Probiotic properties of Lactobacillus strains isolated ...

Anaerobe 16 (2010) 578e585

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Clinical Microbiology

Probiotic properties of Lactobacillus strains isolated from the feces of breast-fed infants and Taiwanese pickled cabbage Chung-Yi Wang a, Pei-Rong Lin b, Chang-Chai Ng b, Yuan-Tay Shyu b, * a b

Biodiversity Research Center, National Taiwan University, Taipei, Taiwan Department of Horticulture, National Taiwan University, No.140, Keelung Road Section 4, Taipei 106, Taiwan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 July 2010 Received in revised form 8 October 2010 Accepted 8 October 2010 Available online 15 October 2010

This study assessed potential probiotic Lactobacillus strains isolated from the feces of breast-fed infants and from Taiwanese pickled cabbage for their possible use in probiotic fermented foods by evaluating their (i) in vitro adhesive ability, resistance to biotic stress, resistance to pathogenic bacteria, and production of b-galactosidase; (ii) milk technological properties; and (iii) in vivo adhesive ability, intestinal survival and microbial changes during and after treatment. Five Lactobacillus isolates identified as Lactobacillus reuteri F03, Lactobacillus paracasei F08, Lactobacillus rhamnosus F14, Lactobacillus plantarum C06, and Lactobacillus acidophilus C11 that showed resistance to gastric juice and bile salts were selected for further evaluation of their probiotic properties. All the strains demonstrated the ability to adhere to Caco-2 cells, particularly, strain L. plantarum C06 and L. reuteri F03 showed satisfactory abilities, which were similar to that of the reference strain L. rhamnosus GG. The strains L. paracasei F08 and L. acidophilus C11 had the highest b-galactosidase activity. Most of the strains were resistant to aminoglycosides and vancomycin but sensitive to ampicillin, erythromycin, and penicillin. All the 5 strains elicited antibacterial activity against both Gram-positive (Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus) and enegative (Escherichia coli and Salmonella enterica) pathogens. Moreover, the strains L. reuteri F03, L. paracasei F08, and L. plantarum C06 could grow rapidly in milk without nutrient supplementation and reached 108 cfu/mL after 24 h of fermentation at 37 C. The viable cell counts of the 3 strains remained above 107 cfu/mL after 21 d of storage at 4 C. In the animal feeding trial, the number of intestinal lactobacilli increased significantly after administration of milk fermented with the 3 strains, and the counts of fecal coliforms and Clostridium perfringens were markedly reduced. Lactobacillus strains could also survive in the ileal intestinal tissue of the treated rats. Technologically interesting Lactobacillus isolates may be used in the future as probiotic starter cultures for manufacturing novel fermented foods. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Lactobacillus spp. Probiotic potential Infant feces Pickled cabbage

1. Introduction Probiotics have been defined as biopreparations that contain living cells or metabolites of stabilized autochthonous microorganisms that optimize the colonization and composition of gut microbiota in both animals and humans and have a stimulatory effect on the digestive process and immunity of the host [1]. Various lactic acid bacteria (LAB), particularly strains of Lactobacillus and Bifidobacterium, are normally found in the human adult gastrointestinal (GI) tract, and have been extensively used as probiotics, especially in fermented dairy products [2]. Over the past few decades, probiotic LAB have been very intensively investigated. Most of the LAB isolated from the GI tracts of

* Corresponding author. Tel.: þ886 2 33664850; fax: þ886 2 23661441. E-mail address: [email protected] (Y.-T. Shyu). 1075-9964/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2010.10.003

animals and humans constitute an important source of new functional bacteria that can play roles in GI transit or food processing [3]. Since LAB isolated from GI tracts must withstand low pH, gastric juice bile, phenol, lysozyme, as well as antibiotics, these characteristics may serve as suitable criteria for probiotic culture selection. Lactic acid bacteria are also generally associated with the gut microbiota of infants although not as dominant microorganisms. The origin of the LAB that colonize the neonatal gut is controversial. The method for feeding infants may influence the relative proportions of bacteria that can establish themselves in the gut of infants. Breast milk appears to be the source of maternal LAB in an infant’s gut [4]. In healthy infants, breastfeeding induces the development of a microbiota that is rich in Lactobacillus spp. and Bifidobacterium spp. to a greater extent than that induced by bottle-feeding. The LAB predominance in breast-fed infants appears to be among the most significant and seems to provide protection against enteric as well as systemic disorders caused by bacterial pathogens [5].

