot, Huys, & Swings, 2002). In the present study, the probiotic pharmaceutical products and drinks studied were of good quality. All the products contained the probiotic bacterium indicated in the label, and the levels of living bacteria were reasonable per dose (which according to Donnet-Hughes, Rochat, Serrant, Aeschlimann, & SchiVrin (1999) is about 109 cfu/day). Considering that Xuorescent stains and culture measure diVerent parameters in cells – the former either membrane integrity or enzymatic activity and latter the capability to multiply on a given growth medium – the results obtained were mostly in good agreement with each other rendering Xuorescent techniques applicable alternatives for rapid viability assessment of probiotic products. The drawback is that no bacterial identiWcation can be performed with the Xuorescent techniques applied. In addition, it should be bared in mind that the accuracy of culture technique with three replicates is usually within »0.5 log. Therefore, the signiWcance of the diVerence between most of the experiments (p < 0.05), although statistically observed, may not accurately reXect the real situation. In conclusion, Xuorescent techniques proved suitable for rapid (approx. 1 h) viability assessment of probiotics

31

in commercial pharmaceutical products and non-dairy drinks. Fluorescent techniques are not universal and successful application necessitates careful tailoring of the method (regarding especially pretreatments and stain– buVer pairs) for targeted probiotic bacterial species and product types.

Acknowledgments Research scientist Riikka Juvonen is greatly acknowledged for her assistance and knowledge of Xow cytometric analysis, Research scientist Arja Laitila for ideas and guidance, PhD Maija-Liisa Suihko for RiboPrinter® analysis, and MScTech Juha Toivari for his help concerning the performed statistical analysis. In addition, the technical assistance of Helena Hakuli, Erja Järvinen, and Niina Torttila is greatly acknowledged.

References Alakomi, H.-L., Mättö, J., Virkajärvi, I., & Saarela, M. (2005). Application of a microplate scale Xuorochrome staining assay for the assessment of viability of probiotic preparations. Journal of Microbiological Methods, 62, 25–35. Auty, M. A. E., Gardiner, G. E., McBrearty, S. J., O’Sullivan, E., Mulvihill, D. M., Collins, J. K., et al. (2001). Direct in situ viability assessment of bacteria in probiotic dairy products using viability staining in conjunction with confocal scanning laser microscopy. Applied and Environmental Microbiology, 67, 420–425. Björneholm, S., Eklöw, A., Saarela, M., & Mättö, J. (2002). Enumeration and identiWcation of Lactobacillus paracasei subsp. paracasei F19. Microbial Ecology in Health and Disease(Suppl. 3), 7–13. Breeuwer, P., & Abee, T. (2000). Assessment of viability of microorganisms employing Xuorescence techniques. International Journal of Food Microbiology, 55, 193–200. Bunthof, C. J., Bloemen, K., Breeuwer, P., Rombouts, F. M., & Abee, T. (2001a). Flow cytometric assessment of viability of lactic acid bacteria. Applied and Environmental Microbiology, 67, 2326– 2335. Bunthof, C. J., van Schalkwijk, S., Meijer, W., Abee, T., & Hugenholtz, J. (2001b). Fluorescent method for monitoring cheese starter permeabilization and lysis. Applied and Environmental Microbiology, 67, 4264–4271. Bunthof, C. J., & Abee, T. (2002). Development of a Xow cytometric method to analyze subpopulations of bacteria in probiotic products and dairy starters. Applied and Environmental Microbiology, 68, 2934–2942. Camper, A. K., & McFeters, G. A. (1979). Chlorine injury and enumeration of waterborne coliform bacteria. Applied and Environmental Microbiology, 37, 633–641. Canganella, F., Paganini, S., Ovidi, M., Vettraino, A. M., Bevilacqua, L., Massa, S., et al. (1997). A microbiological investigation on probiotic pharmaceutical products used for human health. Microbiological Research, 152, 171–179. Daly, C., & Davis, R. (1998). The biotechnology of lactic acid bacteria with emphasis on applications in food safety and human health. Agricultural and Food Science in Finland, 7, 251–265. Diaper, J. P., & Edwards, C. (1994). The use of Xuorogenic esters to detect viable bacteria by Xow cytometry. Journal of Applied Bacteriology, 77, 221–228.

