Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Biotyping of cultivable lactic acid bacteria isolated from donkey milk D. Carminati, F. Tidona, M. E. Fornasari, L. Rossetti, A. Meucci and G. Giraffa Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie (CRA-FLC), Lodi, Italy
Significance and Impact of the Study: There is increased interest in using donkey’s milk as a source of human nutrition. The large amounts of antimicrobial components and defence factors present in donkey’s milk provide protection from microbial infections and distinguish donkey’s milk from the milks of other mammals. However, the microbiota in donkey’s milk has so far been poorly characterized, specifically with regard to the lactic acid bacteria (LAB). This study has identified cultivable, acidifying and thermoduric LAB that could be used to develop starter cultures. This is the first study to investigate the culturable LAB microbiota present in donkey’s milk.
Keywords dairy, diversity, lactic acid bacteria. Correspondence Domenico Carminati, CRA-FLC, Via A. Lombardo 11, 26900 Lodi, Italy. E-mail: [email protected] 2013/2357: received 26 November 2013, revised 24 March 2014 and accepted 11 April 2014 doi:10.1111/lam.12275
Abstract The diversity of lactic acid bacteria (LAB) species in donkey’s milk was analysed by culture-dependent microbial techniques. Dominant strains were isolated on agar media generally used for enumerating LAB. To enrich the number of acidifying LAB present, the milk samples were incubated at 37°C for 24 h (cultured milk samples, CM). One of the CM samples was heattreated at 63°C for 10 min before incubation at 37°C (heat-treated and cultured milk sample, TCM) to select thermophilic LAB. The microflora in these CM and TCM samples was then compared to that of the raw milk samples (RM). Among the 129 LAB isolates, 10 different species (four Enterococcus, five Streptococcus and one Pediococcus) were identified by molecular methods. Although the 10 LAB species were present in the RM samples, only three and two isolates were found in CM and TCM samples, respectively. Despite the selection protocol being set up to favour the isolation of all LAB isolates present in donkey milk, relatively few species and biotypes were isolated. No LAB isolates belonging to the most technologically important dairy starter species were detected. The possible factors related to the limited LAB diversity in donkey’s milk have been discussed below.
Introduction Recently, donkey’s milk has attracted increasing research interest due to its nutritional and functional properties. Its chemical composition is similar to that of human milk, and therefore, donkey’s milk can be used as an alternative food source for infants who cannot tolerate bovine milk (Monti et al. 2007). The enzymes in donkey’s milk possess some unique characteristics, such as bactericidal activity, which make this milk different from the milk of other mammals (Malacarne et al. 2002; Tidona et al. 2011). Specifically, lysozyme and lactoferrin are the most important naturally occurring inhibitory
components present at high concentration in donkey’s milk and play a role in the preservation of its hygienic quality (Coppola et al. 2002; Chiavari et al. 2005; Zhang et al. 2008; Saric et al. 2012). An antiviral activity of donkey’s milk protein fractions has also recently been demonstrated (Brumini et al. 2013). However, the direct relationship between the high levels of antimicrobial components and the reduced bacterial load in donkey’s milk is not well established and requires further studies (Conte et al. 2012). Despite the high content of lysozyme, the abundance of lactose seems to favour the growth and survival of adapted probiotic lactobacilli, which has been reported previously by some authors (Coppola et al.
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2002; Chiavari et al. 2005). Although the chemical composition and nutritional properties of donkey’s milk have been widely investigated, the microbiota in this milk is less well characterized (Quigley et al. 2013). A few studies have focused on the detection of pathogens in or the evaluation of the hygienic quality of donkey’s milk (Salimei et al. 2004; Naccari et al. 2009; Pilla et al. 2010; Conte et al. 2012; Sarno et al. 2012), but studies on donkey’s milk lactic acid bacteria (LAB) are even fewer. The LAB content as evaluated by plate counts on specific agar media was found to range between 95% identification accuracy (Rossetti and Giraffa 2005), a few isolates representing each RAPD cluster were selected for confirmatory identification by 500-bp 16S rRNA sequencing. This method provided reliable identification of the isolates (henceforth considered as individual strains in this manuscript) belonging to the genera Enterococcus, Streptococcus and Pediococcus. Consequently, all isolates clustering with the representative strains mentioned above were indirectly assigned to the corresponding species. Most of Enterococcus were isolated from M17 agar plates (37 strains, 56%), while 16 (24%) and 13 (20%) strains were isolated from LBD and MRS agar plates, respectively. Similarly, Streptococcus strains were mainly isolated from M17 (26 strains, 42%), while 20 (32%) and 16 (26%) were isolated from LBD and MRS, respectively. The single Pediococcus pentosaceus strain was recovered
biotypes belonging to the same species could be discriminated (Rossetti et al. 2008). Using the BIONUMERICS software, presumptive identification was obtained by Table 2 Source of bacterial isolates according to the different types of milk samples and culture media Culture media (No. isolates) Samples RM Bulk milk Martina Franca milk Romagnolo milk Total RM CM Bulk milk Martina Franca milk Romagnolo milk Total CM TCM Bulk milk Total
M17
LBD
MRS
Total
14 5 4 23
0 7 8 15
10 4 3 17
24 16 15 55
16 5 6 27
0 10 11 21
4 5 0 9
20 20 17 57
13 63
0 36
4 30
17 129
RM, raw milk; CM, cultured milk; TCM, heat-treated cultured milk.
