RAPID METHOD FOR SELECTIVE ENUMERATION OF BIFIDUS ESSENSIS IN ACTIVIA YOGURTS

Acta Alimentaria, Vol. 36 (2), pp. 173 183 (2007) DOI: 10.1556/AAlim.2007.0006 First published online 18 April 2007 RAPID METHOD FOR SELECTIVE ENUMERATION OF BIFIDUS ESSENSIS IN ACTIVIA YOGURTS O. REICHART a *, B. NAGY b, Á. JOZWIAK b and K. SZAKMÁR a a Department of Food Hygiene, Faculty of Veterinary Science, Szent István University, H-1078 Budapest, István u. 2. Hungary b Central Agriculture Office and Feed Safety Directorate Laboratory Coordination Department, H 1095 Budapest, Mester u. 81. Hungary (Received: 28 February 2006; accepted: 30 November 2006) The aim of this study was to develop simple, rapid and reliable methods for the selective determination of Bifidus essensis from ACTIVIA (Danone) yogurts. The methods are based on a modified MRS medium (B-broth), which does not contain inhibitory additives. The sugar source of the medium is maltose, which is metabolized by the bifidobacteria applied in the probiotic products, and not by the normal microflora of yogurt (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus). The redox potential of the medium was reduced with cysteine-hcl. Due to its reduced redox potential, the new bouillon is suitable for aerobic cultivation of bifidobacteria, while in agar form it needs anaerobic incubation. In bouillon form (MPN method) the incubation time is only 2 days compared to the 5-day requirement of the classical anaerobic plate counting methods. The B-broth in diluted form was successfully used in a RABIT (Don Whitley) equipment for selective impedimetric determination of bifidobacteria in Danone yogurts. The exact detection time of the Bifidobacterium counts in a good quality probiotic yogurt, containing bifidobacteria at a concentration of 10 7 to 10 8 CFU ml 1 is not more than 10 to 12 h (in contrast to the 5 days of classical anaerobic plate counting methods). Keywords: Bifidobacterium, selective medium, impedimetric method The consumption of fermented probiotic milk products has been continuously increasing. This effect is not only due to the expanding variety, but also to sensory aspects as well as to proposed therapeutic properties of these products (LOURENS- HATTING & VILJOEN, 2001; CASTEELE et al., 2006). Particularly in last decades, specific benefits have been attributed to Lactobacillus acidophilus and to different * To whom correspondence should be addressed. Fax: +36 1 207 3033; e-mail: microtest1@t-online.hu 0139-3006/$ 20.00 2007 Akadémiai Kiadó, Budapest

174 REICHART et al.: RAPID DETECTION OF BIFIDUS ESSENSIS species of Bifidobacterium. L. acidophilus normally dominates in the small intestine and bifidobacteria in the large intestine (HOLZAPFEL et al., 1998). In probiotic yogurtrelated milk products, these microorganisms are used in addition to or instead of the classical yogurt microflora. Dietary supplementation with Bifidobacterium spp. may encourage resistance against enteropathogenic bacteria such as Salmonella enteritidis, Shigella dysenteriae, Clostridium perfringens, etc. (GOMES et al., 1998). It may also result in helping to improve lactose utilization by lactose malabsorbers (KHALIL & MANSOUR, 1998), in absorption of calcium, amelioration of diarrhoea and constipation, in synthesis of B- complex vitamins (TANNOCK, 1999), and might also help in lowering the levels or reducing the activities of faecal bacterial enzymes, nitroreductase and azoreductase involved in procarcinogenic activation (REDDY et al., 1983). In order to provide beneficial effects on the human intestine, in the fermented milk products high initial viable cell number (>10 6 CFU ml 1 ) is necessary (SHAH, 2000; ROY, 2001; TALWALKAR & KAILASAPATHY, 2004). However, Bifidobacteria spp. are susceptible to acids and oxygen, so viable count in yogurts may easily decrease during storage (IWANA et al., 1993; VINDEROLA & REINHEIMER, 1999; DONKOR et al., 2006). The methods to detect the accurate viable counts of Bifidobacteria in fermented milk products are controversial; a few selective and differential agar media were developed to enumerate Bifidobacteria in fermented dairy products, which contain many lactic cultures for fermentation (WIJMAN et al., 1989; SOZZI et al., 1990; CHEVALIER et al., 1991; VINDEROLA & REINHEIMER, 1999; SHAH, 2000; ROY, 2001; TALWALKAR & KAILASAPATHY, 2004). These media contain bifidogenic sugars or antibiotics, which may cause lower viable numbers (IWANA et al., 1993; SIMPSON et al, 2004). Another problem is the long incubation time required of the anaerobic plate counting methods. The typical methods for selective determination of Bifidobacteria in dairy industry are MRS-dicloxacillin agar and GL-agar with LiCl, both of which take 5 days of anaerobic incubation at 37 C. The aim of the present study was to develop simple, rapid and reliable methods for selective determination of Bifidobacteria in commercial yogurt-related products containing Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Bifidobacterium spp. 1.1. Culture media 1. Materials and methods 1.1.1. MRS medium (Merck). The MRS-agar composition: Peptone 10 g l 1, meat extract 8 g l 1, yeast extract 4 g l 1, dextrose 20 g l 1, Tween 80 1 ml l 1, K 2 HPO 4 2 g l 1, Na-acetate 3H 2 O 5 g l 1, (NH 4 ) 3 citrate 2 g l 1, MgSO 4 7H 2 O 0.2 g l 1, MnSO 4 4H 2 O 0.05 g l 1, agar 10 g l 1.