32

J. Maukonen et al. / Food Research International 39 (2006) 22–32

Diaper, J. P., Tither, K., & Edwards, C. (1992). Rapid assessment of bacterial viability by Xow cytometry. Applied Microbiology and Biotechnology, 38, 268–272. Donnet-Hughes, A., Rochat, F., Serrant, P., Aeschlimann, J. M., & SchiVrin, E. J. (1999). Modulation of nonspeciWc mechanisms of defense by lactic acid bacteria: eVective dose. Journal of Dairy Science, 82, 863–869. Glaasker, E., Konings, W. N., & Poolman, B. (1996). The application of pH-sensitive Xuorescent dyes in lactic acid bacteria reveals distinct extrusion systems for unmodiWed and conjugated dyes. Molecular Membrane Biology, 13, 173–181. Gomes, A. M. P., & Malcatam, F. P. (1999). BiWdobacterium spp. and Lactobacillus acidophilus: biological, biochemical, technological and therapeutical properties relevant for use as probiotics. Trends in Food Science & Technology, 10, 139–157. Karwoski, M., Venelampi, O., Linko, P., & Mattila-Sandholm, T. (1995). A staining procedure for viability assessment of starter culture cells. Food Microbiology, 12, 21–29. Kopp-Hoolihan, L. (2001). Prophylactic and therapeutic uses of probiotics: a review. Journal of American Dietetic Association, 101, 229–241. Laplace-Builhé, B. C., Hahne, K., Hunger, W., Tirilly, Y., & Drocourt, J. L. (1993). Application of Xow cytometry to rapid microbial analysis in food and drink industries. Biology of the Cell, 78, 123–128. Mulard, Y. (1995). Flow cytometry: real time microbiology testing. Food Technology Europe, 2, 72–76. Nebe-von Caron, G., & Badley, R. A. (1995). Viability assessment of bacteria in mixed populations using Xow cytometry. Journal of Microscopy, 179, 55–66.

Porter, J., Diaper, J., Edwards, C., & Pickup, R. (1995). Direct measurement of natural planctonic bacterial community viability by Xow cytometry. Applied and Environmental Microbiology, 61, 2783– 2786. Saarela, M., Mogensen, G., Fonden, G., Mättö, J., & Mattila-Sandholm, T. (2000). Probiotic bacteria: safety, functional and technological properties. Journal of Biotechnology, 84, 197–215. Salminen, S., Bouley, C., Boutron-Ruault, M.-C., Cummings, J. H., Franck, A., Gibson, G. R., et al. (1998). Functional food science and gastrointestinal physiology and function. British Journal of Nutrition, 80, S147–S171. Temmerman, R., Pot, B., Huys, G., & Swings, J. (2002). IdentiWcation and antibiotic susceptibility of bacterial isolates from probiotic products. International Journal of Food Microbiology, 81, 1–9. Ueckert, J., Breeuwer, P., Abee, T., Stephens, P., Nebe-von Caron, G., & ter Steeg, P. F. (1995). Flow cytometry applications in physiological study and detection of food borne microorganisms. International Journal of Food Microbiology, 28, 317–326. Ueckert, J. E., Nebe-von Caron, G., Bos, A. P., & ter Steeg, P. F. (1997). Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell division and injury. Letters in Applied Microbiology, 25, 295–299. Wallner, G., Erhart, R., & Amann, R. (1995). Flow cytometric analysis of activated sludge with rRNA-targeted probes. Applied and Environmental Microbiology, 61, 1859–1866. Vivas, N., Lonvaud-Funel, A., & Glories, Y. (1997). EVect of phenolic acids and anthocyanins on growth, viability and malolactic activity of a lactic acid bacterium. Food Microbiology, 14, 291–300.