Table 3 Diversity of cultivable lactic acid bacteria isolated from donkey’s milk samples. Biotyping of strains belonging to each species was performed by cluster analysis of the RAPD profiles Origin of isolates
Species (No. genotypes)* Enterococcus faecalis (4)
Total Ent. faecalis Enterococcus faecium/hirae/durans (5)
Total Ent. faecium Enterococcus gilvus (1) Enterococcus casseliflavus (1) Streptococcus macedonicus (2) Total Strep. macedonicus Streptococcus equinus/bovis (1) Streptococcus equi subsp. zooepidemicus (1) Streptococcus criceti (1) Streptococcus spp. (1) Pediococcus pentosaceus (1) Total
RAPD genotypes A B C D E F G H I L M N O P Q R S T 18
Sample
Isolation media
Total
RM
CM
TCM
M17
MRS
LDB
31 1 3 1 36 4 1 8 6 2 21 5 4 57 1 58 1 1 1 1 1 129
25 0 3 0 28 1 0 5 0 2 8 5 4 5 0 5 1 1 1 1 1 55
6 1 0 1 8 3 1 3 3 0 10 0 0 39 0 39 0 0 0 0 0 57
0 0 0 0 0 0 0 0 3 0 3 (177) 0 0 13 1 14 (823) 0 0 0 0 0 17 (100)
10 0 2 1 13 3 1 6 6 2 18 2 4 24 1 25 0 1 0 0 0 63
6 0 1 0 7 1 0 2 0 0 3 3 0 13 0 13 1 0 1 1 1 30
15 1 0 0 16 (444) 0 0 0 0 0 0 0 0 20 0 20 (556) 0 0 0 0 0 36 (100)
(509)†
(145) (91) (74)
(91) (18) (18) (18) (18) (18) (100)
(141)
(175)
(684)
(100)
(206)
(286) (32) (63)
(397) (16)
(100)
(234)
(100) (100)
(434) (33) (33) (33) (33) (100)
*Number of different RAPD-PCR genotypes recognized for each species. †Total number of isolates from each sample or isolation medium (percentage in parenthesis) of the different species identified. RM, raw milk; CM, cultured milk; TCM, heat-treated cultured milk.
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from MRS agar (Table 3). Although this observation is not surprising because the culture media used do not actually have selective properties, it is important to note that only coccus-shaped LAB were found. LAB populations that exist in donkey’s milk are quite unknown (Quigley et al. 2013), and the few data available refer to the isolation and characterization of single strains (Nazzaro et al. 2008; Ashokkumar et al. 2011; Murua et al. 2013) or to the isolation of some presumed but unidentified LAB, most of which are coccus-shaped bacteria (Tadesse 2010). Based on studies exploring the ability of LAB microflora to grow in donkey’s milk, Zhang et al. (2008) speculated that enterococci could be the major portion of the growing bacteria. The presence of only coccus-shaped species could be due to the high lysozyme content in donkey’s milk (average value of 132 012 g l1 in the samples studied, data not shown). It has been reported that LAB cocci are more resistant to lysozyme than lactobacilli, and among lactobacilli, the lysozyme sensitivity is species specific or strain specific (i.e. thermophilic species are more sensitive than hetero-fermentative mesophilic lactobacilli) (Neviani et al. 1991). Our observations are consistent with the limited studies available on the characterization of donkey’s milk where the only lactobacilli isolated and identified belong to the mesophilic species Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus salivarius and Lactobacillus plantarum (Nazzaro et al. 2008; Ashokkumar et al. 2011; Murua et al. 2013). Regarding cocci species, Enterococcus faecalis is known for its extraordinary lysozyme-resisting capacity, although variations in the lytic response of enterococcal cells have been observed (Hebert et al. 2007). In Gram-positive bacteria such as Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus, other mechanisms of lysozyme resistance have been reported (Le Jeune et al. 2010). Despite the selection protocol adopted to facilitate the isolation of all cultivable LAB present in the donkey milk, relatively few species and strains were isolated. Moreover, none of the isolated strains were found to belong to the most technologically important dairy starter LAB species. Streptococcus macedonicus (recently reclassified as Streptococcus gallolyticus subsp. macedonicus, Schlegel et al. 2003) was found to be the most frequently recovered species (58 isolates) followed by Ent. faecalis (36) and Enterococcus faecium/hirae/durans (21) (species not differentiated on the basis of sequence alignment of the first 500 bps of the 16S rRNA). These were the only species found in CM and TCM samples, while other enterococci (i.e. Enterococcus gilvus and Enterococcus casseliflavus) and streptococci were also isolated from the RM samples (Table 3). Among the coccus-shaped species, Strep. macedonicus is known to be a moderate acidifier in milk, 302
although strains isolated from Italian cheeses and natural milk cultures are also good acidifiers (Lombardi et al. 2004). The acidifying activity of many Strep. macedonicus strains isolated in this study was evaluated. Only few strains were able to coagulate cow’s milk in 65 h and most did so in 8–9 h (data not shown), thus confirming the moderate acidifying capability of this species. These characteristics make Strep. macedonicus unsuitable as a primary starter. However, strains that are known to be bacteriocin producers or possess intracellular peptidases have been proposed as adjunct cultures in cheese production (Anastasiou et al. 2007). Elucidation of the potential abilities of the strains recovered needs further investigation. A variety of species belonging to the genus Enterococcus is found in dairy products, but Ent. faecium and Ent. faecalis are the most important ones (Giraffa 2003). Enterococci are usually found to exhibit low milk acidifying capability, but strains of Ent. faecalis and Ent. faecium with high acidifying capability have been isolated from artisan Italian cheeses (Giraffa 2003). In addition, their recovery from the TCM samples confirmed the thermotolerance of these enterococcal species. They are present in natural milk cultures traditionally obtained by incubating a previously heat-treated raw milk to promote the selection of thermophilic and heat-resistant LAB (Giraffa et al. 1997; Giraffa 2003; Lombardi et al. 2004). Among the streptococci isolated from the RM samples, opportunistic pathogens for both humans and animals such as Streptococcus equi subsp. zooepidemicus and Streptococcus equinus/bovis (Timoney 2004) were found. The presence of undesirable Streptococcus species, such as Strep. equi, Streptococcus equisimilis and other potentially pathogenic bacteria in donkey’s milk, has been reported previously (Pilla et al. 2010; Salimei and Fantuz 2012). The relatively poor diversity of the LAB population observed in this study could be attributed to several factors: (i) the origin of the samples that were collected from a single farm, (ii) the selection process, which primarily favoured dominant cultivable LAB isolates, (iii) the higher lysozyme resistance of coccus-shaped LAB than lactobacilli. Other than these factors, the enrichment/selection procedures applied to the raw milk samples could have reduced the recovery of bacterial diversity significantly. Our data confirmed that although donkey’s milk has low bacterial content, the presence of some potentially pathogenic species underlines the importance of providing a suitable heat treatment before it is used for human consumption or for the production of fermented milk products. This study reports the first approach to studying the cultivable LAB community in donkey’s milk. A more representative sampling and the use of culture-independent
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DNA-based techniques will be needed to study the entire LAB microbiota also including noncultivable and nondominant populations present in donkey’s milk. Material and methods Sampling and treatment of milk Two donkey’s milk samples from single-breed animals (Martina Franca and Romagnolo breeds) and one bulk milk sample were kindly provided by the farm Azienda Montebaducco (Salvarano di Quattro Castella, Reggio Emilia, Italy). After milking, the samples were immediately cooled, transported to the laboratory under refrigerated conditions and stored at 4°C for 12–24 h before use. The samples were divided into two equal parts, one analysed immediately as raw milk (RM), and the other incubated at 37°C for 48 h (CM). Another aliquot of the bulk milk sample was subjected to heat treatment at 63°C for 10 min and then incubated at 37°C for 48 h (TCM). Taken together, a total of seven samples were analysed. Microbiological analysis Serial dilutions of RM, CM and TCM samples were prepared in Ringer solution (Oxoid, Basingstoke, UK). Dilutions were plated on MRS agar, with a final pH of 57, and M17 agar (Merck, Darmstadt, Germany) and on LBD agar (Lactic Bacteria Differential; HiMedia, Mumbai, India). To enumerate LAB, RM samples on M17 and LBD plates were incubated under aerobic conditions at 37°C for 48 h, and MRS plates were incubated under anaerobic condition at 37°C for 72 h. CM and TCM samples, plated and incubated on the different media as described above, were used only for strain isolation after application of enrichment and selective treatments. Colonies representative of all morphologies and sizes were randomly picked from the three different media and purified twice in the same isolation medium. Each single colony was then inoculated in the corresponding liquid medium and incubated for 24 h at 37°C. These cultures were stored at 80°C after addition of 15% sterile glycerol. Identification of bacteria The morphology of the bacteria in the purified cultures was examined by phase-contrast optical microscopy, and they were tested for catalase activity. The bacterial DNA was extracted from all the catalase-negative strains for further genotyping and identification using the Chelex method described in the MicroSeq protocol following the manufacturer’s instruction (Life Technologies, Monza, Italy).
