Brazilian Journal of Microbiology (2011) 42: 158-171 ISSN 1517-8382 CHARACTERIZATION OF LACTOBACILLUS FROM ALGERIAN GOAT’S MILK BASED ON PHENOTYPIC, 16S rDNA SEQUENCING AND THEIR TECHNOLOGICAL PROPERTIES Ahmed Marroki1 *; Manuel Zúñiga2; Mabrouk Kihal3; Gaspar Pérez- Martínez2 1 Laboratory of Applied Microbiology, Department of Biotechnology (IGMO), University of Oran Es-Sénia, Oran, 31100 , Algeria; 2Department of Biotechnology, Instituto de Agroquímica y Tecnología de los Alimentos (C.S.I.C), polígono de la Coma s/n, Burjassot (Valencia), Spain; 3Laboratory of Applied Microbiology, Department of Biology, University of Oran Es-Sénia, Oran, Algeria. Submitted: February 18, 2010; Returned to authors for corrections: February 24, 2010; Approved: August 01, 2010. ABSTRACT Nineteen strains of Lactobacillus isolated from goat’s milk from farms in north-west of Algeria were characterized. Isolates were identified by phenotypic, physiological and genotypic methods and some of their important technological properties were studied. Phenotypic characterization was carried out by studying physiological, morphological characteristics and carbohydrate fermentation patterns using API 50 CHL system. Isolates were also characterized by partial 16S rDNA sequencing. Results obtained with phenotypic methods were correlated with the genotypic characterization and 13 isolates were identified as L. plantarum, two isolates as L. rhamnosus and one isolate as L. fermentum. Three isolates identified as L. plantarum by phenotypic characterization were found to be L. pentosus by the genotypic method. A large diversity in technological properties (acid production in skim milk, exopolysaccharide production, aminopeptidase activity, antibacterial activity and antibiotic susceptibility) was observed. Based on these results, two strains of L. plantarum (LbMS16 and LbMS21) and one strain of L. rhamnosus (LbMF25) have been tentatively selected for use as starter cultures in the manufacture of artisanal fermented dairy products in Algeria. Key words: Lactic acid bacteria; Lactobacillus; identification; goat’s milk; technological properties; Algeria. INTRODUCTION the considerable variations in biochemical attributes between strains currently considered to belong to the same species. In The identification of lactobacilli has been based mainly on fact, some species are not readily distinguishable in terms of fermentation of carbohydrates, morphology, and Gram staining phenotypic characteristics (12). In recent years, the taxonomy and these methods are still being used. However, the has changed considerably with the increasing knowledge of the characterization of some Lactobacillus to species level by genomic structure and phylogenetic relationships between biochemical methods alone is not reliable (27, 40), because of Lactobacillus spp. (27, 43, 47). This novel taxonomy based on *Corresponding Author. Mailing address: Laboratory of Applied Microbiology, Department of Biotechnology (IGMO), University of Oran Es-Sénia, Oran, 31100, Algeria.; Tel: +213 772 142 345 Fax: + 213 41 58 19 41.; E-mail address: [email protected] 158 Marroki, A. et al. Characterization of Lactobacillus and their technological properties DNA analysis offers a variety of advantages over other more MATERIAL AND METHODS conventional typing procedures, such as the stability of the genomic DNA analysis, the capacity to discriminate bacteria at Milk samples the strain level, and the amenability to automation and Five samples of goat’s milk collected from farms located statistical analysis (21). These methods have been employed in the region north-west of Algeria were used in this study. The for differentiation or identification and typing of different samples were collected aseptically in sterile bottles kept in an species of Lactobacillus. The species of lactobacilli most ice-box, and transported immediately to the laboratory. commonly found in milk, and dairy product, especially in goat’s milk are L. plantarum, L. rhamnosus, L. casei or L. Phenotypic characterization paracasei. For this reason, the selection of Lactobacillus One milliliter of each milk sample was homogenized with strains from goat’s milk has been considered in the search for 9 ml of sterile Ringer’s solution 1:4 and mixed thoroughly for new industrially important cultures, in order to select those 60s. Serial dilutions were made and aliquots (100 µl) of each with the highest potential for industrial