Selective enumeration of Lactobacillus acidophilus, Bifidobacterium spp., starter lactic acid bacteria and non-starter lactic acid bacteria from Cheddar cheese

Selective enumeration of Lactobacillus acidophilus, Bifidobacterium spp., starter lactic acid bacteria and non-starter lactic acid bacteria from Cheddar cheese

ARTICLE IN PRESS International Dairy Journal 16 (2006) 439–445 www.elsevier.com/locate/idairyj Selective enumeration of Lactobacillus acidophilus, B...

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ARTICLE IN PRESS

International Dairy Journal 16 (2006) 439–445 www.elsevier.com/locate/idairyj

Selective enumeration of Lactobacillus acidophilus, Bifidobacterium spp., starter lactic acid bacteria and non-starter lactic acid bacteria from Cheddar cheese Jyothsna Darukaradhya, Michael Phillips, Kasipathy Kailasapathy Probiotics Research Unit, Centre for Advanced Food Research, University of Western Sydney, SPDC 1797, NSW 1797, Australia Received 23 December 2004; accepted 23 June 2005

Abstract Twelve media were evaluated for selective and/or differential enumeration of Lactobacillus. acidophilus, Bifidobacterium spp., starter lactic acid bacteria (SLAB) and non-starter lactic acid bacteria (NSLAB) from Cheddar cheese. All media showed variation in counts and selectivity. Some reported selective media failed to inhibit SLAB and NSLAB. The media that were selective and/or differential and also gave better recovery were Reinforced Clostridium Agar with bromocresol green and clindamycin (RCABC), which was selective for L. acidophilus spp. and Reinforced Clostridium Agar with aniline blue and dicloxacillin (RCAAD), which was differential for Bifidobacterium spp. and SLAB. Reinforced Clostridium Agar with bromocresol green and vancomycin (RCABV) was found suitable for NSLAB. Apart from pure cultures, these media were also tested with commercial Cheddar cheese containing L. acidophilus. Additionally, Cheddar cheese containing L. acidophilus and B. lactis was manufactured and the selected media were used to monitor the initial survival of probiotic bacteria, SLAB and NSLAB present. r 2005 Elsevier Ltd. All rights reserved. Keywords: Probiotic cheddar cheese; Selective media; Differential media; Starter lactic acid bacteria (SLAB); Non-starter lactic acid bacteria (NSLAB)

1. Introduction Fermented milk and yoghurt are the most popular means of delivering probiotic bacteria in food, but there can be difficulties in maintaining the level of 106–107 cfu g1 (cfu, colony forming units) viable bacteria recommended by food organizations (LourensHattingh & Viljoen, 2001) over the shelf-life of products (Talwalkar & Kailasapathy, 2004). These problems, plus the increasing popularity of cheese, especially Cheddar cheese (Farkye, 2004), in Western countries has meant that this food could be considered as an alternate vehicle for delivering probiotic bacteria (Ross, Fitzgerald, Collins, & Stanton, 2002; Stanton et al., 1998). Corresponding author. Tel.: +61 2 45701653; fax: +61 2 45701954.

E-mail address: [email protected] (K. Kailasapathy). 0958-6946/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2005.06.009

Cheese contains a complex combination of microorganisms that changes with time; initially containing large numbers of starter lactic acid bacteria (SLAB) and then with maturation, an increasing number of nonstarter lactic acid bacteria (NSLAB) (Ross et al., 2002). To enumerate probiotic bacteria in such a mixed population, selective media should be employed that would allow the growth of the organisms of interest and inhibit other microorganisms encountered in a particular food product. Alternatively, differential media that will allow easy identification of probiotic colonies in the presence of other colonies can be used, provided the probiotic bacteria are in sufficient numbers. In the absence of standard selective or differential media, reliable enumeration of probiotic bacteria in the less complex microbial populations found in yoghurt has been reported to be difficult (Talwalkar & Kailasapathy,