C.-Y. Wang et al. / Anaerobe 16 (2010) 578e585

In searching for interesting strains with probiotic potential, studies on fermented food products as a source of new isolates are rapidly accumulating. In Asia, pickling, which involves spontaneous fermentation by epiphytic microorganisms, is an important and commonly used method to preserve vegetables. In Taiwan, pickled cabbage is a popular traditional snack and the process for pickling varies from region to region. Normally NaCl concentration and the final pH of products are around 10% and from 3.5 to 4.0, respectively. Lactic acid bacteria are known to play an essential role in the preservation and production of wholesome foods including various fermented fresh vegetables, which leads to the degradation of some components of the raw materials, thereby decreasing their pH and causing coupling to produce different organic acids that characterize lactic acid fermentation [6]. Some researchers have characterized the lactic acid microbiota responsible for pickled vegetables and Leuconostoc mesenteriodes, Pediococcus pentosaceus, Lactobacillus brevis, and Lactobacillus plantarum have been the most frequently isolated species [7]. The objective of this study was to identify and establish the functional and technological characteristics of potential probiotic Lactobacillus strains isolated from the feces of breast-fed infant as well as from traditional Taiwanese pickled cabbages. The isolates were preliminarily selected on the basis of acid and bile tolerance, and the selected isolates were further screened for various functional properties such as antimicrobial activity against food pathogenic organisms, adhesion ability, and b-galactosidase production ability. In addition, the probiotic candidate isolates were then used as starters in the production of fermented milks and in animal feeding trials to evaluate their potential probiotic effect in the GI microbiota of rats. 2. Materials and methods 2.1. Isolation of LAB For the isolation of LAB, fresh feces of 6-month-old healthy, breast-fed infants were collected. Pickled cabbage was purchased from a local market in Taipei, Taiwan. Each sample (10 g) was blended with 50 mL of 0.85% NaCl solution and further diluted in a 10-fold dilution series with 0.85% NaCl solution (101e108). Each diluted solution was spread-plated onto de Man, Rogosa and Sharpe (MRS) agar (Difco, Sparks, MD, USA). The plates were incubated in an anaerobic chest with AnaeroPack (Mitsubishi Gas Chemical Co., Inc.) at 37 C for 48 h. The bacterial colonies that developed on the plates were individually picked and streaked on fresh MRS agar plates by dilution streaking to obtain single colonies, which were maintained on MRS agar for immediate use and in 20% glycerol for storage at 80 C. The isolates were first screened for catalase activity and Gram staining, and only those that were catalase-negative and Gram-positive were selected for further studies. 2.2. Tolerance to simulated gastric juice The acid tolerance of isolated LAB was studied in simulated gastric juices as described by Charteris et al. [8]. The simulated gastric juices prepared by PBS buffer solution with pepsin (0.3%, w/ v). The buffer solutions were prepared by adjusting the pH to 2.0, 3.0 and 6.2 (control) with HCl, and sterilized by autoclaving at 121 C for 15 min. After thorough mixing, 10 mL of each pH solution was taken in a sterilized test tubes. Each cell suspension of the selected LAB cultures containing about 109 cells/mL was added to pH solution of 2.0, 3.0 or control (6.4) and mixed. After 3 h of incubation, 1 mL from each pH solution was serially diluted with

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0.85% sterile saline. Appropriate dilutions were spread-plated onto MRS agar and incubated in the anaerobic chest at 37 C for 72 h. 2.3. Bile salt tolerance The ability of the isolates to grow in the presence of bile was determined using the method of Vinderola and Reinheimer [2]. The bile salt solutions were prepared using oxgall (Sigma) powder, at the final concentrations of 0.3%, 0.5%, and 1%. Sterile doubledistilled water without oxgall (pH 6.2) was used as the control. All the solutions were autoclaved, and 10 mL of each solution was transferred into sterile test tubes. Cell suspensions containing w109 cells/mL were added to each solution, i.e., 0.3%, 0.5%, 1% and control, and incubated at 37 C in the anaerobic chest. After 12 h incubation, 1 mL of each culture was diluted in sterile 9 mL of 0.85% saline blanks. Plates were incubated in the anaerobic chest at 37 C for 72 h. The bile salt hydrolase (BSH) activity of the isolates was determined by the method described by Dashkevicz and Feighner [9]. 2.4. Identification and biochemical characterization of the isolates The isolates were identified by using the commercially available API 50CHL system (bioMérieux) and 16S rRNA gene sequence. The nucleic acids of each isolate were extracted using a DNA Purification kit (Promega, USA), according to the manufacturer’s instructions. Two universal primers, namely 27f and 1492r, were used for the amplification of the 16S rRNA gene [10]. Amplicons were later sequenced (Mission Biotech, Taiwan) and compared on the National Center for Biotechnology Information (NCBI) database. The genes then were submitted to NCBI. Lactic acid isomers were determined using an enzymatic D-lactic acid/L-lactic acid analysis kit with UV detection (R-Biopharm; Roche, Italy), according to the manufacturer’s instructions. The primary isomer was defined as that present at percentage values higher than 90%. 2.5. In vitro adhesion assays The ability of the isolates to adhere to intestinal epithelial cells of the Caco-2 cell line was evaluated using the method described by Martín et al. [4]. Briefly, the cells were grown in Dulbecco’s minimal essential medium (DMEM) (Invitrogen, Germany) containing 25 mM glucose, 1 mM sodium pyruvate supplemented with 10% heat-inactivated (30 min at 56 C) fetal calf serum, 2 mM L-glutamine, 1% non-essential amino acid preparation, 100 U/mL penicillin, and 100 mg/mL streptomycin. For the adherence assays, Caco-2 cells were cultured to confluence in 2 mL of the medium, which was devoid of antibiotics. Approximately 10 d after confluence, 1 mL of the medium was replaced with 1 mL of Lactobacillus suspension (108 cfu/mL in DMEM). The inoculated cultures were incubated for 3 h at 37 C in 5% CO2. The infected cells were washed 3 times with sterile PBS (pH 7.8), fixed in methanol, Gram-stained, and observed microscopically. Adherent lactobacilli in 20 random microscopic fields were counted for each test. The probiotic strain Lactobacillus rhamnosus GG (ATCC 53103) was used as a control. The enumeration of the adhered lactobacilli cells was performed in triplicate and the values were expressed as mean SD. 2.6. b-Galactosidase activity