applications. In Algeria, dilution were streaked on MRS agar (Oxoid, UK) (17). The goat’s milk plays a vital role in human consumption, most MRS plates were incubated at 30 °C and 45°C for 24 to 48h being consumed by the rural community, while little is under anaerobic conditions (Anaerogen, Oxoid). Ten colonies available on the market (5). Algerian people make various from plates corresponding to the highest dilutions were fermented dairy products using goat’s milk. The transformation randomly selected and purified by subculturing. Gram-positive, of goat’s milk into traditional Algerian dairy products, such as catalase negative cultures were stored at -80 °C in MRS El – Klila, a traditional cheese which is popular in the country supplemented with 20% glycerol. Isolates were phenotypically side and is made from unpasteurised cow or goat surplus milk assigned to the genus level on the basis of: cell morphology, (7), Jben (local traditional fresh cheese), Raïb, and Lben (local Gram-positive and catalase-negative, according to the methods traditional fermented milks), is achieved through spontaneous and criteria described by Sharpe (42) and Kandler and Weiss fermentation without the addition of any selected starter. Such (26); CO production from glucose in MRS broth containing 2 products generally present irregular sensorial qualities. The aim inverted Durham tubes (32); hydrolysis of arginine, growth at of the present study was to characterize Lactobacillus isolated 15 °C and 45 °C, tolerance to 20, 40, 65 g L-1 NaCl. The acid from goat’s milk from north-west of Algeria, using production from carbohydrates (fructose, glucose, mannitol, physiological, phenotypic and genotypic methods. There are no lactose, mannose, rhamnose, glycerol, arabinose, sorbose, previous reports concerning the genetic identification of dulcitol, amygdalin, melibiose, melezitose, starch, tagatose, Lactobacillus or studies that combined the phenotypic and the arabitol, ribose, maltose, galactose, and xylose) was evaluated genotypic identification of Lactobacillus isolated from goat’s by using a miniaturized assay in microtiter plates, as described milk in Algeria. Additionally, in order to select adequate by Jayne-Williams (25). Ability to ferment carbohydrate strains susceptible to be used as starter cultures for the substrates was studied, using the API 50 CHL system manufacture of artisanal fermented dairy products in Algeria, (BioMérieux, Lyon, France), following the manufacturer some important technological properties, including the capacity recommendations. of acidification/coagulation of skim milk, exopolysaccharide production, aminopeptidase, antibacterial activity, and DNA extraction and 16S rDNA sequencing antibiotics resistance, were also studied. Isolates were grown in MRS broth at 30 °C until OD of 159 Marroki, A. et al. Characterization of Lactobacillus and their technological properties 1.6 - 1.8 at 600 nm. A 1.5 mL aliquot of each overnight culture pH changes were measured with a pH meter (glass electrode, was centrifuged at 10000 × g for 30 s at room temperature in Crison, Spain) after 6, 12, and 24 h of incubation at 30°C. order to pellet cells. Bacterial DNA was isolated by using the Acidification activity was measured by following the change in UltraCleanTM Microbial DNA isolation Kit (MoBio the pH during time. Coagulation of milk was determined after Laboratories Inc., Solana Beach, CA, USA), following the 24 h of incubation at 30°C. Screening of exopolysaccharide instructions of the manufacturer. (EPS) was carried out in ruthenium red milk plates, as The 5¢ end variable region of the 16S rDNA was PCR- described by Stingele et al. (44). amplified with primers 27F (5¢-AGAGTTTGATCCTGGCTC Aminopeptidase activity of the strains was determined AG-3¢) and 558R (5¢-GTATTCCGCGGCTG-3) or with the using the synthetic substrates L-alanine r-nitroaniline (Ala-r- primers 27F and 1525R (5¢-AAGGAGGTGWTCCARCCG NA) (Sigma,USA), and L-leucine r-nitroaniline (Leu-r-NA) (Sigma,USA) as described by Zotta et al. (51). Stationary CA-3¢) using a total volume of 50 µl containing 50 ng of DNA, phase cells grown overnight in MRS broth were harvested by 25 pmol of each primer, 1.6 mM of dNTPs, 2 mM MgCl and 2 centrifugation at 10000 × g for 5 min, washed twice with sterile 1U of Taq DNA polymerase (Biotools Lab, Spain), using the 50 mM potassium phosphate buffer, pH 7.0, and re-suspended reaction buffer supplied by the manufacturer. in the same buffer to obtain cell suspensions of (A = 1.0). Amplifications were carried out in a Thermal Cycler 650 Aminopeptidase activity was measured, according to Macedo (PTC-100 Peltier Themal Cycler, MJR), using the following et al.