Bile salt and acid tolerance of Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese

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1 FEMS Microbiology Letters 244 (2005) Bile salt and acid tolerance of Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese M. Succi a, P. Tremonte a, A. Reale a, E. Sorrentino a, L. Grazia b, S. Pacifico a, R. Coppola a, * a Dipartimento di Scienze e Tecnologie Agro-alimentari, Ambientali e Microbiologiche, Università degli Studi del Molise, Via De Sanctis, Campobasso, Italy b Dipartimento di Protezione e Valorizzazione Agro-alimentare, Università degli Studi di Bologna, Via Rosselli, Reggio Emilia, Italy Abstract Received 8 June 2004; received in revised form 9 September 2004; accepted 20 January 2005 First published online 1 February 2005 Edited by W. Kneifel This study aimed to compare phenotypic and genetic characteristics of Lactobacillus rhamnosus strains isolated at the end of the ripening of Parmigiano Reggiano cheese and to investigate an important prerequisite of probiotic interest, such as the capability to survive at low ph and in presence of bile salts. The use of API 50 CH, RAPD-PCR analysis and species-specific PCR allowed to ascertain the identity of 63 L. rhamnosus strains. Three L. rhamnosus strains isolated from Parmigiano Reggiano cheese, L. rhamnosus ATCC 7469 T and the commercial strain L. GG were assayed to estimate the resistance to various stress factors reproducing in vitro some conditions of the gastro-intestinal environment such as low ph and different amounts of bile salts and acids. The behaviour of almost all the tested strains isolated from Parmigiano Reggiano cheese resulted analogous to that showed by L. GG. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Lactobacillus rhamnosus; Cheese; Bile salts; Acidity; Parmigiano Reggiano 1. Introduction During recent years, numerous studies have been undertaken to obtain scientific evidences for the beneficial effects of fermented foods containing probiotic bacteria [1,2]. Traditionally, probiotics have been utilised in dairy products such as milk or yoghurt and it has been hypothesized that milk enhances probiotic efficacy by providing lactose as a substrate [3]. * Corresponding author. Tel.: ; fax address: coppola@unimol.it (R. Coppola). At the present, a large number of dairy products are present on the market and are being promoted with health claims based on several characteristics of selected strains of lactic acid bacteria, particularly belonging to the genera Lactobacillus and Bifidobacterium [4]. To provide health benefits, the suggested concentration for probiotic bacteria is 10 6 CFU/g of a product [5]. However, some studies have ascertained low viability of probiotics in the market preparations [6,7]. Viability and survival of probiotic bacteria are the most important parameters in order to provide therapeutic functions. A number of factors have been claimed to affect the viability of probiotic bacteria in dairy foods such as yoghurt and fermented milks, including low ph and refrigerated storage [4] /$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi: /j.femsle

2 130 M. Succi et al. / FEMS Microbiology Letters 244 (2005) Moreover, the resistance to human gastric transit constitutes an important selection criterion for probiotic bacteria [8,9]. Micro-organisms ingested with food begin their journey to the lower intestinal tract via the mouth and are exposed during their transit through the gastrointestinal tract to successive stress factors that influence their survival [10,11]. The time reported from entrance to release from the stomach is about 90 min [12], but further digestive processes have longer residence times. Cellular stress begins in the stomach, which has ph as low as 1.5 [13]. Bile secreted in the small intestine reduces the survival of bacteria by destroying their cell membranes, whose major components are lipids and fatty acids and these modifications may affect not only the cell permeability and viability, but also the interactions between the membrane and the environment [14,15]. Therefore before a probiotic can benefit human health it must fulfil several criteria such as the ability to tolerate acid and bile salts as well as to grow in the lower intestinal tract [16 19]. So, the first tool in the selection of a strain of probiotic interest is represented by in vitro methods aiming to ascertain the ability to survive passage through the upper gastro-intestinal tract and arrive alive at its site of action [20]. From this point of view, concrete advantages could be also obtained by using delivery vehicles with high ph values [21]. In