b-Galactosidase activity was assayed according to the method of Hsu et al. [11]. The isolated cells were first harvested by centrifugation (10,000g for 10 min at 4 C). The cells were washed twice with 0.03 M sodium phosphate buffer (pH 6.8), and suspended in PBS. The suspension, which was maintained in an ice

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bath, was sonicated using a sonicator (Model 3000, Misonix, Farmingdale, NY, USA) and centrifuged (15,000g, 10 min). The resultant supernatant served as the enzyme source. The reaction mixture contained 0.5 mL each of the enzyme source and 15 mM o-nitrophenyl b-D-galactopyranoside (OPNG) in 0.03 M sodium phosphate buffer (pH 6.8). After incubation for 10 min at 37 C, the reaction was stopped using 2.0 mL of 0.1 M sodium carbonate. Absorbance was measured at 420 nm using a spectrophotometer (Model Helios a; Unicam Co., Cambridge, UK). A unit of bgalactosidase was defined as the amount of enzyme required for catalyzing the formation of 1 mmol/min of o-nitrophenyl under the assay conditions.

2.7. Antimicrobial activity assay The agar-well diffusion test as described by Mishra and Prasad [12] was used for assaying the antimicrobial activity of the isolates against a variety of food pathogenic bacteria. Briefly, the cultural brothes of the isolates were obtained from overnight producer cultures grown in MRS broth at 37 C. Cell free supernatants were obtained by centrifuging the culture broth at 8000g for 15 min. The supernatants were neutralized to pH 6.5 0.1 and filter-sterilized by membrane filtration (0.22 mm membrane; Millipore). The supernatants were tested against the indicator strains, Escherichia coli BCRC 51745, Bacillus cereus BCRC 10446, Listeria monocytogenes BCRC 14845, Staphylococcus aureus BCRC 10451, Vibrio parahaemolyticus BCRC 12863, and Salmonella enterica BCRC 10242, which were obtained from the Bioresources Collection and Research Center (BCRC, Hsinchu, Taiwan).

2.8. Antibiotic susceptibility assay Antibiotic susceptibility of the selected LAB isolates was determined using agar overlay diffusion method, as described by Cebeci and Gürakan [13]. Cells were grown in MRS broth at 37 C for 18 h to obtain a density of 106e107 cfu/mL. Previously prepared MRS agar plates containing 15 mL of MRS agar were overlaid with 4 mL of cell suspension at soft agar (1%), containing 200 mL of 106e107 cfu/mL. The plates were maintained at room temperature for 1 h. Antibiotic discs were dispensed onto the plates and incubated under anaerobic conditions at 37 C for 24 h. All of the antibiotic discs were purchased from Sigma. Inhibition zones were measured, and susceptibility was expressed in terms of resistant (R), moderately susceptible (MS), and susceptible (S).

2.9. Production of fermented milk The feasibility of using the 3 Lactobacillus strains in lactic acid fermented products, was assessed by producing fermented milk samples. The preparation and fermentation of milk were performed according to the method of Chiu et al. [14]. Briefly, skimmed milk powder was weighed and dissolved in water to obtain 4% skimmed milk (w/v), which was sterilized in an autoclave at 121 C for 15 min and cooled to room temperature. The milk was then inoculated with 1% bacterial solution (v/v) so as to obtain an initial population of about 104 cfu/mL milk. Inoculated milks were fermented in sealed containers that were incubated at 37 C for 24 h. After 24 h of fermentation, the fermented milk was stored at 4 C for 21 d, and the viability of bacteria in the fermented milk was determined at the beginning (starting level) and the end of the fermentation process, and once every week during the storage period. Changes in pH during the fermentation process and the storage period were also monitored.