Lactic acid bacteria in donkey’s milk
RAPD (randomly amplified polymorphic DNA) PCR All isolates were preliminarily identified by RAPD-PCR fingerprinting according to Rossetti and Giraffa (2005). The isolated bacterial DNA was used as a template in PCR fingerprinting using the M13 minisatellite core sequence as the primer (Huey and Hall 1989) with the sequence 50 -GAGGGTGGCGGTTCT-30 (Biotez ChemProgress, Milano, Italy). After amplification, PCR products were analysed by electrophoresis on 15% (w/v) agarose gels (Sigma-Aldrich, Milano, Italy) at 100 V for 2 h in 19 TAE buffer and visualized by GelRed (Biotium, Hayward, CA) staining. One-kb plus DNA Ladder (Invitrogen, Milan, Italy) was used as the DNA molecular weight marker. Gel images from RAPD analysis were captured using the Kodak Electrophoresis Documentation and Analysis System 290 (EDAS 290; Celbio, Milan, Italy) and processed with the pattern analysis software package BIONUMERICS (ver. 6.6; Applied Maths, Sint-Martens-Latem, Belgium). Similarities were calculated based on Pearson correlation coefficients. Dendrograms were deduced from the matrices of similarities by the unweighted pair group method using arithmetic average (UPGMA) clustering algorithm (Vauterin and Vauterin, 1992). The reproducibility of the RAPD-PCR fingerprinting patterns was evaluated by repeated running of DNA samples from duplicate amplifications of a control strain. RAPD-PCR fingerprints were compared with previously mapped genotypic libraries to obtain preliminary strain identification on the basis of RAPD-PCR profile similarity (Rossetti and Giraffa 2005). 16S rRNA gene sequence determination Molecular identification was performed by sequencing a 500-bp fragment of the 16S rRNA of a subset of strains including strains with a unique RAPD profile and representative strains among those that clustered together for each cluster. The 500-bp 16S rRNA fragment was amplified from the 50 end of the gene using the forward primer GCYTAACACATGCAAGTCGA (46 Escherichia coli numbering) and reverse primer GTATTACCGCGGCTGCTGG (536 E. coli numbering). PCR was carried out in a 50 ll volume containing 200 lmol l1 of each dNTP, 50 ll of 109 Taq reaction buffer, 05 lmol l1 each of the two primers (Biotez, Berlin, Germany), 15 mmol l1 of MgCl2, 125 U of AmpliTaq Gold DNA polymerase (Life Technologies) and 50 ng of genomic DNA extracted as described above. PCRs consisted of an initial denaturation step at 95°C for 10 min followed by 30 cycles of: 95°C for 30 s, 59°C for 30 s and 72°C for 45 s and a final elongation step of 72°C for 10 min. Prior to sequencing, the amplified products were purified using Exo-SAP-IT (GE Healthcare, Milan, Italy) according to the manufacturer’s recommendations. The PCR extension reactions
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were performed in 20 ml with 32 pmol l1 of each primer, 4 ml of sequencing mix (Life Technologies) and 7 ml of purified, amplified product. PCR extension consisted of 25 cycles at 96°C for 10 s, 50°C for 5 s and 60°C for 4 min, including rapid thermal ramps (1°C s1) between steps. Amplified products were purified using Big-Dye X-Terminator (Life Technologies) according to the supplier’s instructions. The DNA sequencing was performed using an ABI PRISM 310 automated DNA sequencer (Life Technologies) as previously described (Suarez et al. 2009). The sequence data were processed, and the alignments were performed using the Sequence Navigator software and CLUSTALW algorithm (Life Technologies). Strain identification was performed using the online BLAST software (www.ncbi.nlm.nih.gov/BLAST). Acknowledgements This work was funded by the Italian Ministry of Agriculture through the CANADAIR Project (D.M. MIPAF 27240/7303/2011). Conflicts of Interest The authors have no conflicts of interest to declare. References Anastasiou, R., Georgalaki, M., Manolopoulou, E., Kandarakis, I., De Vuyst, L. and Tsakalidou, E. (2007) The performance of Streptococcus macedonicus ACA-DC 198 as starter culture in Kasseri cheese production. Int Dairy J 17, 208–217. Ashokkumar, S., Krishna, R. S., Pavithra, V., Hemalatha, V. and Ingale, P. (2011) Production and antibacterial activity of bacteriocin by Lactobacillus paracasei isolated from donkey milk. Int J Curr Sci 1, 109–115. Brumini, D., Furlund, C. B., Comi, I., Devold, T. G., Marletta, D., Vegarud, G. E. and Jonassen, C. M. (2013) Antiviral activity of donkey milk protein fractions on echovirus type 5. Int Dairy J 28, 109–111. Carminati, D., Giraffa, G., Quiberoni, A., Binetti, A., Suarez, V. and Reinheimer, J. (2010) Advances and trends in starter cultures for dairy fermentations. In Biotechnology of Lactic Acid Bacteria: Novel Applications ed. Mozzi, F., Raya, R. R. and Vignolo, G. M. ch. 10, pp. 177–192. Ames, IA, USA: Wiley-Blackwell Publishing. ISBN: 978-0-8138-1583-1 Chiavari, C., Coloretti, F., Nanni, M., Sorrentino, E. and Grazia, L. (2005) Use of donkey’s milk for a fermented beverage with lactobacilli. Le Lait 85, 481–490. Conte, F., Foti, M., Malvisi, M., Giacopello, C. and Piccinini, R. (2012) Assessment of antibacterial activity of donkey