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2004). Presently, there are no standard techniques reported to selectively enumerate probiotic bacteria in the presence of SLAB and NSLAB in Cheddar cheese. Therefore, there is a need to examine several enumeration media to select suitable media that will selectively or differentially enumerate probiotic bacteria in the presence of SLAB and NSLAB in Cheddar cheese. Although the manufacture of cheese containing Lactobacillus acidophilus and/or Bifidobacterium spp. has been reported (Dinakar & Mistry, 1994; Yilmaztekin, Ozer, & Atasoy, 2004), there are no reports of both L. acidophilus and Bifidobacterium spp., being incorporated into Cheddar cheese. Considering the increasing global popularity of Cheddar cheese, having both L. acidophilus and Bifidobacterium spp. in Cheddar cheese could be a favourable option for cheese manufacturers and consumers. Considering the regulations for minimum number of probiotic bacteria in fermented milk products, monitoring the survival rate of probiotic bacteria in probiotic Cheddar cheese is essential. The aim of this study was therefore to examine several enumeration media to select media that will reliably enumerate probiotic bacteria in the presence of SLAB and NSLAB in Cheddar cheese. After choosing the selective and/or differential enumeration media, the next aim was to test the media on a commercial probiotic Cheddar cheese and then finally evaluate experimental probiotic Cheddar cheese with both L. acidophilus and B. lactis.

2. Materials and methods 2.1. Cultures and chemicals The probiotic strains used in this study were L. acidophilus LAFTI L10, B. lactis LAFTI B94 (DSM Food Specialities, Moorebank, NSW, Australia), L. acidophilus CSCC 2400, L. acidophilus CSCC 2422, B. breve CSCC 1900, B. bifidum CSCC 1903, B. bifidum CSCC 1909, B. infantis CSCC 1912, B. longum CSCC 5188 (Australian Starter Culture Research Centre Ltd. Werribee, Victoria, Australia). The SLAB strains used were Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis (LL50C, DSM Food Specialities). The NSLAB strains used were L. casei subsp. casei CSCC 2603, L. rhamnosus CSCC 2625, L. paracasei subsp. paracasei CSCC 5437 (Australian Starter Culture Research Centre Ltd., Werribee, Victoria, Australia), and L. paracasei LAFTI L26 (DSM Food Specialities, Moorebank, NSW, Australia). All chemicals were obtained from Sigma–Aldrich (Castle Hill, NSW, Australia) and bacteriological media were obtained from Oxoid (Therbarton, South Australia, Australia) unless stated otherwise.

2.2. Selective and differential media The media tested for L. acidophilus strains were deMan, Rogosa, & Sharpe (MRS) Salicin (Dave & Shah, 1996) and Reinforced Clostridium Agar (RCA) with bromocresol green and clindamycin (RCABC). RCA was prepared following the manufacturer’s recipe but the pH of the agar was adjusted to 5.5. Bromocresol green stock solution was prepared at 0.2% (w/v), autoclaved at 121 1C for 15 min and added at the rate of 20 mL L1 to the autoclaved molten RCA agar base. Clindamycin (7-chlora-7-deoxylincomycin) stock solution, prepared by dissolving 5 mg in 100 mL distilled water, was filter-sterilized and added at a rate of 2 mL L1 to the autoclaved molten RCA agar base before pouring into plates. The media tested for Bifidobacterium spp. were MRSNPNL (neomycin, paromomycin, nalidixic acid and lithium chloride) agar (Wijsman, Johanna, & Groote, 1989), MRS Ox-bile (Bibiloni, Zavaglia, & Antoni, 2001), Wilkins Chalgren Mupirocin agar (WCM) (Rada & Koc, 2000) and Arroyo Martin and Cotton agar (AMC); Arroyo, Cotton, & Martin, 1995). In addition, a new medium based on RCA with the addition of aniline blue and dicloxacillin (RCAAD) was assessed (Sozzi, Brigidi, Mignot, & Matteuzzi, 1990). Aniline blue (0.3 g L1) was added to the RCA agar base, the pH was adjusted to 7.1, and the agar was then sterilized. Dicloxacillin stock solution (0.2% w/v; and filter-sterilized) was added at a rate of 1 mL L1 to the autoclaved molten agar before pouring into plates. RCA with bromocresol green and vancomycin (RCABV) was tested for enumerating NSLAB. The RCABV agar was developed using an RCA agar base. The pH of the base was adjusted to 5.5 prior to autoclaving and then bromocresol green stock 0.2% (w/v) (prepared as previously described) added at a rate of 20 mL L1. Vancomycin stock solution (2% w/v) was prepared with distilled water and filter-sterilized using a 0.45 mm membrane, and added at a rate of 0.5 mL L1of molten agar. M17 agar (Terzaghi & Sandine, 1975), LBS agar (Becton and Dickinson, USA) (Mc Brearty et al., 2001) and Rogosa agar (Rogosa, Mitchell, & Wiseman, 1951) were also tested for SLAB and NSLAB. All media were sterilized by autoclaving at 121 1C for 15 min before pouring into the plates. All the media were tested to find if they were selective, non-selective or differential for Bifidobacterium spp., L. acidophilus, SLAB and NSLAB. The various media were tested for both selectivity and reliability, based on the recovery of the individual pure cultures on them. The recovery of pure cultures was calculated by comparing cell counts of individual cultures obtained on the various selective/differential media with those obtained on the control MRS agar.