2.10. Animal feeding trials Forty 4-week-old female Wistar rats weighing 73.6 4.3 g (mean SD) were obtained from the Laboratory Animal Center of National Taiwan University. Rats were housed under conventional conditions, at a temperature of 23 2 C and a relative humidity of 55 5% in a controlled room with a 12-h of light/dark cycle and maintained under the abovementioned condition for 1 week before the trials. Feed and water were provided ad libitum daily. Standard measures were taken to prevent coprophagia (using a wire mesh floor). The general health, appearance, and weight of the rats were recorded daily. Rats were randomly divided into 4 groups (n ¼ 8) as follows: (A) daily dose of non-fermented sterile milk (control); (B) daily dose of 108 cfu/day of milk fermented by Lactobacillus reuteri F03; (C) daily dose of 108 cfu/day of milk fermented by Lactobacillus paracasei F08; and (D) daily dose of 108 cfu/day of milk fermented by L. plantarum C06. Rats were fed by the fermented milk via a stomach tube for 14 d. All fermented milks were prepared each week and stored at 20 C. Fecal samples were aseptically obtained from each rat in the morning before feeding the fermented milk, on days 0, 3, 7, and 14. On day 14, the rats were sacrificed, and intestinal tissue samples from the ileum were obtained (1 cm long fragments) in order to assess the presence of lactic acid bacteria on the tissue surface. 2.11. Microbial analysis Fecal samples from the rats collected on days 0, 3, 7, and 14 were serially diluted with 0.85% NaCl solution from 101 to 108. Serial dilutions and painting of the producers were performed in an anaerobic glove box (Modular Atmosphere Controlled system: AES CHEMUNEX, France) containing a mixture of 80% v/v N2, 10% v/v CO2, and 10% v/v H2. Total anaerobe counts were obtained by using CDC anaerobe blood agar [15], and the plates were incubated anaerobically for 3 d at 37 C. Lactobacilli MRS medium containing vancomycin and bromocresol green (LAMVAB) was used for the enumeration of lactobacilli. The medium was prepared according to the instructions by Hartemink et al. [16]. Results were expressed as log10 cfu/g of wet feces. Phenotypic identification of the LAB was achieved using the API 50CHL kit (BioMérieux), on the basis of 16S rRNA similarity, and DNA homology was determined fluorometrically by using the method of Ezaki et al. [17]. For coliform counts, a single aliquot (1 mL) from each dilution was spread on PetrifilmÔ E. coli/Coliform Count Plates (3 M Corporation, St Paul, Minnesota). The plates were incubated at 37 C for 2 d. Colonies were identified and counted in accordance with the manufacturer’s instructions. Tryptose-sulfite-D-cycloserine (TSC) medium was used for counting Clostridium perfringens, as per the method described by Cheng et al. [18]. The ileac intestinal tissue samples (1 cm length) were longitudinally cut to expose the internal surface and gently washed 3 times with physiological saline to eliminate the fecal contents. The presence of Lactobacillus spp., was evaluated by placing the intestinal fragments on MRS agar plates for 6 h at 37 C. Next, the tissues were removed and the inoculated media were incubated at 37 C for 48 h. Isolation and identification of the colonies were performed according to standard microbiological methods, as described above. 2.12. Statistical analysis All data were expressed as mean SD. Student’s t test was used for statistical analysis by comparing treatment groups versus control group. Results were regarded as statistically significant at p < 0.05.


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Table 1 Origin, simulated gastric juice and bile tolerance and bile salt hydrolase (BSH) activity of LAB strains (log cfu/ml). Isolate name

Isolation origin

Initial mean countsa

Resistance to gastric juice

Bile tolerance

pH 2

pH 3

0.3%

0.5%

1%

F03 F07 F08 F11 F14 F19 F21 C02 C06 C10 C11

Infant feces Infant feces Infant feces Infant feces Infant feces Infant feces Infant feces Pickled cabbage Pickled cabbage Pickled cabbage Pickled cabbage

8.73 0.17 9.19 0.12 8.81 0.18 9.08 0.16 8.84 0.13 9.20 0.21 8.92 0.19 9.23 0.26 9.06 0.15 9.17 0.18 8.93 0.20

7.39 0.32 eb 7.77 0.28 e 7.13 0.36 2.59 0.22 e 4.83 0.37 8.86 0.34 e 7.72 0.32

8.26 0.22 6.21 0.29 8.58 0.36 7.71 0.33 8.59 0.21 6.86 0.19 6.37 0.27 6.43 0.18 8.88 0.20 e 8.56 0.24

7.24 0.34 3.79 0.27 7.84 0.42 4.51 0.25 7.78 0.38 3.75 0.27 2.86 0.23 4.51 0.34 7.14 0.31 3.81 0.44 6.28 0.42

6.81 0.37 e 7.73 0.41 3.84 0.29 6.18 0.38 2.49 0.39 e 3.73 0.43 7.67 0.32 e 6.61 0.33

6.72 0.40 e 7.62 0.28 e 6.87 0.35 e e e 7.73 0.45 e 6.81 0.37

a b c

BSH activityc

＋＋＋＋＋

Each value represents the mean value stand deviation (SD) from three trials. No growth. －, negative for BSH activity; þ, positive for BSH activity.

3. Results

shown in Table 3. The isolates F03, F08 and C06 expressed strong adhesive abilities to Caco-2 cells compared with the control strain L. rhamnosus GG, with the exception of strain F14 that showed a low level of adhesion to the tested cells.