REICHART et al.: RAPID DETECTION OF BIFIDUS ESSENSIS 175 1.1.2. MRS-dicloxacillin agar (SHAH, 1997). MRS base supplied with 5 ml l 1 of dicloxacillin (100 ml l 1 in demineralised water), 10 ml l 1 of LiCl (10% solution in demineralised water) and 5 ml l 1 of cysteine-hcl (10% solution in demineralised water). 1.1.3. GL-agar with LiCl (IWANA et al., 1993). Proteose peptone 10 g l 1, trypticase peptone 10 g l 1, NaCl 2 g l 1, L-cysteine-HCl 0.5 g l 1, K 2 HPO 4 1.5 g l 1, MgSO 4 0.2 g l 1, LiCl 0.4 g l 1, yeast extract 10 g l 1, Bacto agar 15 g l 1 were sterilized at 121 C for 15 min. After cooling, this mixture was supplied with membrane filtered galactose solution of 10% concentration. The end concentration of the galactose in the medium was 10 g l 1. 1.1.4. Modified MRS medium (B-medium). Peptone 10 g l 1, meat extract 10 g l 1, yeast extract 5 g l 1, Tween 80 1 g l 1, Na-citrate 2 g l 1, Na-acetate 5 g l 1, MgSO 4 7H 2 O 0.1 g l 1, MnCl 2 0.05 g l 1, Na 2 HPO 4 2 g l 1, maltose 10 g l 1, cysteine- HCl 0.5 g l 1. ph 6.5, sterilization at 0.5 bar for 18 min. The sugar source of the medium was maltose which is metabolized by the bifidobacteria (except for B. bifidum) and not by the normal microflora of yogurt. Applying galactose as sugar source, which can be utilized by most bifidobacteria including B. bifidum, in the preliminary experiments resulted in slower growth compared to that of maltose. The redox potential of the medium was reduced with cysteine-hcl. Due to its decreased redox potential, the B-bouillon is suitable for aerobic cultivation of bifidobacteria, while in agar form it needs anaerobic incubation. 1.2. Bacterial strain Bifidus essensis (declared name by the producer). The strain has been isolated from the commercial ACTIVIA yogurt (Danone) containing bifidobacteria. Isolation process was similar to the MPN determination; 1 ml of commercial ACTIVIA yogurt was diluted in decimal steps to 10 9 level. From each of the dilution series, 3 tubes containing B-broth were inoculated. After 2 days of incubation at 37 C, the last positive replicate was controlled microscopically and subcultured in B-broth. B- bouillon was used for the isolation and cultivation. Strains were maintained in B- bouillon at 4 6 C, and subcultured weekly. The identification of the isolated strain was based on its carbohydrate utilization (SCARDOVI, 1986). 1.3. Determination of viable cell concentration For the determination of viable cell concentration, plate counting and MPN methods were used. Decimal dilutions of the samples were prepared in a solution of reduced redox potential (peptone 1 g l 1, NaCl 8.5 g l 1, L-cysteine-HCl 0.3 g l 1 ). In case of plate counting, the Petri dishes were placed in anaerobic jars in H 2 /CO 2 atmosphere (OXOID, BR 38) and incubated at 37 C for 5 days. The inoculated tubes of the MPN method were incubated aerobically at 37 C for 3 days. The results (characteristic turbidity of the test tubes) practically developed after 2 days. The reason for the further incubation was the necessity of cell mass for microscopic investigation.

