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/lp/springer-journals/in-vitro-assessment-of-probiotic-potential-of-lactobacillus-plantarum-HSh5T5iaSH
- Publisher
- Springer Journals
- Copyright
- Copyright © 2017 by Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan
- Subject
- Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
- ISSN
- 1590-4261
- eISSN
- 1869-2044
- DOI
- 10.1007/s13213-017-1318-7
- Publisher site
- See Article on Publisher Site
Abstract
Samples of fermented herbs were used to isolate lactic acid bacteria (LAB). Of a total of 19 isolates, eight were resistant both to gastric acid and bile salts (glycocholic acid, GCA; taurocholic acid, TCA; glycodeoxycholic acid, GDCA; and taurodeoxycholic acid, TDCA). Most isolates exhibited a pH-dependent surface hydrophobicity: a pH of 4 conferred a greater hydrophobicity compared to a pH of 7. Based on the hydrophobicity characteristics, the LAB isolate WU-P19 from the traditional fermented herb Oroxylum indicum was selected for further study. WU-P19 was identified as Lactobacillus plantarum WU-P19. The presence of bile salts GCA and GDCA in the culture medium induced production of the relevant bile salt hydrolase. Relative to controls, the presence of the bile salts in the culture medium affected the carbon and nitrogen contents of the cells and their hydrophobicity. Cells grown in a medium free of bile salts were morphologically different to cells grown in the presence of GCA and GDCA. WU-P19 was resistant to several antibiotics. It produced β-galactosidase and inhibited growth of the tested pathogenic bacteria at various levels. In vitro, L. plantarum WU-P19 adapted well to conditions typical of the various zones of the human gastrointes- tinal tract. In view of the promising results, in vivo evaluations are planned for the isolate WU-P19. . . . . Keywords Bile salt hydrolase β-Galactosidase Probiotic lactobacilli Hydrophobicity Lactic acid bacteria Introduction potential inhibitors of bacterial growth. Fermented Roselle and starfruit are used as digestive aids by indigenous people Lactic acid bacteria (LAB) have been commonly isolated from in Thailand. A typical preparation involves mixing the herb fermented foods because the fermentation conditions allow (3 kg), sugar (1 kg) and water (5 L). This mixture is packed them to dominate (Argyri et al. 2013;Wanget al. 2014; in plastic containers and incubated at ambient temperature (30– Yang et al. 2014; Elizaquível et al. 2015; Han et al. 2017). 35 °C) for 12 to 24 months. The fermentation involves micro- Pickled vegetables have been the source of some isolates organisms naturally present in the herbs and no starter cultures (Argyri et al. 2013; Wang et al. 2014), but isolation from are added. The fermented product has a pH of 3.0–3.5. Such fermented herbs has been rare. fermented preparations are generally consumed after the main Unlike many other fermented foods, herbs generally have a meal. Isolation of LAB from fermented herbs is of interest as low nutritional value. Furthermore, herbs such as Roselle or these herbs have a long history of use as digestive aids and the sour tea (Hibiscus sabdariffa) and starfruit (Averrhoa bacteria in the fermented products are tolerant to the low pH of carambola) are rich in polyphenolic flavonoids that are the stomach. Acid-tolerant safe isolates are potential probiotics. To survive and colonize the human gastrointestinal tract a probiotic species must have certain characteristics. It must * Wanna Choorit withstand gastric acidity, be resistant to bile salts and adhere cwanna35@gmail.com to mucus and/or human epithelium (FAO/WHO 2006). Any 1 in vitro assessment of bacteria as potential probiotics must Biotechnology Program, Agricultural Technology, Walailak consider their ability to survive in the gastrointestinal tract University, Tha Sala District, Nakhon Si Thammarat 80161, Thailand 2 and adhere to suitable surfaces. In addition, a probiotic must School of Engineering, Massey University, Private Bag 11 222, provide some health benefit. For example, it may have anti- Palmerston North, New Zealand 3 microbial activity against pathogens, or reduce their adhesion Biomass and Oil Palm Center of Excellence, Walailak University, to surfaces of the gastrointestinal tract. Diverse health benefits Tha Sala District, Nakhon Si Thammarat 80161, Thailand 80 Ann Microbiol (2018) 68:79–91 have been claimed for LAB, including a lowering of the risk Biochemical Reactions, Protocols and Applications Guide; of gastrointestinal disease; prevention or reduction of the prev- www.promega.com). The ninhydrin reagent had been alence of allergies in susceptible individuals (Isolauri 2001; prepared by mixing 25 mL of 1% (w/v, g/100 mL) ninhydrin Joo et al. 2009; Chiang and Pan 2012); and a lowering of the in 0.5 M citrate buffer (pH 5.5) with 60 mL of 30% v/v glycerol blood cholesterol (Anandharaj and Sivasankari 2014). and 10 mL of 0.5 M citrate buffer, pH 5.5. The Z buffer, pH 7, This work reports on isolation and characterization of used for assaying β-galactosidase activity contained the follow- potentially probiotic LAB from Asian fermented herbs. ing chemicals (mM): Na HPO 60, NaH PO 40, KCl 10, 2 4 2 4 The bacteria able to survive the conditions of the gastroin- MgSO ⋅7H O1and β-mercaptoethanol 50 (Lee et al. 2016). 