3.1. Acid and bile tolerance In all, 11 Gram-positive, catalase-negative, rod-shaped isolates were obtained from infant feces and pickled cabbage. Table 1 shows the survival of the 11 isolates under low pH and bile salt conditions. All the strains, except C10, consistently showed tolerance to pepsin at pH 3, and the residual counts were greater than 106 cfu/mL after 3 h of incubation. Only 7 isolates survived at pH 2, and the isolates F03, F08, F14, C06, and C11 exhibited fairly good acid tolerance with maintenance levels greater than 75% after exposure to pH 2. In the bile salts test, all the tested isolates were able to grow in 0.3% bile, but only F03, F08, F14, C06, and C11 showed resistance at 1% bile concentration for up to 12 h. The F11, F19, and C02 isolates were resistant to only 0.5% bile. The isolates F08 and C11 showed comparatively better tolerance with viable counts greater than 107 cfu/mL in 1% bile. On the basis of the screening results for tolerance to low pH and bile salts, 5 isolates, namely, F03, F08, F14, C06, and C11, were found to be able to survive at levels of 106 cfu/mL under pH 2 or 1% bile salts conditions and were selected for further analyzing their probiotic properties. 3.2. Identification of lactic acid bacteria Table 2 details the identification by API 50CHL kit, 16S rRNA gene-based identification, and the type of the lactic acid isomer produced by the 5 isolates. On the basis of the 16S rRNA gene analysis, the 5 isolates were identified as L. reuteri F03, L. paracasei F08, L. rhamnosus F14, L. acidophilus C06, and L. plantarum C11. Identification of isolates by API 50CHL was confirmed by molecular identification, except for strain F03, which was misidentified as L. fermentum by API 50CHL. Strains F08, and F14 produced only the L-form of the lactic acid. 3.3. Adhesion properties The adhesion ability of the isolates was tested using the Caco-2 cell line. The isolates demonstrated variable adhesion abilities as Table 2 Identification results of selected strains after screening for acid and bile tolerance. Isolate name

Lactic acid isomer produced

Identification by API 50CHL kit

Identification by 16S rRNA

Accession number

F03 F08 F14 C06 C11

DL L L DL DL

L. L. L. L. L.

L. L. L. L. L.

GQ202839 GQ202836 GQ202840 GQ202837 GQ202838

fermentum paracasei rhamnosus plantarum acidophilus

reuteri paracasei rhamnosus plantarum acidophilus

3.4. b-Galactosidase activity Fig. 1 shows the b-galactosidase activity of the lactobacilli isolates. All the isolates produced b-galactosidase after 24 h of culture at 37 C b-galactosidase activities detected in the cultures of F08 (3.14 U/mL) and C11 (2.89 U/mL) did not significantly differ (p > 0.05), but were significantly higher than those of the other 3 isolates. F14 had the lowest activity of 1.23 U/mL. 3.5. Antimicrobial activity assay The inhibitory abilities of the lactobacilli isolates in the form of cell free spent broth tested against common food pathogens are shown in Table 4. Most of the Lactobacillus isolates showed inhibitory activities against the indicator bacteria, although the inhibitory extents were variable. In particular, strains F03 and C06 showed strong (>9 mm zone of inhibition) antibacterial activity against E. coli and S. aureus. In addition, most of the isolates exhibited a slight inhibitory activity towards B. cereus, and few showed weak to medium activity against S. enterica. Taken together, these findings suggest that the V. parahaemolyticus strain was more tolerant to the inhibitory effect of the Lactobacillus isolates compared to the other pathogenic bacteria. E. coli and S. aureus growth was suppressed by all of the Lactobacillus isolates. 3.6. Antibiotic resistance Table 5 lists the antibiotic susceptibility results of the Lactobacillus isolates. Different strains were resistant to antibiotics

Table 3 Number of lactobacilli strains that adhered to Caco-2 cells in 20 random microscopic fields. Strain

Caco-2

L. L. L. L. L. L.

325.7 177.6 384.2 146.8 94.6 78.4 408.4 126.7 227.8 96.7 451.3 197.4

reuteri F03 paracasei F08 rhamnosus F14 plantarum C06 acidophilus C11 rhamnosus GG

Each value represents the mean value stand deviation (SD) from three trials.

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was lower (pH 4.21) than that of the milk fermented with F03 and F08 (4.39 and 4.35, respectively), although not significantly different (p < 0.05). During the cold storage period, the pH values of the fermented milks remained fairly constant. 3.8. In vivo feeding trials

Fig. 1. b-Galactosidase activity of lactobacilli isolates in units. The values shown represent averages from triplicate experiments. Error bars represent the standard deviation. Data bearing different letter were significantly different (p < 0.05).

belonging to different classes. All the 5 selected isolates were resistant to kanamycin, streptomycin, tetracyclin, and vancomycin. However, almost all the strains were interpreted to be sensitive toward ampicillin, cephalexin, erythromycin, penicillin, and streptomycin.