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The growth of the various cultures on the different media was tested as follows: 1 g of pure freeze-dried culture was suspended in 10 mL of sterile 0.1% (w/v) peptone water to form a suspension. The suspension was serially diluted in 0.1% (w/v) peptone water and 100 mL of the appropriate dilution was spread-plated on six replicates of each selective and differential media. The plates were incubated anaerobically in gas jars using the GasPak System (Oxoid, Therbarton, South Australia, Australia) for 48 h at 37 1C prior to observation.

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commenced while maintaining the temperature of the curd at 38 1C. When the acidity increased to 0.55–0.65%, the curd was milled and left for 10–15 min before salting. The salted curd was placed in cheesecloth and pressed overnight in a stainless steel mould. The next day, cheese was cut into segments and, vacuum packed and stored for maturation at 10 1C. This cheese will be referred to as UWS (University of Western Sydney) Cheddar cheese for the remainder of this paper. Six individual batches of UWS Cheddar cheese with L. acidophilus and B. lactis were manufactured for analysis.

2.3. Enzyme-based colorimetric assay 2.5. Microbiological analysis of Cheddar cheese To confirm that colonies obtained on differential media were that of Bifidobacterium spp., an enzyme-based colorimetric assay was also performed (Bibiloni, Perez, & Antoni, 2000). The assay employs the detection of fructose-6-phosphate phosphoketolase (F6PPK) activity as an indicator for the presence of Bifidobacterium spp. (Bibiloni et al., 2001). The enzyme assay was tested with all the strains listed in Section 2.1 with L. acidophilus, SLAB and NSLAB strains serving as controls. Briefly, the bacteria were disrupted using a French Press (Specronic, Garforth, UK) at 20,000 psi to release the enzyme. The samples were centrifuged for 10 min at 10,000  g at room temperature and the supernatants were then assayed according to the enzyme assay protocol (Bibiloni et al., 2001). Development of purple colour in the wells confirmed the presence of Bifiodobacterium spp. The assay was carried out in 6-microtitre plates for each sample. 2.4. Manufacture of probiotic Cheddar cheese with L. acidophilus and B. lactis Cheddar cheese was manufactured in a 10 L waterjacketed mini-vat (Armfield, Ringhood, England). Ten litres of raw milk obtained from the University of Western Sydney Dairy was pasteurized (72 1C, 15 s) and added to the vat and heated to 31 1C with constant agitation. After the addition of calcium chloride (2 mL 10 L1 of milk), annatto (colour) was added to the milk. The freeze-dried starter cultures (LL50C) were added at a rate of 0.4 manufacturers units per 300 L milk. Probiotic cultures were also added at the same time at 1 g 10 L1 for B. lactis (LAFTI B94) and 10 g 10 L1 for L. acidophilus (LAFTI L10). The milk was stirred for 10 min and left for 30 min before recommencing stirring. Calf rennet (2 mL of 10% v/v) (Home Cheese Making Supplies, Werribee, Australia) solution was then added. After stirring for 4 min, the stirrers were removed and the milk was left to coagulate for 35–40 min. The curd was cut with wire knives and left for 10 min without stirring for healing. The vat was then heated to 38 1C over a period of 55 min. The pH and acidity were monitored from this step. Once the acidity reached 0.16–0.17%, the whey was drained. Cheddaring

The media selected from the study described in Sections 2.2 and 2.3 were applied for enumerating probiotic bacteria from UWS and a commercial Cheddar cheese containing L. acidophilus (Nimbin Cheese, Nimbin, NSW, Australia). Ten grams of duplicate samples of each batch of cheese were homogenized in 100 mL of 2% (w/v) tri-sodium citrate solution for 10 min. Microbiological counts of the cheese suspension were obtained as described in Section 2.2. Plates containing 20–200 colonies were enumerated and the colony forming units per gram (cfu g1) of the cheese sample was calculated. 2.6. Statistical analysis A student’s t-test was employed to determine the significant differences (po0:05) between the recovery obtained on MRS and on the chosen selective and differential media. Analysis of variance (ANOVA) was used to determine significant (po0:05) variation between counts obtained on all 12 media.