(31). The assay mixture contained: 30µl of 20 mM program: for primers 27F and 558R, the 16S rDNA was amplified as described by Linaje et al. (30); for the primers 27F aminoacyl r-nitroanilide substrates dissolved in methanol, 195 and 1525R, the PCR mixtures were subjected to an initial µL of 50 mM potassium phosphate buffer (pH 7.0), 95 µL of denaturing step of 95 °C for 5min, followed by 30 PCR cycles 0.05% (w/v) sodium azide solution, and 75 µL of cell (94 °C, 15s; 52 °C, 30s; 72 °C, 2min) and final cycle at 72 °C suspension. After incubation at 30 °C for 1 to 4 h, the reaction for 5min. The PCR products were subjected to gel was stopped by the addition of 900 µL of 1% (v/v) acetic acid. electrophoresis in 1% agarose gel, followed by staining with The release of r-nitroaniline (r-NA) (Sigma, USA) was ethidium bromide and visualization under UV light. A Lambda measured spectrophotometrically (Hewlett Packaro, Diod DNA (Biorad) digested with PstI ladder was used as a Array Spectrophotometer, Germany) at 410 nm after molecular mass marker. centrifugation of the mixture at 10000 × g for 5min. Data Polymerase chain reaction products were purified by using obtained were compared to a calibration curve prepared using the GFX PCR DNA and a gel band purification Kit (General r-NA (Sigma, USA) dilutions ranging from 0.1 to 20.0 mM. Electric Healthcare, Spain), following the manufacturer’s One unit of enzyme activity was defined as the amount of instructions. DNA sequencing was carried out by the Central enzyme required to release 1 µmol of r-NA min-1 under the Service of Research Support of the University of Valencia assay conditions. (Spain), by using the dideoxynucleotide DNA chain Antibacterial activity of Lactobacillus strains was tested termination method. with the well diffusion method described by Linaje et al. (30). Cells were grown overnight in MRS broth. Cell-free Technological properties supernatants were obtained by centrifugation at 10000 × g for Acidifying activity in skim milk was assayed as described 10 min at 4 °C, adjusted to pH 6.5 with 1N NaOH, and then by Psono et al. (35). Sterile skim milk samples (100 mL; 1.0%) filtered through 0.22µm Durapore membrane filters were inoculated with overnight cultures which had been (Millipore). The supernatants were adjusted to pH 6.5, in order previously activated by two successive transfers in milk. The to eliminate the eventual antimicrobial activity linked to 160 Marroki, A. et al. Characterization of Lactobacillus and their technological properties organic acids. A 100 µL aliquot of an overnight culture of the RESULTS AND DISCUSSION indicator bacteria Listeria monocytogenes CECT 932T, Bacillus cereus INRA AVZ 421, Staphylococcus aureus CECT 86T and Phenotypic identification of isolates Staphylococcus aureus UT 602, was mixed with 5 mL of soft Isolated strains were identified based on their agar (Trypticase Soya Broth for Bacillus cereus and physiological and biochemical characteristics given by Kandler Staphylococcus aureus and Brain Heart Infusion for Listeria and Weiss (26), Dellaglio et al. (15) and Stiles and Holzapfel monocytogenes, supplemented with 0.8% agar). Aliquots (50 (43), to the species level, and also by using API 50 CHL test µl) of supernatant of overnight cultures were poured in the strips (BioMérieux, Lyon, France) for confirmation of species wells digged in the soft agar. After 24 h of incubation at 37 °C, of selected strains. inhibition zones were read. A clear zone of inhibition >1 mm All isolates (19 Lactobacillus strains) were rod shaped around a well was scored as positive. In order to check the cells, Gram-positive, catalase-negative, non motile and thermoresistance of the bacteriocins, cell free supernatant facultative anaerobic bacteria. Isolates were classified as samples were heated at 100°C for 10 min, prior the belonging to the genus Lactobacillus. All isolates were able to antibacterial assay. The proteinaceous nature of the inhibitory grow at 15°C, 2%, and 4% NaCl. They were divided into two activity was tested by the addition of 0.5 µg of proteinase K preliminary groups (I and II), according to the results for CO 2 (Roche Molecular Biochemicals) to the concentrated culture production from