3.7. Production of fermented milk Basis on the adhesion properties and antimicrobial activity, the strains L. reuteri F03, L. paracasei F08 and L. plantarum C06 were selected for production fermented milk for future animal feeding trials. The three strains, F03, F08, and C06, grew well on sterilized milk. Fig. 2 presents the time-dependent changes in the viable counts of the Lactobacillus isolates under the pH of the fermented milk. The three strains grew rapidly on the fermented milk and reached 10 108 cfu/mL after 24 h of fermentation at 37 C, but extending the fermentation beyond 24 h did not result in a significant increase. Among the 3 isolates tested, strain F08 showed the fastest growth, with maximum viable counts of 9.1 108 cfu/mL, which was greater than that of F03 and C06, with viable counts of 3.2 108 and 1.4 108 cfu/mL, respectively. The three strains were capable of surviving in the fermented milk at 4 C for several weeks. However, the viable counts of the isolates diminished by approximately 0.4e0.9 101 cfu/mL during the 21 d of storage at 4 C, most markedly for F08 which showed a decrease of 0.59 101 cfu/mL between day 14 and day 21 of storage. Moreover, depending upon the strain used, the isolates were able to acidify milk during the 24 h fermentation period. The initial pH of the milk was 6.72. After 24 h of fermentation, the pH of the milk fermented with strain C06

In the fermented milk feeding trials, the Lactobacillus colony count on LAMVAB increased on day 3 as compared to day 0. Table 6 summarizes the microbial population composition in the fecal samples of the treated and control groups, as determined by count on selective media. The total anaerobe counts were significantly higher in each treated group than in the control group. We observed a significant increase of circa 3 log10 cfu/mL in the mean count of the fecal lactobacilli in the treated rats compared to that in the control rats, for each tested isolate. On the other hand, for each treated group, the counts of coliform organisms and C. perfringens in rat feces significantly decreased between 0 and 7 days of administration of fermented milk, especially with the strains L. paracasei F08 and L. plantarum C06. After 7 days of administration, the counts of coliform organisms and C. perfringens were remained stabilized until the end of 14 days. In addition, a large proportion of the lactobacilli obtained from the cultures of the ileal intestinal tissue samples of the control group were identified as L. acidophilus (46%), L. casei (16%), L. paracasei (6%), and L. reuteri (32%) by API 50CHL system and DNAeDNA hybridization. The distribution of Lactobacillus isolates in the ileal intestinal tissue samples of all the treatment groups was clearly affected after administration of the fermented milk for 14 days, as shown in Fig. 3. That is, the number of L. reuteri, L. paracasei, and L. plantarum increased in the ileal intestinal tissues of rats of groups B, C, and D, respectively, compared with those in the control group. In particular, L. plantarum was detected in the rat feces only in group D (Fig. 3), suggesting that the strain L. plantarum C06 survived the transit through the stomach and small intestine. 4. Discussion Thus far, significant efforts have been made to identify potential probiotic LAB isolated from humans or from fermented foods because of their benefits. Stringent selection criteria for the identification of probiotic strains are now being considered essential. For specifically selecting highly potent probiotic strains, the safety and functional properties of LAB, such as antibiotic resistance, adhesion to intestinal cells, antimicrobial activity, and production of b-galactosidase, as well as survival in the GI tract, are highly important and should be studied using reliable in vitro and/or in vivo screening methods [2,19,20]. Therefore, this study aimed to direct the selection of potentially probiotic Lactobacillus strains that

Table 4 Antimicrobial activities of cell free spent broth of lactobacilli strains against common pathogens. Microbe tested

Inhibition of growth by L. reuteri F03

L. paracasei F08

L. rhamnosus F14

L. plantarum C06

L. acidophilus C11

E. coli BCRC 51745 B. cereus BCRC 10446 L. monocytogenes BCRC 14845 S. aureus BCRC 10451 V. parahaemolyticus BCRC 12863 S. enterica BCRC 10242

þþþ þþ e

þþ þ þ

þþ e e

þþþ þ þ

þþ e e

þþþ e

þþ e

þþ e

þþþ e

þ þ

þ

e

e

þþ

e

Symbols: e, no inhibition zone; þ, inhibition zone up to 3 mm; þþ, inhibition zone up to 6 mm; þþþ, inhibition zone over 9 mm.


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Table 5 Antibiotic susceptibility profilesa. Antibiotic

L. reuteri F03

L. paracasei F08

L. rhamnosus F14

L. plantarum C06

L. acidophilus C11

Cephalexin Chloramphenicol Clindamycin Enrofloxacin Erythromycin Gentamicin Kanamycin Polymyxin Rifampicin Streptomycin Tetracycline

MS S S R S S MS R MS S S

S S S MS S R R MS R S R

S R MS R S S R S R S MS

S MS R S S MS R R R MS MS

S MS MS R S MS R R R S R

a

All the strains are aminoglycosides and vancomycin resistant, and are susceptible to ampicillin and penicillin. R: resistant, MS: moderately susceptible, S: susceptible.