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milk lysozyme. Safety and hygiene issues. Large Anim Rev 18, 13–16. Coppola, R., Salimei, E., Succi, M., Sorrentino, E., Nanni, M., Ranieri, P., Belli Blanes, R. and Grazia, L. (2002) Behaviour of Lactobacillus rhamnosus strains in ass’ milk. Ann Microbiol 52, 55–60. Delgado, S., Rachid, C. T. C. C., Fernandez, E., Rychlik, T., Alegrıa, A., Peixoto, R. S. and Mayo, B. (2013) Diversity of thermophilic bacteria in raw, pasteurized and selectivelycultured milk, as assessed by culturing, PCR-DGGE and pyrosequencing. Food Microbiol 36, 103–111. Giraffa, G. (2003) Functionality of enterococci in dairy products. Int J Food Microbiol 88, 215–222. Giraffa, G., Carminati, D. and Neviani, E. (1997) Enterococci isolated from dairy products: a review of risks and potential technological use. J Food Prot 60, 732–738. Hebert, L., Courtin, P., Torelli, R., Sanguinetti, M., ChapotChartier, M.-P., Auffray, Y. and Benachour, A. (2007) Enterococcus faecalis constitutes an unusual bacterial model in lysozyme resistance. Infect Immun 75, 5390–5398. Huey, B. and Hall, J. (1989) Hypervariable DNA fingerprinting in E. coli minisatellite probe from bacteriophage M13. J Bacteriol 171, 2528–2532. Le Jeune, A., Torelli, R., Sanguinetti, M., Giard, J.-C., Hartke, A., Auffray, Y. and Benachour, A. (2010) The extracytoplasmic function Sigma factor SigV plays a key role in the original model of lysozyme resistance and virulence of Enterococcus faecalis. PLoS ONE 5, e9658. Lombardi, A., Gatti, M., Rizzotti, L., Torriani, S., Andrighetto, C. and Giraffa, G. (2004) Characterization of Streptococcus macedonicus strains isolated from artisanal Italian raw milk cheeses. Int Dairy J 14, 967–976. Malacarne, M., Martuzzi, F., Summer, A. and Mariani, P. (2002) Protein and fat composition of mare’s milk: some nutritional remarks with reference to human and cow’s milk. Int Dairy J 12, 869–877. Monti, G., Bertino, E., Muratore, M. C., Coscia, A., Cresi, F. and Silvestro, L. (2007) Efficacy of donkey’s milk in treating highly problematic cow’s milk allergic children: an in vivo and in vitro study. Pediatr Allergy Immunol 18, 258–264. Murua, A., Todorov, S. D., Vieira, A. D. S., Martinez, R. C. R., Cencic, A. and Franco, B. D. G. M. (2013) Isolation and identification of bacteriocinogenic strain of Lactobacillus plantarum with potential beneficial properties from donkey milk. J Appl Microbiol 114, 1793–1809. Naccari, F., Foti, M., Giacopello, C., Mariavitale, P., Passantino, A., Russo, C. and Conte, F. (2009) Methicillin resistant Staphylococcus sp isolated from donkey’s milk in Sicily: preliminary study. J Vet Pharmacol Ther 32, 150. Nazzaro, F., Anastasio, M., Fratianni, F. and Orlando, P. (2008) Isolation and identification of probiotic lactic acid bacteria from raw donkey milk. Int J Probiotics Prebiotics 3, 174.
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Neviani, E., Carminati, D., Veaux, M., Hermier, J. and Giraffa, G. (1991) Characterization of Lactobacillus helveticus strains resistant to lysozyme. Le Lait 71, 65–73. Pilla, R., Dapra, V., Zecconi, A. and Piccinini, R. (2010) Hygienic and health characteristics of donkey milk during a follow-up study. J Dairy Res 77, 392–397. Quigley, L., O’Sullivan, O., Stanton, C., Beresford, T. P., Ross, R. P., Fitzgerald, G. F. and Cotter, P. D. (2013) The complex microbiota of raw milk. FEMS Microbiol Rev 37, 664–698. Rossetti, L. and Giraffa, G. (2005) Rapid identification of dairy lactic acid bacteria by M13-generated, RAPD-PCR fingerprint databases. J Microbiol Methods 63, 135–144. Rossetti, L., Fornasari, M. E., Gatti, M., Lazzi, C., Neviani, E. and Giraffa, G. (2008) Grana Padano cheese whey starters: microbial composition and strain distribution. Int J Food Microbiol 127, 168–171. Salerno, M., Paterlini, F. and Martino, P. A. (2011) Microbiologia e attivita battericida del latte di asina. In Latte di asina: produzione, caratteristiche e gestione dell’azienda asinina, ed. Milonis, E. and Polidori, P. pp. 193–205. Brescia (Italy): Fondazione Iniziative Zooprofilattiche e Zootecniche. ISBN 978-88-904416-6-0. Salimei, E. and Fantuz, F. (2012) Equid milk for human consumption. Int Dairy J 24, 130–142. Salimei, E., Fantuz, F., Coppola, R., Chiofalo, B., Polidori, P. and Varisco, G. (2004) Composition and characteristics of ass’s milk. Anim Res 53, 67–78. Saric, L., Saric, B. M., Mandic, A. I., Torbica, A. M., Tomic, J. M., Cvetkovic, D. D. and Okanovic, D. G. (2012) Antibacterial properties of Domestic Balkan donkeys’ milk. Int Dairy J 25, 142–146. Sarno, E., Santoro, A., Di Palo, R. and Costanzo, N. (2012) Microbiological quality of raw donkey milk from Campania Region. Ital J Anim Sci 11, 266–269.