glucose, and NH production from arginine: 3 supernatants (50 µL) distributed among the wells of the assay Group I, facultatively heterofermentative, and arginine- plates. negative lactobacilli (94.73%); group II strictly heterofermentative, and arginine-positive strain (one Antibiotic susceptibility testing Lactobacillus strain). Susceptibility testing was based on the agar overlay disc Table 1 shows the carbohydrate utilization patterns and diffusion test described by Charteris et al. (10), as modified by other physiological and biochemical characteristics of the Aymerich et al. (4). Briefly, Lactobacillus strains were grown lactobacilli isolates. The analysis of data compared with those overnight in MRS broth at 30°C under anaerobic conditions of the criteria given by several authors, resulted in four (Anaerogen, Oxoid). Eight ml of MRS soft agar kept at 50°C subgroups (A-D). Group I was subdivided into three subgroups were inoculated with 200 µL of the grown culture. Petri dishes (A-C) and group II, comprised only one subgroup (D). Isolates containing 15 mL of MRS were overlaid with 8.2 mL of the belonging to the four subgroups were able to ferment fructose, inoculated MRS and allowed to solidify at room temperature. glucose, mannitol, lactose, mannose, ribose, maltose, and Antibiotic discs were placed onto the overlaid plates and all galactose and unable to ferment xylose. plates were incubated at 30°C for 24 h under anaerobic • Subgroup A was the largest one, with 11 strains (58%) conditions. All isolates were screened for their susceptibility to identified as L. plantarum. The strains were able to ferment penicillin G (10 µg), ampicillin (10 µg), vancomycin (30 µg), amygdalin, melibiose, melezitose, and arabitol, but unable to tetracycline (30 µg), erythromycin (15 µg), kanamycin (30µg) ferment rhamnose, glycerol, sorbose, dulcitol, and tagatose. gentamicin (10 µg), and chloramphenicol (30 µg). Inhibition However, variations in fermentation patterns were observed for zones diameters of antibiotics were compared to those defined some sugars: arabinose was fermented by 36%, starch by 18%, by Charteris et al. (10) for lactobacilli. sorbose by 9%, and dulcitol by 9% of the strains. Most of the 161 Marroki, A. et al. Characterization of Lactobacillus and their technological properties lactobacilli reported as being able to produce amylase are (40%) to ferment glycerol resulted in their classification either strictly homofermentative (26), although some L. plantarum as L. plantarum or as L. pentosus (8, 50). Growth at 45°C was have been reported as starch fermenting strains (33). Eighteen observed for 40% of isolates. percent of isolates from this subgroup were able to grow at • Subgroup C, included two isolates able to ferment 45°C. Some of the L. plantarum strains were capable of rhamnose, arabinose, amygdalin, and melezitose. However, growing at 45°C, in contrast to the characteristics given in glycerol, sorbose, dulcitol, melibiose, starch, tagatose, and Bergey’s Manual (26). Other studies have also reported the arabitol were not fermented. All the isolates were able to grow isolation of L. plantarum strains capable of growing at this at 45°C and identified as L. rhamnosus. temperature (20, 39). • Subgroup D, with one isolate able to ferment • Subgroup B, comprised 5 strains (26%) identified as rhamnose, arabinose amygdalin, melibiose and tagatose. In belonging to L .plantarum/L. pentosus species. The isolates contrast, this strain was incapable of fermenting glycerol, only differed from those of subgroup A in the inability to sorbose, dulcitol, melezitose, starch and arabitol. This isolate ferment the starch and arabitol. The ability of some strains was able to grow at 45°C and was classified as L. fermentum. Table 1. Biochemical and physiological characteristics of Lactobacillus strains isolated from Algerian goat’s milk. Group I II Subgroup A B C D Number of isolates 11 5 2 1 CO from glucose - - - + 2 Arginine Hydrolysis - - - + Growth at 15°C + + + + 45°C 18 40 + + Growth in 2 % NaCl + + + + 4 % NaCl + + + + 6.5 % NaCl - - - - Sugar fermentation Fructose + + + + Glucose + + + + Mannitol + + + + Lactose + + + + Mannose + + + + Rhamnose - - + + Glycerol - 40 - - Arabinose 36 + + + Sorbose 9 - - - Dulcitol 9 20 - - amygdalin + 20 + + Melibiose + + - + Melezitose + + + - Starch 18 - - - Tagatose - - - + Arabitol + - - - Ribose + + + + Maltose + + + + Galactose + + + + Xylose - - - - +: Positive reaction; -: Negative reaction; 20: 20% of isolates showed a positive result; Subgroup A: LbMA9, LbMF13, LbMF33, LbMS4, LbMS9, LbMS14, LbMS16, LbMS20, LbMS21, LbMS24, LbMO16; Subgroup B: LbMO27, LbMO42, LbMS40, LbMT9, LbMT10; Subgroup C: LbMF24, LbMF25; Subgroup D: LbMA47. 