176 REICHART et al.: RAPID DETECTION OF BIFIDUS ESSENSIS 1.4. Comparative studies on commercial products In the experiments, the bifidobacterium concentrations of ACTIVIA yogurts were determined with several media and methods. ACTIVIA yogurts (Danone) containing Bifidus essensis 1. Natural yogurt; 2. Yogurt with muesli; 3. Yogurt with strawberry. Media and methods A) MRS-dicloxacillin agar, anaerobic incubation at 37 C for 4 days; B) GL agar with LiCl, anaerobic incubation at 37 C for 4 days; C) B-agar, anaerobic incubation at 37 C for 4 days; D) B-bouillon, aerobic incubation at 37 C for 2 days The selectivity of the media was confirmed microscopically. Determination of bifidobacteria in yogurts was performed in 3 replicates at every combination. 1.5. Impedimetric measurements The impedimetric measurements were performed in a RABIT (Don Whitley) equipment, containing 32 test cells. The test parameters were as follows: test duration 48 h, time resolution 15 and 12 min, temperature 37 C, detection criterion +10, +5 μs. The cells were incubated aerobically. Inoculum: 0.1 ml diluted microbial suspension (or yogurt) in 9 ml diluted B-broth. The components of the diluted B-broth for 100 ml: 30 ml B-broth without cysteine-hcl+70 ml distilled water+0.05 g cysteine-hcl. In the experiments, decimal dilution series of the original suspension of bifidobacteria (or yogurt) were used. The cell concentration in the test tubes ranged from 10 0 to 10 8 with steps of 1 order of magnitude. The concentration of bifidobacteria in the original suspension was determined by plate counting and MPN method using B-medium. The TTD values (time to detection, when the increase in conductivity exceeds the limit of +10 or +5 μs), were given by the RABIT equipment. The relationship between the TTD values and the logarithmic cell concentrations of the inoculum was determined by linear regression (STATGRAPHICS 5.1 program package). On the basis of the calibration curve from the TTD values, the cell concentration can be calculated without any classical viable cell determination method. 2.1. Selectivity of the B-medium 2. Results and discussion The selectivity of the new medium was tested by comparing the MPN determination results of ACTIVIA (Danone) yogurt to that of a bifidobacterium-free yogurt. As the selectivity of the B-medium is based on the sugar source utilised by bifidobacteria and not by the normal microflora of yogurt, in order to avoid false positive growth caused

REICHART et al.: RAPID DETECTION OF BIFIDUS ESSENSIS 177 by lactose being present in the yogurt, the members of the dilution series were evaluated from the 10 2 level. The concentration of lactic acid bacteria and bifidobacteria was determined by plate counting on MRS agar incubated anaerobically at 37 C for 5 days. The results were as follows. SOLE natural yogurt (Bifidobacterium-free) Total count on MRS agar: N=2.8 10 8 CFU ml 1, Bifidobacterium on B-agar: negative in B-bouillon: negative. ACTIVIA-yogurt Total count on MRS agar N=4.8 10 8 CFU ml 1, Bifidobacterium on B-agar N=5.2 10 7 CFU ml 1 in B-bouillon MPN=9.3 10 7 ml 1. Comparing the results of the two yogurts, the selectivity of the B-medium to Bifidobacterium is accepable. 2.2. Carbohydrate utilisation of the selected Bifidus essensis strain For the identification of the selected strain, MRS base media were used with several sugar sources. The results are summarized in Table 1. Table 1. Carbohydrate metabolism of the isolated Bifidus essensis strain Carbohydrate Bifidus essensis isolate Bifidobacterium breve (SCARDOVI, 1986) Arabinose Fructose + + Galactose + + Glucose + + Lactose + + Maltose + + Mannit weak d Melecitose weak d Melibiose Raffinose + + Salicin + + Sorbit + d Sucrose + + Trehalose + d Xylose d: 11 89% strains positive On the basis of the high similarity in carbohydrate utilisation, the isolated strain proved to be very similar to the species of Bifidobacterium breve.