this field, cheeses such as Parmigiano Reggiano could play the leading role, not only for their chemical and physicochemical properties but also for the particular technology of production, which is based on the use of cowõs raw milk as a source of fermenting micro-organisms, and for the presence of high amounts of viable lactic acid bacteria at the moment of consumption [22]. The aim of this study was to obtain a reliable identification of Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese by using API 50 CH test, RAPD-PCR and species-specific PCR and to ascertain the resistance of tested strains to bile salts and low ph. 2. Materials and methods 2.1. Isolation of lactobacilli from Parmigiano Reggiano cheese Microbiological analyses were performed on 20 g of Parmigiano Reggiano cheese at the end of the ripening (24 months) withdrawn from 18 different producers. Portions (20 g) from the centre of each cheese were first diluted in 180 ml of RingerÕs solution and than homogenized (1 min homogenisation, 1 min break, 1 min homogenisation) in a blender (Stomacher 400, Seward Medical, London SE1 1PP, UK). Subsequent Table 1 Phenotypic identification of 63 lactobacilli isolated from Parmigiano Reggiano cheese and of commercial strain Lactobacillus GG Amygdaline Arabinose D-Arabitol Arbutine Cellobiose Gentibiose Gluconate Glucose Glycerol Inositol Identification Growth at 45 C Growth at 15 C CO2 production 53 strains Lactobacillus rhamnosus 10 strains Lb. paracasei ssp. paracasei Lb. GG Lb. casei Lactose Maltose Mannitol Melezitose Rhamnose Ribose Saccharose Salicine Sorbitol L-Sorbose Trehalose D-Turanose Xylose Identification 53 strains Lactobacillus rhamnosus 10 strains Lb. Paracasei ssp. paracasei Lb. GG Lb. casei

3 M. Succi et al. / FEMS Microbiology Letters 244 (2005) serial dilutions were made in RingerÕs solution and plated in MRS agar (Oxoid) at 37 C for 48 h under anaerobiosis (Anaerogen, Oxoid). After incubation, five colonies with different morphologies were randomly picked from each colony count plate of MRS agar. All isolates that were Gram-positive, catalase-negative, very short to very long rods, occurring singly, in pairs or in chains, grown on MRS agar under anaerobic conditions, were taken as presumptive lactobacilli. Isolates were stored at 4 C in MRS (5 g/l agar) Phenotypic identification Gas production from glucose was observed in MRS broth with citrate omitted and containing inverted vials (Durham). Growth at 15 and 45 C was examined in MRS broth. Amygdaline, arabinose, D-arabitol, arbutine, cellobiose, gentibiose, gluconate, glucose, glycerol, inositol, lactose, maltose, mannitol, melezitose, rhamnose, ribose, saccharose, salicine, sorbitol, L-sorbose, trehalose, D-turanose and xylose fermentation was determined on the isolated strains and on the commercial strain Lactobacillus GG, isolated from a pharmaceutical preparation (Valio LTD, Helsinki, Finland), as described by Coppola et al. [23]. Results were confirmed by API 50 CH fermentation assays as described by Nigatu [24] RAPD-PCR analysis Sixty three strains, phenotypically identified as L. rhamnosus or as L. paracasei ssp. paracasei, and the commercial strain L. GG were genetically characterized by RAPD-PCR as described by Andrighetto et al. [25]. The following type and reference strains provided by the American Type Culture Collection (ATCC, Rockville, USA) were used: L. alimentarius ATCC T, L. casei ATCC 393 T, L. coryniformis subsp. coryniformis ATCC T, L. coryniformis subsp. torquens ATCC T, L. paracasei subsp. paracasei ATCC T, L. plantarum ATCC T, L. rhamnosus ATCC 7469 T, L. sakei ATCC T. PCR reaction was performed in a Mastercycler gradient (Eppendorf, Hamburg, Germany), using the following primers and amplification conditions: M13: 5 0 -GAGGGTGGCGGTTCT-3 0 [26]. Thirty five cycles of: 94 C for 1 min, 40 C for 20 s, ramp to 72 C at 0.5 Cs 1,72 C for 2 min. Table 2 Phenotypic and API 50 CH identification of 63 lactobacilli isolated from Parmigiano Reggiano cheese at the end of ripening and of the commercial strain Lactobacillus GG (some strains were grouped on the basis of similar API profiles) Strains Preliminary identification (Table 1) Species identification by API Quality of identification by API 9 strains L. rhamnosus L. rhamnosus Excellent AM185 L. rhamnosus L. rhamnosus Excellent VT1/1 L. rhamnosus L. rhamnosus Excellent 10 strains L. rhamnosus L. rhamnosus Very good 3 strains L. rhamnosus L. rhamnosus Very good 2 strains L. rhamnosus L. rhamnosus Very good 2 strains L. rhamnosus L. rhamnosus Very good AT181 L. rhamnosus L. rhamnosus Very good 5 strains L. rhamnosus L. rhamnosus Very good VM1/3 