162 Marroki, A. et al. Characterization of Lactobacillus and their technological properties To establish the final phenotypic identification, all strains C and D as L. rhamnosus and L. fermentum, respectively. tested for their biochemical and physiological characteristics A clear identification of species, especially within the (Table 1), were submitted to further biochemical genus Lactobacillus, based on phenotypic methods, such as characterization, using API 50 CHL galleries (BioMérieux, fermentation patterns, may sometimes be difficult, due to an Lyon, France). The programme of identification (Cox and increasing number of lactic acid bacteria species which vary on Thomson, Biochemistry institute, Odense University) plus a small number of biochemical traits (36). Commercially database was used for the interpretation of the strains available systems based on carbohydrate fermentation should fermentation profiles (Table 2). The strains of the subgroups A be combined with conventional phenotypic properties other and B were classified as L. plantarum and those of subgroups than carbohydrate fermentation or with genotypic techniques. Table 2. Fermentation of carbohydrates by Lactobacillus strains from Algerian goat’s milk, tested by the API 50 CHL system Groups A B C D Isolates LbMA9 LbMF13 LbMF33 LbMS4 LbMS9 LbMS20 LbMS14 LbMS16 LbMS24 LbMS21 LbMO16 LbMO27 LbMO42 LbMS40 LbMT9 LbMT10 LbMF24 LbMF25 LbMA47 Glycerol - - - - - - - - - - - - - - + + - - - L-Arabinose + + + + - - - - - - - - + + + + + + + L-Sorbose - - - - - - - - - - - + - - - - - - - Dulcitol - - - - - - - - - - - + - - - - - - - Mathyl- D- + + + + + + + + + + + + + + + + - - + Mannopyranoside Mathyle-D- + + + + + + - - + - + - + + - - + + + Glucopyrannoside N-Acetylglucosamine + + + + + + + + + + + - + + + + + + + Amygdalin + + + + + + + + + + + - + + - - + + + D-Melibiose + + + + + + + + + + + + + + + + - - + D-Melezitose + + + + + + + + + + + + + + + + + + - Starch - - - - + + - - - - + - - - - - - - - D-Tagatose - - - - - - - - - - - - - - - - - - + D-Arabitol - - - - - - + + - + - - - - - - - - - L-Arabitol + + + + + + + + + + + - - - - - - - - +: Positive reaction; -: Negative reaction. All isolates were able to ferment D-ribose, D-galactose, D-glucose,D-fructose,D-mannose, D-mannitol, D-sorbitol, arbutin, esculin, salicin, D-celibiose, D- maltose, D-lactose, D-sucrose, D-trehalose, D-raffinose, gentibiose, D-turanose and gluconate. None fermented erythriol, D-arabinose, D-xylose, L-xylose, D- adonitol, methyl-(cid:1)-D-xylopyranoside, inositol, inulin, glycogen, xylitol, D-rhamnose, D-fucose, L-fucose, 2-ketogluconate and 5-ketogluconate. 163 Marroki, A. et al. Characterization of Lactobacillus and their technological properties Genotypic identification of the isolates by 16S rDNA acknowledged that L. plantarum and L. pentosus belong to the sequencing same 16S rRNA phylogenetic group and could only be The 16S rDNA of the 11 strains of subgroup A and 5 distinguished using phylogenetic analysis of sequences of the isolates of the subgroup B was amplified by 27F and 558R 16S-23S large spacer region (22). primers, as reported by Acedo-Félix and Pérez-Martinez (1). The comparative evaluation of phenotypic and genotypic By partial sequencing of 16S rDNA all strains belonging to the results confirmed that the phenotypic test, in spite of giving subgroup A were classified as L. plantarum. Of the 5 isolates information on the biochemical and metabolic traits of LAB, forming subgroup B, 60% were identified as L. pentosus, are not reliable enough for the identification of these (LbMS40, LbMT9 and LbMT10) and 40% as L. plantarum microorganisms, although it is a useful tool for presumptive (LbMO42, and LbMO27). The 16S rDNA of two isolates of classification. Lactobacillus UFV H2B20, a probiotic strain, subgroup C were amplified with primers 27F and 1525R and for example, which was first identified as L. acidophilus based identified as L. rhamnosus (LbMF24 and LbMF25). The 16S on its sugar fermentation profile (38), was afterwards classified rDNA of the isolate of subgroup D was amplified by the as L. delbrueckii using molecular methods (16). One major primers used for subgroup C and