3. Results and discussion A range of selective and/or differential media were compared with non-selective media for lactic acid bacteria, such as MRS to assess the growth of probiotic bacteria, SLAB and typical NSLAB. The counts of L. acidophilus, Bifidobacterium spp., SLAB and NSLAB are listed in Table 1. Generally, the media showed variation in counts and selectivity. Some media that have been reported as selective were not found to be so in this study, demonstrated by a failure to inhibit nontarget bacteria. In addition, some media did not give a good recovery of the target strains as compared with that obtained on the control medium (MRS). 3.1. Media for enumeration of L. acidophilus MRS-Salicin is reported to be selective for L. acidophilus (Dave & Shah, 1996). However in our

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Table 1 Recovery of probiotic, SLAB and NSLAB on various selective and differential media Cultures

Bifidobacteria 1900a 1903 1909 1912 5188 LAFTI B94

Media (log10 cfu mL1) MRSa

MRS Salicin

RCABC

MRS Ox-Bile

WCM

NPNL

AMC

RCAAD

RCABV

Rogosa

LBS

M17

8.90 8.87 9.11 8.80 8.81 9.04

Ng 7.45 7.81 7.08 7.58 8.00

Ng Ng Ng Ng Ng Ng

8.08 7.34 7.26 7.68 8.15 7.45

8.41 8.08 7.83 7.76 7.90 7.76

8.36 8.53 8.92 8.51 8.71 8.28

8.15 7.90 8.79 7.70 8.74 8.81

8.88 8.85 9.01 8.81 8.82 8.99

Ng Ng Ng Ng Ng Ng

7.89 7.63 8.32 7.91 7.79 Ng

7.79 7.70 8.23 7.81 8.63 Ng

Ng Ng Ng Ng Ng 8.00

8.45 8.56 8.85

Ng 6.67 Ng

7.69 Ng Ng

Ng 7.87 Ng

Ng Ng Ng

Ng Ng Ng

Ng Ng Ng

8.11 7.41 8.54

Ng 7.04 8.66

8.04 Ng 8.76

Ng Ng Ng Ng

8.79 Ng 7.59 Ng

Ng 5.73 6.98 Ng

8.67 Ng Ng Ng

Ng 7.70 7.18 8.36

Ng Ng Ng Ng

9.99 7.85 8.11 8.24

9.74 Ng 8.16 Ng

10.08 7.71 8.15 8.34

6.74 7.94 8.08 8.15

Ng

Ng

Ng

Ng

9.11

9.98

Ng

Ng

Ng

9.98

Lactobacillus acidophilus 2400b 8.53 7.95 2422 8.59 8.15 LAFTI L10 8.90 8.80 NSLAB LAFTI L26c 10.11 8.66 2625 7.93 Ng 2603 8.20 Ng 5437 8.40 Ng SLAB LL50Cd 10.04 8.00

MRS, deMan, Rogosa, & Sharpe; RCABC, reinforced clostridium agar with bromocresol green and clindamycin. a Abbreviations: SLAB, starter lactic acid bacteria; NSLAB, non-starter lactic acid bacteria; WCM, Wilkins Chalgren Mupirocin agar; RCAAD, reinforced clostridium agar with aniline blue and dicloxacillin; NPNL, neomycin, paromomycin, nalidixic acid; Ng, no growth on any plates. n ¼ 6 (counts are a mean of six readings). b 1900 – B. breve, 1903 – B. bifidum, 1909 – B. bifidum, 1912 - B. infantis, 5188 - B. longum, LAFTI B94 – B. lactis. c LAFTI L26 – L. paracasei, 2625 – L. rhamnosus, 2603 - L. casei subsp. casei, 5437 – L. paracasei subsp. paracasei. d LL50C – Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris.