L. rhamnosus L. rhamnosus Very good 2 strains L. paracasei ssp. paracasei L. paracasei ssp. paracasei Very good AT185 L. rhamnosus L. rhamnosus Very good CLT2/1 L. rhamnosus L. rhamnosus Very good 2 strains L. paracasei ssp. paracasei L. paracasei ssp. paracasei Very good CLT2/2 L. rhamnosus L. rhamnosus Very good FLT3/1 L. rhamnosus L. rhamnosus Good MM1/1 L. paracasei ssp. paracasei L. paracasei ssp. paracasei Very good 2 strains L. paracasei ssp. paracasei L. paracasei ssp. paracasei Very good LLT2/3 L. rhamnosus L. rhamnosus Good LLT2/2 L. rhamnosus L. rhamnosus Good 2 strains L. paracasei ssp. paracasei L. paracasei ssp. paracasei Very good CT182 L. rhamnosus L. rhamnosus Good 8 strains L. rhamnosus L. paracasei ssp. paracasei Good MLT2/1 L. rhamnosus L. rhamnosus Good RBM728 L. rhamnosus L. paracasei ssp. paracasei Doubtful TT2/1 L. rhamnosus L. paracasei ssp. paracasei Doubtful Lb. GG L. casei L. paracasei ssp. paracasei Doubtful CTl/3 L. paracasei ssp. paracasei L. paracasei ssp. paracasei Doubtful

4 132 M. Succi et al. / FEMS Microbiology Letters 244 (2005) D8635: 5 0 -GAGCGGCCAAAGGGACGAGAC-3 0 [27]. An initial step of 94 C for 2 min, and then 35 cycles of: 94 C for 1 min, 42 C for 1 min, 72 C for 1.5 min, and a final step at 72 C for 10 min. Amplification products were separated by electrophoresis on 1.5% (w/v) agarose gel in 0.5 TBE buffer. RAPD-PCR profiles were obtained directly using the digital camera ImageMaster VDS (Amersham Pharmacia Biotech, Milano) and analysed with the pattern analysis software package, Gel Compar Version 4.1 (Applied Maths, Kortrijk, Belgium). Calculation of similarities in the profiles of bands was based on Pearson product-moment correlation coefficient. Dendrograms were obtained by means of the Unweighted Pair Group Method using Arithmetic Average (UPGMA) clustering algorithm [28] Lactobacillus rhamnosus species-specific PCR The specific PCR was performed on 17 strains previously identified by RAPD-PCR as L. rhamnosus and belonging to different clusters and on 8 strains of L. paracasei ssp. paracasei from the DISTAAM collection (University of Molise), isolated from different cheeses. The conditions for specificity testing were the same of those reported for RAPD-PCR. For comparative purpose the following type strains were used: L. casei ATCC 393 T, L. paracasei subsp. paracasei ATCC T, and L. rhamnosus ATCC 7469 T. PCR reaction was performed in a Mastercycler gradient (Eppendorf, Hamburg, Germany), using the following primers: Y2: 5 0 -CCCACTGCTGCCTCCCGTAGGAGT-3 0 [29]. rhamn: 5 0 -TGCATCTTGATTTAATTTTG-3 0 [29]. The amplification profile was an initial step of 94 C for 3 min, and then 30 cycles of: 94 C for 45 s, 55 C for 45 s, 72 C for 1 min. Amplification products were separated by electrophoresis on 1.5% (w/v) agarose gel in 0.5 TBE buffer Bile salt and acid tolerance Bile salt tolerance of 63 lactobacilli identified as L. rhamnosus was ascertained in MRS broth and in MRS broth containing 1.0%, 1.5% or 2.0% bile salts (Oxoid LP0055) incubated at 37 C for 3, 6, 24 and 48 h. Overnight MRS broth cultures (in the early stage of stationary phase) were utilized as an inoculum at 1.0% (v/v). Three strains (GT1/1, OT1/3 and VT1/1 selected for their high ability to lower ph in presence of bile salts), the type strain L. rhamnosus ATCC 7469, and the commercial strain L. GG were also tested for their resistance to different stress factors. Fig. 1. RAPD-PCR analysis performed with primers M13 and D8635 on 63 Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese and on the commercial strain Lactobacillus GG. For this purpose, overnight MRS broth cultures were 1.0% inoculated in 100 ml of MRS broth acidified to ph 2.0 or 3.0 with only 1.0 N hydrochloric acid or first to ph 4.5 with lactic acid and than to ph 2.0 or 3.0 with 1.0 N hydrochloric acid. The broths were incubated at 37 C and the survival at 2 and 4 h (strains GT 1/1, L. rhamnosus ATCC 7469 and L.GG) or at 1 4 h (strains