could survive at a low pH and in the presence of bile salts from the feces of breast-fed infants and from pickled cabbage. The in vitro survival test revealed that several Lactobacillus strains (F03, F08, F14, C06, and C11) were resistant to pH 2 even after 3 h of exposure, while most of the isolates showed reduced viability after being exposed to pH 2. These results are in agreement with those obtained from similar previous studies, where Lactobacillus strains were viable even after being exposed to pH values of 2.5e4.0, but showed reduced viability at lower pH values [12,21]. Acid tolerance of bacteria is important not only for withstanding gastric stresses, but is also a prerequisite for their use as dietary adjuncts. It enables the strains to survive for longer periods in highacid carrier foods, such as yogurt, without large reduction in their numbers [19]. Bile plays a fundamental role in specific and nonspecific defense mechanisms of the gut; the magnitude of its inhibitory effects is determined primarily by the concentration of bile salts [8]. The relevant physiological concentrations of human bile range from 0.3% to 0.5% [22,23]. There was considerable variability in resistance to bile salts among the different species of Lactobacillus, supporting the importance of assessing the bile tolerance of isolates in selecting potential probiotics. In this study, strains F08 and C06 showed the highest bile salt tolerance and BSH enzyme activity. It has been suggested that the BSH enzyme might be a detergent shock protein that enables lactobacilli to survive intestinal bile stress [24]. Probiotic Lactobacillus strains have several mechanisms that enable them to adhere to the intestinal epithelial cells. This property could confer a competitive advantage, which is an important criterion for colonization of Lactobacillus strains in the human GI tract. Caco-2 cell line is a colonic adenocarcinoma cell line that

Fig. 2. Viable Lactobacillus strain counts and pH changes during milk fermentation (24 h at 37 C) and cold storage (21 days at 4 C). Viable counts (cfu/mL) are shown in ramp bars for L. reuteri F03, in white bars for L. paracasei F08, and in shaded bars for L. plantarum C06; pH changes are indicated with square for L. reuteri F03, triangle for L. paracasei F08, and circle for L. plantarum C06. Bars on data points represent standard errors.

expresses the morphological and physiological characteristics of normal mature human enterocytes [25], and is thus extensively used as an in vitro model to select and assess the adhesive properties of Lactobacillus strains [4]. In our study, the adhesion abilities of L. reuteri F03, L. paracasei F08, and L. plantarum C06 to Caco-2 cells were generally similar to that of the reference strains L. rhamnosus GG. The adhesion of the Lactobacillus strains to the Caco-2 cells may indicate the competitive exclusion of other pathogenic bacteria from the epithelial surface [26]. Although the conclusions drawn from results of the in vitro studies cannot be directly applied to the in vivo situations, there is evidence that shows an association between adhesion ability and temporary colonization of the human intestinal tract [27]. Hence, the strains L. reuteri F03, L. paracasei F08, and L. plantarum C06 show good adhesive abilities were selected for fermentation of milk for the animal feeding trials. Use of LAB as biopreservatives has been approved in several studies, owing to their antagonistic activities toward various foodborne pathogens such as S. aureus, E. coli, and L. monocytogenes, etc [25]. The Lactobacillus strains isolated in this study showed antagonistic potential toward all of the abovementioned pathogenic bacteria; however, they did not significantly inhibit the growth of V. parahaemolyticus (Table 4). Xanthopoulos et al. [28] reported that L. paracasei and L. acidophilus strains isolated from infant feces had weak antibacterial activity against E. coli, while our findings showed that L. reuteri F03 and L. plantarum C06 had high antibacterial activity against E. coli. S¸ims¸ek et al. [29] conducted a similar study and reported the antibacterial activity of lactobacilli isolated from sourdough against Gram-positive and enegative pathogenic bacteria, including L. monocytogenes, Bacillus subtilis, E. coli, S. aureus, and C. perfringens. In this study, strain L. reuteri F03, L. paracasei F08 and L. plantarum C06 showed satisfactory abilities inhibit various pathogenic bacteria, these antagonistic Lactobacillus strains may be effectively used in various foods to protect against pathogenic bacterial contamination that may occur during the manufacturing processes. A recent study reported that lactobacilli isolated from dairy products had high levels of resistance to vancomycin and teicoplanin [30]. Similar results of vancomycin resistance have also been reported by several other authors [20,31]. These findings were confirmed by our study; the majority of the Lactobacillus isolates examined were found to be resistant to aminoglycosides and vancomycin (Table 5). In addition, almost all the isolates were resistant to kanamycin and rifampicin. Previous reports also confirm the general susceptibility of the lactobacilli strains studied here towards ampicillin, cephalexin erythromycin, penicillin, and streptomycin [26,28]. Danielsen and Wind [32] conducted a survey and found that transferable resistance genes may be present among LAB, such as those for chloramphenicol, erythromycin, and

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Table 6 Microbial composition of fecal samples assayed in rats before and after 3, 7 and 14 of treatment with fermented milk containing different Lactobacillus strain or non-fermented sterile milk*. Treatment

Total anaerobes

Clostridium perfringens

Coliforms

Lactobacillus spp.