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Schlegel, L., Grimont, F., Ageron, E., Grimont, P. A. and Bouvet, A. (2003) Reappraisal of the taxonomy of the Streptococcus bovis/Streptococcus equinus complex and related species: description of Streptococcus gallolyticus subsp. gallolyticus subsp. nov., S. gallolyticus subsp. macedonicus subsp. nov. and S. gallolyticus subsp. pasteurianus subsp. nov. Int J Syst Evol Microbiol 53, 631– 645. Suarez, V., Zago, M., Giraffa, G., Reinheimer, J. and Quiberoni, A. (2009) Evidence for the presence of restriction/modification systems in Lactobacillus delbrueckii. J Dairy Res 76, 433–440. Tadesse, T. (2010) Investigation on nutritional and microbiological properties of Abyssinian donkey milk from Adea woreda. Thesis, Addis Ababa University, School of Graduate Studies, Faculty of Science, Food Science and Nutrition Program. Available at: http://etd.aau.edu.et/ dspace/bitstream/123456789/2103/1/ 106269243304001441109869231529309209426 Tidona, F., Sekse, C., Criscione, A., Jacobsen, M., Bordonaro, S., Marletta, D. and Vegarud, G. E. (2011) Antimicrobial effect of donkeys’ milk digested in vitro with human gastrointestinal enzymes. Int Dairy J 21, 158–165. Timoney, J. F. (2004) The pathogenic equine streptococci. Vet Res 35, 397–409. Vauterin, L. and Vauterin, P. (1992) Computer aided objective comparison of electrophoresis patterns for grouping and identification of microorganisms. Eur Microbiol 1, 37–41. Zhang, X.-Y., Zhao, L., Jiang, L., Dong, M.-L. and Ren, F.-Z. (2008) The antimicrobial activity of donkey milk and its microflora changes during storage. Food Control 19, 1191– 1195.
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ORIGINAL ARTICLE
Biotyping of cultivable lactic acid bacteria isolated from donkey milk D. Carminati, F. Tidona, M. E. Fornasari, L. Rossetti, A. Meucci and G. Giraffa Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie (CRA-FLC), Lodi, Italy
Significance and Impact of the Study: There is increased interest in using donkey’s milk as a source of human nutrition. The large amounts of antimicrobial components and defence factors present in donkey’s milk provide protection from microbial infections and distinguish donkey’s milk from the milks of other mammals. However, the microbiota in donkey’s milk has so far been poorly characterized, specifically with regard to the lactic acid bacteria (LAB). This study has identified cultivable, acidifying and thermoduric LAB that could be used to develop starter cultures. This is the first study to investigate the culturable LAB microbiota present in donkey’s milk.
Keywords dairy, diversity, lactic acid bacteria. Correspondence Domenico Carminati, CRA-FLC, Via A. Lombardo 11, 26900 Lodi, Italy. E-mail: [email protected] 2013/2357: received 26 November 2013, revised 24 March 2014 and accepted 11 April 2014 doi:10.1111/lam.12275
Abstract The diversity of lactic acid bacteria (LAB) species in donkey’s milk was analysed by culture-dependent microbial techniques. Dominant strains were isolated on agar media generally used for enumerating LAB. To enrich the number of acidifying LAB present, the milk samples were incubated at 37°C for 24 h (cultured milk samples, CM). One of the CM samples was heattreated at 63°C for 10 min before incubation at 37°C (heat-treated and cultured milk sample, TCM) to select thermophilic LAB. The microflora in these CM and TCM samples was then compared to that of the raw milk samples (RM). Among the 129 LAB isolates, 10 different species (four Enterococcus, five Streptococcus and one Pediococcus) were identified by molecular methods. Although the 10 LAB species were present in the RM samples, only three and two isolates were found in CM and TCM samples, respectively. Despite the selection protocol being set up to favour the isolation of all LAB isolates present in donkey milk, relatively few species and biotypes were isolated. No LAB isolates belonging to the most technologically important dairy starter species were detected. The possible factors related to the limited LAB diversity in donkey’s milk have been discussed below.
Introduction Recently, donkey’s milk has attracted increasing research interest due to its nutritional and functional properties. Its chemical composition is similar to that of human milk, and therefore, donkey’s milk can be used as an alternative food source for infants who cannot tolerate bovine milk (Monti et al. 2007). The enzymes in donkey’s milk possess some unique characteristics, such as bactericidal activity, which make this milk different from the milk of other mammals (Malacarne et al. 2002; Tidona et al. 2011). Specifically, lysozyme and lactoferrin are the most important naturally occurring inhibitory
components present at high concentration in donkey’s milk and play a role in the preservation of its hygienic quality (Coppola et al. 2002; Chiavari et al. 2005; Zhang et al. 2008; Saric et al. 2012). An antiviral activity of donkey’s milk protein fractions has also recently been demonstrated (Brumini et al. 2013). However, the direct relationship between the high levels of antimicrobial components and the reduced bacterial load in donkey’s milk is not well established and requires further studies (Conte et al. 2012). Despite the high content of lysozyme, the abundance of lactose seems to favour the growth and survival of adapted probiotic lactobacilli, which has been reported previously by some authors (Coppola et al.