5 M. Succi et al. / FEMS Microbiology Letters 244 (2005) OT 1/3 and VT 1/1) was ascertained in MRS agar after incubation at 37 C for 48 h. After 2 or 4 h of incubation in the different acid solutions, ph value was increased to 7.00 with a sodium bicarbonate saturated solution (8 g of sodium bicarbonate in 100 ml of distilled water, sterilised at 121 C for 15 min) and 2.0% bile salts were added to reproduce some conditions of the small intestine environment. After 5 h of incubation at 37 C the viability of the strains was assayed in MRS agar incubated at 37 C for 7 days. 3. Results 3.1. Identification of Lactobacillus rhamnosus strains A total of 63 lactobacilli isolated from Parmigiano Reggiano cheese at the end of the ripening (24 months) and the commercial strain L. GG were preliminarily identified as described in Table 1. All the strains were present at counts higher than 10 5 CFU/g (data not shown). CO 2 production, ability to grow at 15 and 45 C and fermentation profiles allowed to ascribe 53 isolates to L. rhamnosus and 10 to L. paracasei ssp. paracasei while L. GG was identified as L. casei. The strains were identified by API 50 CH as L. paracasei ssp. paracasei (20 isolates) and L. rhamnosus (43 isolates) (Table 2). API system also invalidated the preliminary identification of the commercial strain L. GG, identified as member of L. paracasei ssp. paracasei but with a doubtful identification (Table 2). RAPD-PCR analysis (Fig. 1) and species-specific PCR (Fig. 2) clarified the real identity of tested strains, all of them resulted belonging to L. rhamnosus spp Bile salt and acid tolerance Sixty three L. rhamnosus strains isolated from Parmigiano Reggiano cheese were tested for their ability to lower ph in presence of different percentages of bile salts in an effort to isolate bile-tolerant strains (Fig. 3). Among the tested strains a narrow percentage demonstrated ability to lower ph at most 0.2 or 0.6 units after 3 or 6 h of incubation respectively, but only in Fig. 2. Lactobacillus rhamnosus species-specific PCR performed with primers rhamn and Y2 on 17 lactobacilli isolated from Parmigiano Reggiano cheese. Lane M, molecular weight marker; B, blank; T1, Lactobacillus rhamnosus type strain ATCC 7469; T2, Lactobacillus paracasei ssp. paracasei type strain ATCC 25302; T3, Lactobacillus casei type strain ATCC 393; Lanes 1, 3 13, 16, 20, 23 25, lactobacilli isolated from cheese, identified as Lactobacillus rhamnosus with RAPD-PCR analysis and belonging to different clusters; lanes 2, 14, 15, 17 19, 21, 22, Lactobacillus paracasei ssp. paracasei from the DISTAAM (University of Molise) collection and isolated from food.

6 134 M. Succi et al. / FEMS Microbiology Letters 244 (2005) Fig. 3. Lowering ph ability tested in MRS broth and in MRS broth added of different percentage of bile salts (BS) of 63 Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese. presence of the lower percentages of bile salts (1.0% and 1.5%). DpH resulted higher after 24 h of incubation with values included between 0 and 1 for almost all the tested strains while a higher value (i.e. 1.5) was gained by a slight percentage of strains (6%) incubated in presence of 1.0% bile salts. After 48 h of incubation at 37 C only 1% of strains incubated in MRS broth added of 1.0% and 1.5% bile salts gave a DpH value of 2 while for the majority of them DpH resulted between 0.5 and 1.5. Comparing inhibitory effects of bile salts on the tested strains, it was possible to select three L. rhamnosus strains with the best ability to lower ph in presence of 1.0%, 1.5% and 2.0% bile salts. The results showing the behaviour in presence of stress factors of the strains GT1/1, OT1/3 and VT1/1, of L. rhamnosus ATCC 7469 T and of the commercial strain L. GG are reported in Fig. 4. At low ph values the injury due to the acid environment was revealed by the lowering of 2 3 Log CFU/ml after 2 or 4 h of incubation at ph 3.00 with different acid solutions (only hydrochloric acid or hydrochloric acid plus lactic acid) while a lowering between 6 and 8 Log CFU/ml was evidenced at ph The longer permanence time of 4 h instead of 2 h in presence of acids determined a further lowering of 1 Log CFU/ml for L. GG and for the strain OT1/3 and of Log CFU/ml for the strain VT1/1. The increase of ph to 7.00 with a sodium bicarbonate saturated solution and the addition of 2% bile salts would not seem to represent an adverse environment for almost all the tested strains, showing in these conditions a good survival and, in some cases, high values. Only strains OT1/3 and VT1/1 evidenced a further lowering in microbial counts in this phase, more pronounced at ph At this ph value the effect of the composition in acids resulted more evident than that observed at ph 3.00, in particular for the strain VT1/1 and for the commercial strain L. GG. 4. Discussion Today market offers a large number of preparations claimed to contain probiotic micro-organisms that beneficially affect the host by improving the properties of the indigenous microflora. The popularity of these products aroused interest of both researchers and consumers and the dairy industry have taken into account the requirement to guarantee not only the safety of employed strains, but also that sufficient number of probiotic cells survive throughout the shelf-life of the product, generating desirable sensory qualities in the fermented foodstuff as well as producing beneficial effects on the health of consumers. From this point of view, Parmigiano Reggiano cheese represents an excellent candidate as a functional food, thanks to the presence of up to 10 7 CFU of viable lactic acid bacteria per gram of product at the end of the ripening, i.e. at the moment of consumption [22]. The need to establish an optimal strain identification represented the first step of this study. This goal was attained by the application of both phenotypic and biomolecular tests that allowed to ascertain the identity of