study, MRS-Salicin allowed the growth of some Bifidobacteria spp., NSLAB (L. paracasei) and SLAB, though the latter was at much reduced levels (1% of MRS counts) possibly due to residual sugars in the media components. In comparison, RCABC was found to be a reliable selective medium with only the three L. acidophilus strains, LAFTI L10, CSCC 2400 and CSCC 2422 growing (Table 1). Apart from being selective, RCABC also gave good recovery for all the three L. acidophilus strains. The recoveries of CSCC 2400, CSCC 2422 and LAFTI L10 on RCABC were not significantly different (p40:05, n ¼ 6)) from that obtained on MRS. While the recovery of CSCC 2400, CSCC 2422 and LAFT L10 on RCABC were 8.45, 8.56 and 8.85log10 cfu mL1, respectively, their counts on MRS agar were 8.53, 8.59 and 8.90 log10 cfu mL1, respectively. Therefore, RCABC was used as a selective medium for enumeration of L. acidophilus in Cheddar cheese samples. The colonies of all the L. acidophilus strains on RCABC were of medium size (3 mm), rough, irregular, convex, granular and dark bottle green in colour (Fig. 1A).

(Arroyo et al., 1995) and RCAAD were tested for selectivity of Bifidobacterium spp. Differences were seen in the selectivity and recovery of bifidobacteria among these media. The media MRS Ox- Bile, WCM and NPNL were all found to allow growth of L. acidophilus strains (Table 1.) While AMC inhibited the growth of L. acidophilus, it did not inhibit the growth of SLAB on NSLAB. In comparison, RCAAD inhibited both L. acidophilus and NSLAB and although it allowed the growth of SLAB (LL50C), its colonies could be well-differentiated from those of bifidobacteria (Fig. 1B). The presence of aniline blue (dye) allowed easy distinguishing of bifidobacteria colonies from SLAB colonies on RCAAD. The colonies of Bifidobacterium spp. were of medium size (2.5 mm), smooth and shiny, convex and entire, round and light blue in colour. In contrast, the colonies of SLAB (LL50C) were of varying sizes, matty, rough, flat, irregular, granular and dark blue in colour (Fig. 1B). The recovery of Bifidobacterium spp. on RCAAD was very good as it was not significantly different (po0:05, n ¼ 6) from that obtained on MRS agar.

3.2. Media for enumeration of Bifidobacterium spp. and SLAB

3.3. Media for the enumeration of NSLAB

MRS Ox-Bile (Bibiloni et al., 2001), WCM (Rada & Koc, 2000), MRS-NPNL (Wijsman et al., 1989), AMC

Rogosa and LBS have been reported to be selective for NSLAB (Rogosa et al., 1951; Terzaghi & Sandine,

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1975). However in our study, Rogosa, LBS and M17 allowed the growth of some L. acidophilus, Bifidobacterium spp. and SLAB. In comparison, RCABV only allowed the growth of typical NSLAB such as LAFTI L26, CSCC 2625, CSCC 2603 and CSCC 5437 and inhibited the growth of all L. acidophilus, Bifidobacterium spp. and SLAB strains. Therefore, RCABV was found to be a reliable selective medium for NSLAB. In addition, no significant differences (p40:05) were observed between the recoveries of the NSLAB strains on RCABV as compared with those on MRS (Table 1). The colonies of NSLAB on RCABV were pinpoint, compact, dark green with a thin white or pale green margin, convex, smooth and shiny (Fig. 1C). Our study therefore demonstrated that the most appropriate media for selectively enumerating L. acidophilus and Bifidobacterium spp. in probiotic Cheddar cheese were RCABC and RCAAD, respectively. The findings from our study suggest that the numbers of SLAB in Cheddar cheese could be assessed by their differential counts on RCAAD, as long as there are comparable numbers of SLAB and Bifidobacteria. The NSLAB could be monitored using the selective RCABV medium described. 3.4. Confirmation of Bifidobacterium spp. with the enzyme based colorimetric assay Positive purple colouration, indicative of the presence of bifidobacteria was observed for all the Bifidobacterium spp. tested. In contrast, no purple colouration was observed with L. acidophilus, SLAB and NSALB strains. 3.5. Microbiological analysis of probiotic Cheddar cheese

Fig. 1. (A). Dilutions of probiotic cheese on Reinforced Clostridium Agar with bromocresol green and clindamycin (RCABC) showing L. acidophilus LAFTI L10 colonies. (B) Dilutions of probiotic cheese on Reinforced Clostridium Agar with aniline blue and dicloxacillin (RCAAD) showing LAFTI B94 as large, light-coloured colonies and LL50C as small, dark colonies. (C) Pure culture of L. paracasei LAFTI L26 growing on Reinforced Clostridium Agar with bromocresol green and vancomycin (RCABV).