178 REICHART et al.: RAPID DETECTION OF BIFIDUS ESSENSIS 2.3. Recovery of Bifidus essensis In the recovery experiments, the new selective medium (B-bouillon) was compared to the classical, non-selective MRS- and selective GL-bouillons in order to determine the recovery ratio and the required incubation time. The isolated Bifidus essensis strain was cultured in sterile milk at 37 C and stored in refrigerator at 4 to 6 C. After 3 days of storage, the cell concentration was determined by MPN method in 2 replicates, incubating the tubes at 37 C. The number of positive tubes was recorded after incubation of 21, 45 and 72 h. From the results shown in Fig. 1, it can be established that the recoveries in the GL- and B-bouillons after 3 days were equal to those of the MRS bouillon (which was the control), but there was a difference between the growth rates. The number of the turbid tubes containing the B- and MRS-broths after 48 h remained unchanged, while in the case of GL-bouillon (due to the inhibitory effect of LiCl) the required incubation was 72 h. Fig. 1. Recovery of Bifidus essensis in bouillons. M: MRS-bouillon; G: GL-bouillon; B: B-bouillon 2.4. Results of comparative studies on commercial products The results were evaluated with Analysis of Variance. The logarithmic means are shown in Fig. 2. The effect of the nutrient media on the recovered Bifidobacterium counts is shown in Fig. 3.

REICHART et al.: RAPID DETECTION OF BIFIDUS ESSENSIS 179 Fig. 2. Logarithmic means of the 3 replicate determinations of Bifidus essensis in ACTIVIA yogurts. 95% confidence interval = ±0.385. a: MRS-dicloxacillin agar; b: GL-agar; c: B-agar; d: B-bouillon Fig. 3. The effect of the nutrient media on the recovered Bifidobacterium counts. 95% confidence interval of means = ±0.223. A: MRS-dicloxacillin agar; B: GL-agar; C: B-agar; D: B-bouillon From the results demonstrated by Fig. 3, it can be established that significant difference exists between the results of the recovery of the media. The significantly highest value (lgn=7.89) was obtained in B-bouillon. The MRS-dicloxacillin agar produced the lowest result (lgn=6.15). The difference could be accounted for by the

180 REICHART et al.: RAPID DETECTION OF BIFIDUS ESSENSIS inhibitory effect of the antibiotics content of the latter. The results of GL-agar and B- agar (lgn=7.23 and 7.24, respectively) did not differ from each other. Their results were between those of MRS-dicloxacillin agar and B-bouillon, significantly differing from both. The standard deviation of the determination of bifidobacteria, calculated from the residual mean square of the variance analysis was SD lgn =0.324. 2.5. Impedimetric determination of Bifidus essensis from single culture The effect of the B-broth dilution on the time to detection (TTD) was tested with different dilutions of the single culture of Bifidobacterium essensis. Test parameters were: test duration 48 h, time resolution 15 min, temperature 37 C, and detection criterion 10 μs. Inoculum: 0.1 ml. The results are summarized in Table 2. Table 2. Effect of B-broth concentration on the detection time of Bifidus essensis Bifidobacterium B-broth=10% B-broth=20% B-broth=30% B-broth=40% lgn a TTD (h) 6.79 b 6.75 6.50 6.50 6.79 b 6.50 6.75 6.25 4.79 12.75 11.75 10.50 10.25 4.79 12.75 11.30 10.25 10.50 2.79 20.50 18.25 14.50 14.15 2.79 22.00 18.25 14.50 14.75 (μs) o 2400 2600 3900 4300 5400 6000 6800 7400 Δ(μS) 200 400 600 600 Δ(μS)/(μS) o 8% 9.8% 10.5% 8.5% a Logarithm of the Bifidobacterium number in the inoculum; b Uncertain evaluation; (μs) o : impedance of the broth at beginning (μs); Δ(μS): change in impedance (μs) On the basis of data in Table 2, the following conclusions can be drawn. The detection time decreased with increasing broth concentration in the range of 10 to 30%. At concentrations of 30 and 40%, the detection time was independent of the broth concentration. It only depended on the size of inoculation. The relative change in impedance as a result of the microbial growth had a maximum (10.5%) at the broth concentration of 30%. According to the conclusions summarized above, the applied substrate concentration for the impedimetric measurements of yogurts was 30% B- broth. The calibration curve constructed from the combined data obtained at 30 and 40% broth concentrations together with the results of the ACTIVIA yogurt measurements is demonstrated in Fig. 4.