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2002; Chiavari et al. 2005). Although the chemical composition and nutritional properties of donkey’s milk have been widely investigated, the microbiota in this milk is less well characterized (Quigley et al. 2013). A few studies have focused on the detection of pathogens in or the evaluation of the hygienic quality of donkey’s milk (Salimei et al. 2004; Naccari et al. 2009; Pilla et al. 2010; Conte et al. 2012; Sarno et al. 2012), but studies on donkey’s milk lactic acid bacteria (LAB) are even fewer. The LAB content as evaluated by plate counts on specific agar media was found to range between 95% identification accuracy (Rossetti and Giraffa 2005), a few isolates representing each RAPD cluster were selected for confirmatory identification by 500-bp 16S rRNA sequencing. This method provided reliable identification of the isolates (henceforth considered as individual strains in this manuscript) belonging to the genera Enterococcus, Streptococcus and Pediococcus. Consequently, all isolates clustering with the representative strains mentioned above were indirectly assigned to the corresponding species. Most of Enterococcus were isolated from M17 agar plates (37 strains, 56%), while 16 (24%) and 13 (20%) strains were isolated from LBD and MRS agar plates, respectively. Similarly, Streptococcus strains were mainly isolated from M17 (26 strains, 42%), while 20 (32%) and 16 (26%) were isolated from LBD and MRS, respectively. The single Pediococcus pentosaceus strain was recovered
biotypes belonging to the same species could be discriminated (Rossetti et al. 2008). Using the BIONUMERICS software, presumptive identification was obtained by Table 2 Source of bacterial isolates according to the different types of milk samples and culture media Culture media (No. isolates) Samples RM Bulk milk Martina Franca milk Romagnolo milk Total RM CM Bulk milk Martina Franca milk Romagnolo milk Total CM TCM Bulk milk Total
M17
LBD
MRS
Total
14 5 4 23
0 7 8 15
10 4 3 17
24 16 15 55
16 5 6 27
0 10 11 21
4 5 0 9
20 20 17 57
13 63
0 36
4 30
17 129
RM, raw milk; CM, cultured milk; TCM, heat-treated cultured milk.
Table 3 Diversity of cultivable lactic acid bacteria isolated from donkey’s milk samples. Biotyping of strains belonging to each species was performed by cluster analysis of the RAPD profiles Origin of isolates
Species (No. genotypes)* Enterococcus faecalis (4)
Total Ent. faecalis Enterococcus faecium/hirae/durans (5)
Total Ent. faecium Enterococcus gilvus (1) Enterococcus casseliflavus (1) Streptococcus macedonicus (2) Total Strep. macedonicus Streptococcus equinus/bovis (1) Streptococcus equi subsp. zooepidemicus (1) Streptococcus criceti (1) Streptococcus spp. (1) Pediococcus pentosaceus (1) Total
RAPD genotypes A B C D E F G H I L M N O P Q R S T 18
Sample
Isolation media
Total
RM
CM
TCM
M17
MRS
LDB
31 1 3 1 36 4 1 8 6 2 21 5 4 57 1 58 1 1 1 1 1 129
25 0 3 0 28 1 0 5 0 2 8 5 4 5 0 5 1 1 1 1 1 55
6 1 0 1 8 3 1 3 3 0 10 0 0 39 0 39 0 0 0 0 0 57
0 0 0 0 0 0 0 0 3 0 3 (177) 0 0 13 1 14 (823) 0 0 0 0 0 17 (100)
10 0 2 1 13 3 1 6 6 2 18 2 4 24 1 25 0 1 0 0 0 63
6 0 1 0 7 1 0 2 0 0 3 3 0 13 0 13 1 0 1 1 1 30
15 1 0 0 16 (444) 0 0 0 0 0 0 0 0 20 0 20 (556) 0 0 0 0 0 36 (100)
(509)†
(145) (91) (74)
(91) (18) (18) (18) (18) (18) (100)
(141)
(175)
(684)
(100)
(206)
(286) (32) (63)
(397) (16)
(100)
(234)
(100) (100)
(434) (33) (33) (33) (33) (100)
*Number of different RAPD-PCR genotypes recognized for each species. †Total number of isolates from each sample or isolation medium (percentage in parenthesis) of the different species identified. RM, raw milk; CM, cultured milk; TCM, heat-treated cultured milk.
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from MRS agar (Table 3). Although this observation is not surprising because the culture media used do not actually have selective properties, it is important to note that only coccus-shaped LAB were found. LAB populations that exist in donkey’s milk are quite unknown (Quigley et al. 2013), and the few data available refer to the isolation and characterization of single strains (Nazzaro et al. 2008; Ashokkumar et al. 2011; Murua et al. 2013) or to the isolation of some presumed but unidentified LAB, most of which are coccus-shaped bacteria (Tadesse 2010). Based on studies exploring the ability of LAB microflora to grow in donkey’s milk, Zhang et al. (2008) speculated that enterococci could be the major portion of the growing bacteria. The presence of only coccus-shaped species could be due to the high lysozyme content in donkey’s milk (average value of 132 012 g l1 in the samples studied, data not shown). It has been reported that LAB cocci are more resistant to lysozyme than lactobacilli, and among lactobacilli, the lysozyme sensitivity is species specific or strain specific (i.e. thermophilic species are more sensitive than hetero-fermentative mesophilic lactobacilli) (Neviani et al. 1991). Our observations are consistent with the limited studies available on the characterization of donkey’s milk where the only lactobacilli isolated and identified belong to the mesophilic species Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus salivarius and Lactobacillus plantarum (Nazzaro et al. 2008; Ashokkumar et al. 2011; Murua et al. 2013). Regarding cocci species, Enterococcus faecalis is known for its extraordinary lysozyme-resisting capacity, although variations in the lytic response of enterococcal cells have been observed (Hebert et al. 2007). In Gram-positive bacteria such as Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus, other mechanisms of lysozyme resistance have been reported (Le Jeune et al. 2010). Despite the selection protocol adopted to facilitate the isolation of all cultivable LAB present in the donkey milk, relatively few species and strains were isolated. Moreover, none of the isolated strains were found to belong to the most technologically important dairy starter LAB species. Streptococcus macedonicus (recently reclassified as Streptococcus gallolyticus subsp. macedonicus, Schlegel et al. 2003) was found to be the most frequently recovered species (58 isolates) followed by Ent. faecalis (36) and Enterococcus faecium/hirae/durans (21) (species not differentiated on the basis of sequence alignment of the first 500 bps of the 16S rRNA). These were the only species found in CM and TCM samples, while other enterococci (i.e. Enterococcus gilvus and Enterococcus casseliflavus) and streptococci were also isolated from the RM samples (Table 3). Among the coccus-shaped species, Strep. macedonicus is known to be a moderate acidifier in milk, 302