identified as L. fermentum reason for the mismatch between phenotypic and genotypic (LbMA 47). data might be ascribed to loosing or acquiring plasmids, which Results of the PCR assay correlated with those obtained leads to metabolite inconsistencies, as some carbohydrate using the API 50 CHL system for 13 isolates identified as L. fermentation capacities are plasmid encoded (2). Genotypic plantarum, 2 isolates as L. rhamnosus, and one isolate as L. techniques are doubtlessly rapid and accurate tools for the fermentum. However, three isolates identified as L. plantarum identification of LAB. The advantages of genotyping include by the API system were found to be L. pentosus by sequencing the stability of genomic DNA, its composition being 16S rDNA (Table 3). This result is not surprising, given that independent of cultural conditions or preparation methods, and the two species themselves have very similar 16S rDNA amenability to automation and statistical data analysis (21). sequences that differ only by 2pb (19). In fact, it is widely Table 3. Phenotypic and genotypic identification of Lactobacillus isolated from Algerian goat’s milk. Phenotypic dentification Isolates Genotypic identification (16S rDNA) Most similar sequence (Acc. Nº) Lactobacillus plantarum LbMA9, LbMO16, LbMO27 Lactobacillus plantarum AB362768.1 L. plantarum LbMF13 L. plantarum EF185922.1 L. plantarum LbMF33 L. plantarum AB362758.1 LbMS4, LbMS9, LbMS14, L. plantarum LbMS16, LbMS20, L. plantarum EU257480.1 LbMS21,LbMS24 L. plantarum LbMO42 L. plantarum AB362625.1 L. plantarum LbMS40, LbMT9 L. pentosus AB362758.1 L. plantarum LbMT10 L. pentosus AB362712.1 L. rhamnosus LbMF24,LbMF25 L. rhamnosus AB008211.1 L. fermentum LbMA47 L. fermentum AB362626.1 Technological characteristics of strains skim milk are shown in Table 4. Milk pH after 24 h of Results on acidifying activity of Lactobacillus strains incubation varied between 4.40 and 5.54 for all cultures. All isolated from goat’s milk, after 6, 12 and 24 h of growth in Lactobacillus isolates tested reduced the pH of milk to 6.43 164 Marroki, A. et al. Characterization of Lactobacillus and their technological properties (6.12 - 6.43) after 6 h of growth, except for strains species (49). LbMA47,LbMS21 and LbMS16 which, respectively, were able L. pentosus and L. rhamnosus strains showed a more to decrease milk pH to values 5.31, 5.78 and 5.97. After 12 h, homogenous acidifying behaviour. Strains of the first species the pH values ranged from 5.24 (LbMS21) to 5.94 (LbMT10), had a medium acidifying capacity with (cid:2)pH ranging between while after 24 h, 47.37% of the strains had lowered milk pH to 0.43 and 0.58 pH units, after 6 h of incubation and achieved 4.40 - 4.96. According to their ability to reduce the pH more or 1.18-1.30 pH units until 24 h. Furthermore, the two L. less rapidly, three clusters of L. plantarum isolates were rhamnosus strains showed a fast acidifying activity, with observed. (i) Slow acidifying strains (cluster I, 23% of L. values of (cid:2)pH 0.63 and of 0.69 pH units after 6 h. After 24 h of plantarum strains) showed a slow rate of acidifying ability incubation the (cid:2)pH reached values of 1.87 and 1.96 pH units. during the first 6 h of incubation ((cid:2)pH ranging between 0.27- L. fermentum strain showed the lowest rate of acidifying: (cid:2)pH 0.40 pH units). These L. plantarum strains lowered the (cid:2)pH 0.25 pH units after 6 h and resulted in (cid:2)pH 1.35 pH units until values between 0.86-1.01 pH units and 1.33-1.60 pH units after 24 h of incubation. Generally, L. plantarum strains produce 12 h and 24 h of incubation, respectively. (ii) Medium acid more rapidly, when compared to other lactobacilli (41, acidifying L. plantarum strains (cluster II) showed a faster rate 14). The fast acidifying strains (mostly L. plantarum and L. of acidifying ability after 6 h of incubation, with a (cid:2)pH ranging rhamnosus isolates) should be selected as part of a starter between 0.50 and 0.57 pH units. In this cluster, two subgroups preparation. were observed with different behaviours in their acidifying Seven isolates of L. plantarum strains and two L. capacity. The first subgroup A (31% of L. plantarum strains) in rhamnosus were able to coagulate skim milk, when inoculated which the (cid:2)pH values was achieved, respectively, 0.78-0.98 in skim milk after 24 h at 30 °C (Table 4). None of the L. and 1.16-1.74 pH units, after 12 h and 24 h of incubation. On pentosus and L. fermentum strains were able to coagulate milk. the other hand, the second subgroup B (31% of L. plantarum Coagulation of milk by some strains of L. plantarum and two strains) showed a faster acidification rate. This subgroup strains of L. rhamnosus revealed their potential as starters or included L. plantarum strains showing a similar acidifying adjunct cultures in production of fermented dairy food products activity until 6 h but capable of achieving (cid:2)pH values ranging (34). between 1.05-1.13 and 1.96-2.14 pH units after 12 h and 24 h Many strains of LAB produce exopolysaccharide (EPS), of incubation.