REICHART et al.: RAPID DETECTION OF BIFIDUS ESSENSIS 181 2.6. Impedimetric determination of Bifidus essensis from yogurt The calibration curve was determined by using commercial ACTIVIA yogurts containing Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and the target microorganism, Bifidus essensis. The original concentration of bifidobacteria was tested with B-broth. As a negative control, SOLE natural yogurt (containing Streptococcus thermophilus and Lactobacillus bulgaricus in viable form) was used. Test parameters were: test duration 48 h, time resolution 12 min, temperature 37 C, detection criteria +10 and +5 μs. Inoculum: 0.1 ml. The microflora of natural yogurt did not grow in B-broth either with MPN method or in RABIT tubes. The Bifidobacterium concentration in ACTIVIA yogurt was tested in 3 replicates. The mean value was: N o =7.2 10 7 CFU ml 1, lgn o =7.86. As yogurt contains lactose, which is a good substrate for the normal microflora (Lactobacillus bulgaricus and Streptococcus thermophilus), the RABIT tests were started with diluted yoghurt samples. The dilution levels ranged from 10 2 to 10 8. Negative control was Bifidobacterium-free natural yogurt. The tests were performed in 4 replicates (two with detection criterion of 10 μs, two with 5 μs). SOLE yoghurt (negative control) did not cause change in impedance. The remaining lactose concentration in the tubes was too low for the growth of Streptococcus thermophilus and Lactobacillus bulgaricus. The results of RABIT tests are summarized in Table 3. Table 3. Impedimetric measurement of Bifidobacterium in ACTIVIA yoghurt Bifidobact. Δμ=10 μs Δμ=5 μs TTD Mean lg N a Detection time (h) (h) 5.86 8.5 8.5 8.5 8.3 8.45 4.86 10.1 10.7 10.7 11.3 10.70 3.86 12.9 13.1 12.5 12.5 12.75 2.86 15.1 16.3 14.3 16.1 15.45 1.86 19.1 17.1 17.5 19.1 18.20 0.86 20.3 20.7 20.7 19.5 20.30 1.14 b 28.9 >48 30.3 22.5 a Logarithm of the Bifidobacterium number in the inoculum; Δμ: detection criterion; b The cell number is too low, Poisson distribution, results are unreliable From the data, the following conclusions can be drawn. Decreasing the detection criterion from 10 to 5 μs resulted in no significant difference in the TTD values. The last dilution contained too low cell numbers for obtaining reliable results, therefore, this series was omitted from the evaluation. The relationship between the logarithm of the inoculum cell number and the TTD values, including the results of the single culture measurements, was evaluated with linear regression and is displayed in Fig. 4.

182 REICHART et al.: RAPID DETECTION OF BIFIDUS ESSENSIS Fig. 4. Combined impedimetric calibration curve of Bifidus essensis. TTD= 2.34 lgn + 22.00; R 2 =0.9766. x: Single culture; : ACTIVIA yoghurt As shown in Fig. 4, the combined results of the impedimetric measurements of the single culture of Bifidus essensis and ACTIVIA yogurts could be described with a common calibration curve. On the basis of this curve, the determination of Bifidobacterium levels in the products can be reduced below 20 h. 3. Conclusions The modified MRS broth (B-broth) containing maltose instead of glucose proved to be a useful tool for rapid and reliable determination of Bifidobacterium concentration in the ACTIVIA (Danone) yogurts. The normal microflora of yogurt (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) does not grow in the B- medium, so the selectivity is very good. As the B-broth does not contain inhibitory additives, the recovery of bifidobacteria is 100%. Comparing the selective Bifidobacterium media (MRS-dicloxacillin agar, GL-agar, B-agar and B-bouillon) in quality control of commercial Danone products, the B-bouillon resulted in significantly higher viable bifidobacterium number than the others. Applying the B-bouillon in aerobic incubation, the time requirement decreased to two days from the five-day anaerobic incubation of the commonly used bifidobacterium selective media. Using a RABIT equipment with diluted (30%) B-broth for the impedimetric determination of bifidobacteria in Danone yogurts, in the case of the prescribed bifidobacterium level (>10 6 CFU ml 1 ), the detection time is only 10 12 h in contrast to the 5 days of incubation required by the standardised methods.

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