although strains isolated from Italian cheeses and natural milk cultures are also good acidifiers (Lombardi et al. 2004). The acidifying activity of many Strep. macedonicus strains isolated in this study was evaluated. Only few strains were able to coagulate cow’s milk in 65 h and most did so in 8–9 h (data not shown), thus confirming the moderate acidifying capability of this species. These characteristics make Strep. macedonicus unsuitable as a primary starter. However, strains that are known to be bacteriocin producers or possess intracellular peptidases have been proposed as adjunct cultures in cheese production (Anastasiou et al. 2007). Elucidation of the potential abilities of the strains recovered needs further investigation. A variety of species belonging to the genus Enterococcus is found in dairy products, but Ent. faecium and Ent. faecalis are the most important ones (Giraffa 2003). Enterococci are usually found to exhibit low milk acidifying capability, but strains of Ent. faecalis and Ent. faecium with high acidifying capability have been isolated from artisan Italian cheeses (Giraffa 2003). In addition, their recovery from the TCM samples confirmed the thermotolerance of these enterococcal species. They are present in natural milk cultures traditionally obtained by incubating a previously heat-treated raw milk to promote the selection of thermophilic and heat-resistant LAB (Giraffa et al. 1997; Giraffa 2003; Lombardi et al. 2004). Among the streptococci isolated from the RM samples, opportunistic pathogens for both humans and animals such as Streptococcus equi subsp. zooepidemicus and Streptococcus equinus/bovis (Timoney 2004) were found. The presence of undesirable Streptococcus species, such as Strep. equi, Streptococcus equisimilis and other potentially pathogenic bacteria in donkey’s milk, has been reported previously (Pilla et al. 2010; Salimei and Fantuz 2012). The relatively poor diversity of the LAB population observed in this study could be attributed to several factors: (i) the origin of the samples that were collected from a single farm, (ii) the selection process, which primarily favoured dominant cultivable LAB isolates, (iii) the higher lysozyme resistance of coccus-shaped LAB than lactobacilli. Other than these factors, the enrichment/selection procedures applied to the raw milk samples could have reduced the recovery of bacterial diversity significantly. Our data confirmed that although donkey’s milk has low bacterial content, the presence of some potentially pathogenic species underlines the importance of providing a suitable heat treatment before it is used for human consumption or for the production of fermented milk products. This study reports the first approach to studying the cultivable LAB community in donkey’s milk. A more representative sampling and the use of culture-independent
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DNA-based techniques will be needed to study the entire LAB microbiota also including noncultivable and nondominant populations present in donkey’s milk. Material and methods Sampling and treatment of milk Two donkey’s milk samples from single-breed animals (Martina Franca and Romagnolo breeds) and one bulk milk sample were kindly provided by the farm Azienda Montebaducco (Salvarano di Quattro Castella, Reggio Emilia, Italy). After milking, the samples were immediately cooled, transported to the laboratory under refrigerated conditions and stored at 4°C for 12–24 h before use. The samples were divided into two equal parts, one analysed immediately as raw milk (RM), and the other incubated at 37°C for 48 h (CM). Another aliquot of the bulk milk sample was subjected to heat treatment at 63°C for 10 min and then incubated at 37°C for 48 h (TCM). Taken together, a total of seven samples were analysed. Microbiological analysis Serial dilutions of RM, CM and TCM samples were prepared in Ringer solution (Oxoid, Basingstoke, UK). Dilutions were plated on MRS agar, with a final pH of 57, and M17 agar (Merck, Darmstadt, Germany) and on LBD agar (Lactic Bacteria Differential; HiMedia, Mumbai, India). To enumerate LAB, RM samples on M17 and LBD plates were incubated under aerobic conditions at 37°C for 48 h, and MRS plates were incubated under anaerobic condition at 37°C for 72 h. CM and TCM samples, plated and incubated on the different media as described above, were used only for strain isolation after application of enrichment and selective treatments. Colonies representative of all morphologies and sizes were randomly picked from the three different media and purified twice in the same isolation medium. Each single colony was then inoculated in the corresponding liquid medium and incubated for 24 h at 37°C. These cultures were stored at 80°C after addition of 15% sterile glycerol. Identification of bacteria The morphology of the bacteria in the purified cultures was examined by phase-contrast optical microscopy, and they were tested for catalase activity. The bacterial DNA was extracted from all the catalase-negative strains for further genotyping and identification using the Chelex method described in the MicroSeq protocol following the manufacturer’s instruction (Life Technologies, Monza, Italy).
Lactic acid bacteria in donkey’s milk
RAPD (randomly amplified polymorphic DNA) PCR All isolates were preliminarily identified by RAPD-PCR fingerprinting according to Rossetti and Giraffa (2005). The isolated bacterial DNA was used as a template in PCR fingerprinting using the M13 minisatellite core sequence as the primer (Huey and Hall 1989) with the sequence 50 -GAGGGTGGCGGTTCT-30 (Biotez ChemProgress, Milano, Italy). After amplification, PCR products were analysed by electrophoresis on 15% (w/v) agarose gels (Sigma-Aldrich, Milano, Italy) at 100 V for 2 h in 19 TAE buffer and visualized by GelRed (Biotium, Hayward, CA) staining. One-kb plus DNA Ladder (Invitrogen, Milan, Italy) was used as the DNA molecular weight marker. Gel images from RAPD analysis were captured using the Kodak Electrophoresis Documentation and Analysis System 290 (EDAS 290; Celbio, Milan, Italy) and processed with the pattern analysis software package BIONUMERICS (ver. 6.6; Applied Maths, Sint-Martens-Latem, Belgium). Similarities were calculated based on Pearson correlation coefficients. Dendrograms were deduced from the matrices of similarities by the unweighted pair group method using arithmetic average (UPGMA) clustering algorithm (Vauterin and Vauterin, 1992). The reproducibility of the RAPD-PCR fingerprinting patterns was evaluated by repeated running of DNA samples from duplicate amplifications of a control strain. RAPD-PCR fingerprints were compared with previously mapped genotypic libraries to obtain preliminary strain identification on the basis of RAPD-PCR profile similarity (Rossetti and Giraffa 2005). 