(iii) Fast acidifying L. plantarum strains (15% of which might be a capsule, closely attached as slim (9). The L. plantarum strains) showed the highest acidifying capacity. production of EPS by Lactobacillus strains was examined in This two L. plantarum strains, LbMS16 and LbMS21, showed milk culture and on ruthenium red milk plates. Ruthenium red a fast rate of acidifying ability until 6 h (0.73 and 0.92 pH units stains the bacterial cell wall, thus producing red colonies for respectively). This high capacity of acidifying milk by strains nonropy strains. Production of EPS prevents this staining, and of this cluster was also shown after 12 h and 24 h of hence ropy colonies appear white on the same plates (44). The incubation: (cid:2)pH values were achieved, respectively, 1.31 to results revealed EPS-production by isolated Lactobacillus 2.20 pH units for LbMS16 and 1.46 to 2.30 pH units for strains (Table 4). Among the Lactobacillus strains tested, eight LbMS21. Result of acidifying capacity of L. plantarum strains showed EPS-production. Four L. plantarum (LbMS14, after 6 h and 12 h of incubation is in agreement with several LbMS16, LbMO16 and LbMO27 strains), two L. rharmnosus authors (14, 23). In contrast, the acidifying rate of isolates after (LbMF24 and LbMF25 strains), one L. pentosus (LbMT10 24 h of incubation was lower than those reported in the former strain) and one L. fermentum (LbMA47 strain) produced EPS. studies. The possibility to find groups of strains characterized Exopolysaccharides play a major industrial role in the by different acidifying ability is frequent in L. plantarum production of fermented products, in particular for the 165 Marroki, A. et al. Characterization of Lactobacillus and their technological properties production of yoghurt, drinking yoghurt, cheese, fermented Eleven percent of strains presented high activity to release Ala- cream and milk-based desserts (18). rNA (13.12-14.10 U) and 16% of strains showed high ability Results on aminopeptidase (AP) activity of 19 strains of to release Leu-rNA (10.10-11.37 U). Twenty six percent of Lactobacillus strains tested using (Ala-rNa and Leu-rNa) are tested strains presented medium Ala-aminopeptidase activity shown in Figure 1. Amino acids released from peptides derived (6.14-8.35 U) and 26% Leu-aminopeptidase (5.15-9.70 U). from hydrolysis of casein may contribute directly or indirectly However, 63% of isolates revealed low activity to degrade Ala- for the development of flavour during ripening of cheese (48). rNA (1.02-4.93 U) and 58% to release Leu-rNA (0.95-4.32 The proteolytic activity of dairy LAB is essential for the U). All tested strains of L. pentosus, L. rhamnosus, and L. bacterial growth in milk and it is involved in the development fermentum exhibited low AP activity, except for L. pentosus of sensory properties of different fermented milk products (11). LbMT10 strain, which had medium Leu-aminopeptidase Tested strains exhibiting aminopeptidase activity ranging activity. Concerning the results obtained for L. plantarum between 1.02 and 14.10 U for L-alanine-rNA and 0.95 to strains, two strains (LbMF13 and LbMO16) had high Ala- 11.37 U for L-leucine-rNA. The AP was divided, according to aminopeptidase activity and four strains (LbMS14, LbMS16, the activity of each strain to high, medium and low activity. LbMS24 and LbMO16) had high Leu-aminopeptidase activity. Figure1. Aminopeptidase activity of strains of the Lactobacillus tested in this study. Well diffusion assay was used to screen 19 Lactobacillus 932T) (Table 4). Bacteriocin like antimicrobial compound strains isolated from goat’s milk for their antibacterial activities production was indicated by a zone of clearing of more than 1 against several pathogenic indicator bacteria (Staphylococcus mm against at least one of the indicator bacteria tested. The