Group**

Days

A

0 3 7 14

9.22 0.54a 9.34 0.72a 9.85 0.53a 9.39 0.68a

8.48 0.67b 9.31 0.64c 9.02 0.57bc 8.78 0.81b

6.78 0.61c 7.17 0.47c 7.35 0.34d 6.86 0.55cd

6.58 0.34a 6.82 0.41a 7.37 0.38b 6.91 0.45a

B

0 3 7 14

9.37 0.54a 10.68 0.87b 10.82 0.73b 10.35 0.62b

8.54 0.87a 9.17 0.60bc 8.37 0.73b 7.96 0.85a

6.83 0.35cd 6.09 0.37b 5.16 0.27a 5.10 0.31a

6.62 0.54a 8.27 0.48c 8.32 0.53c 8.24 0.57c

C

0 3 7 14

9.19 0.67a 10.26 0.50b 10.23 0.43b 10.89 0.66b

8.34 0.62b 7.79 0.72a 7.11 0.67a 7.38 0.58a

6.91 0.39cd 5.88 0.42b 4.92 0.26a 5.08 0.24a

6.35 0.46a 8.12 0.51c 8.69 0.41cd 9.24 0.48d

D

0 3 7 14

9.58 0.64a 10.67 0.45b 10.85 0.61b 10.27 0.55ab

8.54 0.47b 7.81 0.57a 7.32 0.66a 7.58 0.52a

6.70 0.35c 6.08 0.21bc 5.10 0.19a 5.38 0.23a

6.20 0.38a 9.67 0.51d 9.15 0.62d 9.41 0.59d

* Values were expressed as log cfu/g wet feces (mean SD, 8 rats per group). Data bearing superscript in lowercase letters in the same column are significantly different (p < 0.05). ** A, administration of non-fermented sterile milk (control group); B, administration of 108 cfu/day of milk fermented by L. reuteri F03; C, administration of 108 cfu/day of milk fermented by L. paracasei F08; and D, administration of 108 cfu/day of milk fermented by L. plantarum C06.

tetracycline. Our study indicated that there are lesser chances of occurrence of such transferable resistance gene in the infant feces and pickled cabbage. Lactose maldigestion and/or intolerance may be treated using LAB from fermented products that contain the lactose hydrolyzing enzyme b-galactosidase. b-Galactosidase is an intracellular enzyme that appears to act when being released from bacterial cells during their transit through the small intestine [33]. In this study, L. paracasei F08 was found to have the highest b-galactosidase activity (Fig. 1). LAB that is able to hydrolyze lactose might also be useful for compensating for the lactase insufficiency, although to a lesser extent compared to yogurt cultures [34]. The health benefits of milk fermented with probiotic LAB are well documented; the extent of their benefits depends upon the high viability of the probiotic microorganisms. A satisfactory fermented probiotic milk product should contain more than 106 cfu/ mL of viable cells at the time of consumption to be effective as a health promoter [35]. In the animal feeding trial, the strains

L. reuteri F03, L. paracasei F08 and L. plantarum C06 were selected for producing probiotic fermented milk on the basis of their technological performance, i.e., acid and bile tolerance and ability to adhere to Caco-2 cells. These 3 isolates grew rapidly on milk and reached 108 cfu/mL after 24 h of fermentation at 37 C. The milks fermented with any of the 3 strains tested could be well tolerated by the rats with no adverse effects. After the consumption of the Lactobacillus-fermented milk, the rats showed increased fecal lactobacilli counts, while the counts of coliform and C. perfringens were significantly decreased. This result may support the use of Lactobacillus isolates in the production of probiotic dairy products. Our results are contrary to those of Minelli et al. [19], who reported that in the rats administered milk fermented with L. casei, the fecal E. coli counts remained stable with a significant decrease in Clostridia counts. Yang et al. [36] also reported decreased fecal coliform counts as one of advantages of Lactobacillus and Bifidobacterium proliferation in the rat gut. Such potentially probiotic bacteria colonizing the intestinal mucosa provide a barrier effect against pathogens by using a variety of mechanisms, occupation of niches, competition for nutrients, and production of antimicrobials [37]. Moreover, the strains L. reuteri F03 and L. paracasei F08 were able to survive under the in vivo conditions, which was not surprising because the strains were originally isolated from infant feces. Interestingly, L. plantarum C06, which was originally isolated from pickled cabbage, could tolerate the GI conditions and was found to persist in the rat ileum. In conclusion, we successfully identified 3 Lactobacillus strains that show satisfactory properties for probiotic applications. The present study indicates that the analyzed Lactobacillus strains can be useful in the production of dairy foods for potential human health benefits. Further studies would be required to focus on improving the technological characteristics of probiotic strains and various properties of fermented milks by the modulation of physicochemical variables commonly used at the industrial level.

Fig. 3. Lactobacillus isolates distribution in ileal intestinal tissue of each treatment group. Percentages were caculated on 100 isolates for each sample. Group A, daily dose of non-fermented sterile milk (control); group B, daily dose of milk fermented by L. reuteri F03; group C, daily dose of milk fermented by L. paracasei F08; and group D, daily dose of milk fermented by L. plantarum C06.

Acknowledgement Part of this research was supported by Chen Yung Memorial Foundation.


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