In order to confirm the applicability of RCABC and RCAAD to cheese, the media were tested with commercial Cheddar cheese (Nimbin) containing the probiotic L. acidophilus. The medium RCABC demonstrated selective isolation of L. acidophilus from commercial Cheddar cheese with a single colony morphology that was indistinguishable from those seen with pure cultures of L. acidophilus. The counts of L. acidophilus were 1.1  105 cfu g1 (Table 2), which was well below the recommended levels of 106–107 cfu g1. The age of the cheese, however, was not known and may have contributed to the low numbers observed. SLAB and NSLAB present in the commercial cheese demonstrated growth on RCAAD and RCABV, respectively (Table 2). The colony morphologies of SLAB and NSLAB were similar to those of pure cultures (Fig. 1B and C). The low numbers of SLAB and the high numbers of NSLAB are consistent with the expected

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Table 2 Counts of L. acidophilus, SLABa and NSLABb in Nimbin probiotic Cheddar cheese Medium Target bacteria cfu g1 d

RCABCc L. acidophilus 1.1  10570.015

RCAAD b

SLAB 2.5  10370.02

RCABV NSLABc 4.5  10670.02

a

SLAB – starter lactic acid bacteria. NSLAB – non-starter lactic acid bacteria. c RCABC; clostridial agar with bromocresol green and clindamycin, RCAAD; clostridial agar with aniline blue and dicloxacillin, RCABV; clostridial agar with bromocresol green and vancomycin. d n ¼ 6 (counts are a mean of six readings). b

balance of these populations in a mature Cheddar cheese (Crow, Curry, & Hayes, 2001). Once the application of RCABC, RCAAD and RCABV in commercial cheese was successful, the media were further used to monitor the initial viability of L. acidophilus, B. lactis, SLAB and NSLAB in UWS Cheddar cheese. The presence of B. lactis in the UWS Cheddar cheese was also confirmed using the enzyme based colorimetric assay. The counts of L. acidophilus were found to be well above the recommended 107 cfu g1 in the first month (6  108 cfu g1) but decreased by a factor of more than 300 in the second month to 2  106 cfu g1. This loss of viability is consistent with the low numbers found in the commercial cheese. Therefore, maintaining long-term viability of this organism in Cheddar cheese becomes an important issue. In contrast, although the initial numbers of B. lactis in the first month were 107 cfu g1, there was only a four-fold decrease in the subsequent month. It will be necessary to monitor both organisms over a much longer period to determine whether they can survive the 6–12 month ripening period normally associated with mature Cheddar cheeses. The colonies of L. acidophilus and Bifidobacterium spp. obtained from the UWS Cheddar cheese were similar to the colonies obtained using pure cultures (Fig. 1A and B). The colonies of SLAB and NSLAB from the cheese samples were similar to those obtained from pure cultures (Fig. 1B and C). This study therefore illustrates that RCABC, RCAAD and RCABV can be used as reliable selective and differential media for the enumeration of L. acidophilus, Bifidobacterium spp., SLAB and NSLAB in Cheddar cheese.

4. Conclusion This study demonstrated that RCABC could be used as a reliable selective medium for L. acidophilus, whereas RCAAD can be used as a reliable differential medium for Bifidobacterium spp. and SLAB, whilst RCABV can be used as a reliable selective medium for NSLAB. Additionally, the successful manufacture of Cheddar

cheese containing L. acidophilus and B. lactis, as demonstrated in this study, lends support to the suggestion that Cheddar cheese has potential as a probiotic vehicle, though further work will be needed to determine the long term viability of these organisms. The enumeration media described above can be used to enumerate counts of L. acidophilus, Bifidobacterum spp., SLAB and NSLAB in such a probiotic Cheddar cheese. Alternatively, the poor survival of probiotic bacteria indicates that they need to be protected physically from being exposed to the deleterious factors in Cheddar cheese during the ripening period. Encapsulation technology for probiotic bacteria or protective cultures provides promising prospects for improved culture performance in terms of viability and protecting the bacteria during digestion in the gastrointestinal tract. This could be a solution to improve the viability of probiotic bacteria in Cheddar cheese and requires investigation.

Acknowledgements We would like to thank Lai Tran for providing assistance in this study. This research was supported by Australian Research Council (LINKAGE grant) and Dairy Farmers, Australia.

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