aureus CECT 86T, Staphylococcus aureus UT 602, Bacillus cell-free supernatants of five L. plantarum strains (38.46 %, cereus INRA AVZ421 and Listeria monocytogenes CECT LbMA9, LbMF13, LbMS14, LbMS24 and LbMO16) produced 166 Marroki, A. et al. Characterization of Lactobacillus and their technological properties an inhibition zone on agar against S. aureus CECT 86T and S. according to Charteris et al. (10). All Lactobacillus strains aureus UT 602 and three L. plantarum strains (23,07%, isolated from goat’s milk were assayed for their susceptibility LbMF13, LbMS24, and LbMO16) against Bacillus cereus to eight antibiotics, using the disk diffusion method. Zone AVZ 421. Antibacterial activity is a relatively frequent feature diameters were measured, and strains were classified as, of L. plantarum natural isolates (46). However, one strain of L. susceptible (S), moderately susceptible (MS), and resistant (R) rhamnosus strain (LbMF25) showed antimicrobial activity (Table 4). No strains of Lactobacillus were totally susceptible against S. aureus CECT 86T and S. aureus UT 602. to all antibiotics tested and multiple resistances to most Nevertheless, none of the tested strains of L. pentostus and L. antibiotics were observed. Most strains were susceptible to (cid:1)- fermentum displayed inhibitory activity against any of the lactam, inhibitors of cell wall synthesis (penicillin G and indicator bacteria. In addition, all strains tested were inactive ampicillin). Some tested strains are moderately susceptible to against Listeria monocytogenes CECT 932T. Complete the former antibiotics. However, two L. plantarum strains, inactivation of the antimicrobial activity was observed after LbMO42 and LbMS24, were resistant to penicillin G or treatment with proteinase K, indicating the proteinaceous ampicillin, respectively, and one L. pentosus strain (LbMT9) nature of the antimicrobial compound, whereas treatment with was resistant to these two antibiotics simultaneously. Several heat did not affect the inhibitory activity. Bacteriocins studies report that species of lactobacilli exhibited produced by LAB are of particular interest because of their susceptibility to almost all penicillins (10). Nevertheless, the potential use as natural food preservatives. The antibacterial resistance of Lactobacillus strains to penicillin G and activity potential of some L. plantarum strains against ampicillin has been described in other studies (45, 13, 24). Staphylococcus aureus (37, 6) and Bacillus cereus (6) has been Our result show that all tested strains were resistant to previously reported. vancomycin, which is equally a cell wall synthesis inhibitor Our results confirm the high incidence of bacteriocin- (non-(cid:1)-lactam). Resistance of Lactobacillus species to producing lactic acid bacteria in milk samples, with inhibitory vancomycin is due to the presence of D-Ala-D-lactate in their activity against both pathogenic and spoilage microorganisms. peptidoglycan, rather than the D-ala-D-ala dipeptide (28). Such Goat’s milk may represent a source of new Lactobacillus resistance is usually intrinsic, that is, chromosomally encoded strains with the potential to inhibit undesirable and pathogenic and nontransmissible (27). Concerning the protein synthesis microorganisms for use in the biopreservation of dairy inhibitors, all strains tested were susceptible to tetracyclin, products. The resistance to high temperature, the proteinaceous erythromycin and resistant to kanamycin and gentamycin. nature, and the spectrum of activity of these antimicrobial Lactobacilli are generally susceptible to antibiotics which compound are advantageous for their use as biopreservatives in inhibit the synthesis of proteins, such as erythromycin and food (37). tetracycline and more resistant to aminoglycosides (kanamycin and gentamicin) (10). Chloramphenicol inhibited most tested Antibiotic resistance strains. Three Lactobacillus strains showed a moderate A key requirement for probiotic strains is that they should susceptibility and one L. plantarum (LbMO42) strain was not carry transferable antibiotic resistance genes. Transferable resistant to this antibiotic. The high natural susceptibility of resistance genes may pose a risk, as they can be transferred to lactobacilli to chloramphenicol (protein synthesis inhibitor) is pathogenic bacteria (4). In this study, antibacterial well known (10, 45). susceptibility testing of Lactobacillus strains was made 167