16S rRNA gene sequence determination Molecular identification was performed by sequencing a 500-bp fragment of the 16S rRNA of a subset of strains including strains with a unique RAPD profile and representative strains among those that clustered together for each cluster. The 500-bp 16S rRNA fragment was amplified from the 50 end of the gene using the forward primer GCYTAACACATGCAAGTCGA (46 Escherichia coli numbering) and reverse primer GTATTACCGCGGCTGCTGG (536 E. coli numbering). PCR was carried out in a 50 ll volume containing 200 lmol l1 of each dNTP, 50 ll of 109 Taq reaction buffer, 05 lmol l1 each of the two primers (Biotez, Berlin, Germany), 15 mmol l1 of MgCl2, 125 U of AmpliTaq Gold DNA polymerase (Life Technologies) and 50 ng of genomic DNA extracted as described above. PCRs consisted of an initial denaturation step at 95°C for 10 min followed by 30 cycles of: 95°C for 30 s, 59°C for 30 s and 72°C for 45 s and a final elongation step of 72°C for 10 min. Prior to sequencing, the amplified products were purified using Exo-SAP-IT (GE Healthcare, Milan, Italy) according to the manufacturer’s recommendations. The PCR extension reactions
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were performed in 20 ml with 32 pmol l1 of each primer, 4 ml of sequencing mix (Life Technologies) and 7 ml of purified, amplified product. PCR extension consisted of 25 cycles at 96°C for 10 s, 50°C for 5 s and 60°C for 4 min, including rapid thermal ramps (1°C s1) between steps. Amplified products were purified using Big-Dye X-Terminator (Life Technologies) according to the supplier’s instructions. The DNA sequencing was performed using an ABI PRISM 310 automated DNA sequencer (Life Technologies) as previously described (Suarez et al. 2009). The sequence data were processed, and the alignments were performed using the Sequence Navigator software and CLUSTALW algorithm (Life Technologies). Strain identification was performed using the online BLAST software (www.ncbi.nlm.nih.gov/BLAST). Acknowledgements This work was funded by the Italian Ministry of Agriculture through the CANADAIR Project (D.M. MIPAF 27240/7303/2011). Conflicts of Interest The authors have no conflicts of interest to declare. References Anastasiou, R., Georgalaki, M., Manolopoulou, E., Kandarakis, I., De Vuyst, L. and Tsakalidou, E. (2007) The performance of Streptococcus macedonicus ACA-DC 198 as starter culture in Kasseri cheese production. Int Dairy J 17, 208–217. Ashokkumar, S., Krishna, R. S., Pavithra, V., Hemalatha, V. and Ingale, P. (2011) Production and antibacterial activity of bacteriocin by Lactobacillus paracasei isolated from donkey milk. Int J Curr Sci 1, 109–115. Brumini, D., Furlund, C. B., Comi, I., Devold, T. G., Marletta, D., Vegarud, G. E. and Jonassen, C. M. (2013) Antiviral activity of donkey milk protein fractions on echovirus type 5. Int Dairy J 28, 109–111. Carminati, D., Giraffa, G., Quiberoni, A., Binetti, A., Suarez, V. and Reinheimer, J. (2010) Advances and trends in starter cultures for dairy fermentations. In Biotechnology of Lactic Acid Bacteria: Novel Applications ed. Mozzi, F., Raya, R. R. and Vignolo, G. M. ch. 10, pp. 177–192. Ames, IA, USA: Wiley-Blackwell Publishing. ISBN: 978-0-8138-1583-1 Chiavari, C., Coloretti, F., Nanni, M., Sorrentino, E. and Grazia, L. (2005) Use of donkey’s milk for a fermented beverage with lactobacilli. Le Lait 85, 481–490. Conte, F., Foti, M., Malvisi, M., Giacopello, C. and Piccinini, R. (2012) Assessment of antibacterial activity of donkey
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milk lysozyme. Safety and hygiene issues. Large Anim Rev 18, 13–16. Coppola, R., Salimei, E., Succi, M., Sorrentino, E., Nanni, M., Ranieri, P., Belli Blanes, R. and Grazia, L. (2002) Behaviour of Lactobacillus rhamnosus strains in ass’ milk. Ann Microbiol 52, 55–60. Delgado, S., Rachid, C. T. C. C., Fernandez, E., Rychlik, T., Alegrıa, A., Peixoto, R. S. and Mayo, B. (2013) Diversity of thermophilic bacteria in raw, pasteurized and selectivelycultured milk, as assessed by culturing, PCR-DGGE and pyrosequencing. Food Microbiol 36, 103–111. Giraffa, G. (2003) Functionality of enterococci in dairy products. Int J Food Microbiol 88, 215–222. Giraffa, G., Carminati, D. and Neviani, E. (1997) Enterococci isolated from dairy products: a review of risks and potential technological use. J Food Prot 60, 732–738. Hebert, L., Courtin, P., Torelli, R., Sanguinetti, M., ChapotChartier, M.-P., Auffray, Y. and Benachour, A. (2007) Enterococcus faecalis constitutes an unusual bacterial model in lysozyme resistance. Infect Immun 75, 5390–5398. Huey, B. and Hall, J. (1989) Hypervariable DNA fingerprinting in E. coli minisatellite probe from bacteriophage M13. J Bacteriol 171, 2528–2532. Le Jeune, A., Torelli, R., Sanguinetti, M., Giard, J.-C., Hartke, A., Auffray, Y. and Benachour, A. (2010) The extracytoplasmic function Sigma factor SigV plays a key role in the original model of lysozyme resistance and virulence of Enterococcus faecalis. PLoS ONE 5, e9658. Lombardi, A., Gatti, M., Rizzotti, L., Torriani, S., Andrighetto, C. and Giraffa, G. (2004) Characterization of Streptococcus macedonicus strains isolated from artisanal Italian raw milk cheeses. Int Dairy J 14, 967–976. Malacarne, M., Martuzzi, F., Summer, A. and Mariani, P. (2002) Protein and fat composition of mare’s milk: some nutritional remarks with reference to human and cow’s milk. Int Dairy J 12, 869–877. Monti, G., Bertino, E., Muratore, M. C., Coscia, A., Cresi, F. and Silvestro, L. (2007) Efficacy of donkey’s milk in treating highly problematic cow’s milk allergic children: an in vivo and in vitro study. Pediatr Allergy Immunol 18, 258–264. Murua, A., Todorov, S. D., Vieira, A. D. S., Martinez, R. C. R., Cencic, A. and Franco, B. D. G. M. (2013) Isolation and identification of bacteriocinogenic strain of Lactobacillus plantarum with potential beneficial properties from donkey milk. J Appl Microbiol 114, 1793–1809. Naccari, F., Foti, M., Giacopello, C., Mariavitale, P., Passantino, A., Russo, C. and Conte, F. (2009) Methicillin resistant Staphylococcus sp isolated from donkey’s milk in Sicily: preliminary study. J Vet Pharmacol Ther 32, 150. Nazzaro, F., Anastasio, M., Fratianni, F. and Orlando, P. (2008) Isolation and identification of probiotic lactic acid bacteria from raw donkey milk. Int J Probiotics Prebiotics 3, 174.
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