7 M. Succi et al. / FEMS Microbiology Letters 244 (2005) Fig. 4. Survival of Lactobacillus rhamnosus strains after incubation in MRS broth for 2 h (A) or 4 h (B) at ph 2.00 (d, only HCl; h, HCl + lactic acid) or 3.00 (s, only HCl; n, HCl + lactic acid), followed by incubation at ph 7.00 in presence of 2% bile salts. L. rhamnosus strains isolated from Parmigiano Reggiano cheese at 24 months of ripening. In particular, API 50 CH test, despite its claimed sensitivity due to the involvement of a large number of fermentable carbohydrates, failed in the identification and 20 L. rhamnosus strains (21 including the commercial strain L. GG), misidentified as L. paracasei ssp. paracasei.

8 136 M. Succi et al. / FEMS Microbiology Letters 244 (2005) The results of phenotypic tests obtained in this study are in agreement with those of other authors [30], revealing that the profile of L. GG differs significantly from that of other L. rhamnosus strains. Although protein electrophoretic patterns and selected gene probes suggest it to be a member of the species L. rhamnosus [31], its taxonomic position has not conclusively proven and it has been proposed that strain GG be given the new species designation L. zeae [32]. RAPD-PCR and species-specific PCR resulted rapid and reliable techniques for the identification of Lactobacillus spp., as described by other authors [24,33], evidencing a good intra-specific biodiversity between tested strains, that constitutes an important character, e.g. to monitor micro-organisms in the industry. L. rhamnosus does not belong to the group of primary starters used in the dairy industry, but this species includes some important probiotic strains, e.g. L. rhamnosus GG [34]. Bile salt tolerance is considered one of the essential properties required for lactic acid bacteria to survive in the small intestine [20,35]. L. rhamnosus strains isolated from Parmigiano Reggiano cheese and considered in this study evidenced a good survival in presence of 1.0%, 1.5% and 2.0% bile salts (Fig. 3). The lowering of ph resulted more evident after 48 h of incubation, showing that the tested strains have an optimal adaptability in unfavourable conditions. The stress caused by incubation in acidic conditions, assayed on three L. rhamnosus strains with the best ability to grow in presence of bile salts, on the type strain ATCC 7469 and on the commercial strain L. GG, was noticeable after the first hour of incubation while after 4 h of incubation a major injury was evident only in some cases and appeared related to the ph value of the incubation medium and to the acids adopted. In fact, the combination of organic (lactic) and inorganic (hydrochloric) acids had a more marked influence on the viability of tested strains but only at ph 2.00 while at ph 3.00 this combination did not seem to have influence. A similar study was performed by Chou and Weimer [36] on L. acidophilus strains from the American Type Culture Collection (Rockville, MD). After 90 min of incubation at ph 3.5 all tested strains showed an optimal survival but no colonies were found in MRS agar incubated for 96 h at the same ph. These results are in agreement with those obtained in the present study: in fact, L. rhamnosus strains from Parmigiano Reggiano cheese exhibited a good survival after 2 and 4 h of incubation at ph 3.00, but the experience reported by Chou and Weimer [36] let suppose that the long-term exposure to acidic environment causes a strong stress with a significant loss of survival. However this data could be explained in a different manner, since in our study the counting of acid and bile salt stressed strains was possible only after 168 h of incubation in MRS agar, owing to a slow recovery from stress conditions. The ability of L. rhamnosus strains isolated from Parmigiano Reggiano cheese to survive stress conditions such as the presence of high amounts of bile salts and low ph was in vitro tested utilising a common substrate such as MRS. 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