4 2 testinal tract are focused on. The bile salts resistance, bile salt hydrolase activity, β-galactosidase activity, hydropho- Medium bicity, antibiotic susceptibility and antimicrobial properties of a selected isolate are reported, as being relevant to any Lactobacillus MRS medium was used for the isolation of bac- probiotic application. teria, maintenance of the isolates, preparation of cultures and cultivation of isolates. In addition to MRS components, the me- dium contained 1 g/L of polysorbate 80. The pH indicator Materials and methods bromocresol purple (0.2 g per L of medium) was added. Sodium azide (0.2 g per L of medium) was added as an inhibitor Source of LAB of gram-negative bacteria (Snyder and Lichstein 1940). The medium was adjusted to pH 7 with 0.1 M HCl and/or 0.1 M LAB were isolated from the following traditional fermented NaOH and sterilized at 121 °C for 15 min and cooled to room herb samples: fermented starfruit (Averrhoa carambola), temperature. The MRS agar medium was prepared by adding fermented Roselle or sour tea (Hibiscus sabdariffa), 15 g of agar to 1 L of the above-mentioned MRS medium. fermented Gac (Momordica cochinchinensis) and fermented Indian trumpet or midnight horror (Oroxylum indicum). These Screening of LAB resistant to gastric acids and bile fermented products were purchased from local markets in salts Trang province, Thailand. The fermented herb samples used in this work had been naturally fermented via the action of The procedure used for isolation of the LAB resistant to gas- endogenous microorganisms (no starter cultures were added), tric acids and bile salts is shown in Fig. 1. Selection of the as typically prepared by the local people. The pH values of LAB resistant to each form of bile salt followed the method of fermented herb samples were in the range 3.0–3.5. Kumar et al. (2012) modified as follows. The bacterium was streaked onto MRS agar medium supplemented with 0.37 g/L Chemicals and reagents CaCl and 5 g/L of the designated bile salt (Fig. 1). The fol- lowing bile salts were used separately: glycocholic acid All chemicals were of analytical grade and had been pur- (GCA), taurocholic acid (TCA), glycodeoxycholic acid chased from Sigma-Aldrich (www.sigmaaldrich.com), unless (GDCA) and taurodeoxycholic acid (TDCA). All bile salts specified otherwise. Phosphate buffered saline (PBS), were ≥ 97% pure by thin layer chromatography (Sigma- artificial gastric juice and artificial small intestinal juice were Aldrich). The LAB that grew in the presence of the four bile prepared as described by Pitino et al. (2010), Mäkeläinen et al. salts were selected. (2009) and Pitino et al. (2010), respectively. In general, the LAB were incubated for the desired period Artificial gastric enzyme solution was prepared by dissolving at 37 °C under anaerobic conditions. Anaerobiosis was 5.44 mg of porcine gastric mucosa pepsin (activity of 3300 U/ achieved by placing the petri plates inside an air-tight chamber mg of protein based on haemoglobin as the substrate) in 1 mL of and lighting a candle inside before sealing the lid. the artificial gastric juice andadjustingthepH to2.5with6M HCl. Artificial enzyme solution of the small intestine part of the Hydrophobicity tests gastrointestinal tract was prepared by dissolving 4.8 mg pancre- atin (4× the United States Pharmacopeia specifications) and The LAB were grown anaerobically in 100 mL of MRS broth 9 mg oxgall (ox bile; Oxoid, www.oxoid.com) in 1 mL of for 24 h. The cells were recovered by centrifugation (Sorvall® artificial small intestinal juice. All of the above enzyme- Biofuge Stratos, Heraeus; www.thermoscientific.com)at containing solutions were filter-sterilizedbypassingthrougha 10,000 × g, 4 °C, 10 min, unless specified otherwise. The sterile membrane filter with a 0.45 μmporesize. recovered cells were washed twice with PBS, pH 7.0, and Sodium phosphate buffer (0.1 M, pH 7.0) was prepared by suspended in PBS, pH 7.0, to an optical density (OD )of mixing 84 mL of 1 M Na HPO with 16 mL of 1 M NaH PO 1 measured at 600 nm (Lambda 20 Spectrophotometer, 2 4 2 4 and making up to 1 L with deionized water (Buffers for Shimadzu, Tokyo, Japan) against a blank of PBS, pH 7.0. Ann Microbiol (2018) 68:79–91 81 suspension was taken to measure the absorbance at 600 nm. The hydrophobicity (H) of the cells was calculated using the following equation (Bautista-Gallego et al. 2013): HðÞ % ¼ðÞ 1−H =H 100 ð1Þ t 0 In the above equation, H is the OD of the cell suspension 0 600 before n-hexadecane was added and H is the OD of the cell t 600 suspension after incubation with n-hexadecane at 37 °C for 1 h. Contact angle measurements The contact angle was measured using the method of Soon et al. (2012). A suspension of the washed selected isolate with an OD of 1 in 0.85% NaCl was prepared and this was used to create bacterial lawns on a glass plate. For this, the suspen- sion was filtered through a sterile cellulose acetate membrane filter (Millipore, www.emdmillipore.com; pore diameter of 0. 45 μm). The cells were blotted from the filter membrane onto a glass slide and allowed to dry for 30 min at room temperature. A droplet of distilled water (6.2 μL) was placed on the bacterial lawn using the syringe probe of a contact angle meter (DM- 300; Kyowa Interface Science, Japan, www.face.kyowa.co.jp) and imaged automatically. Identification of the selected isolate with 16S rDNA sequencing DNA from the selected isolate was extracted as previously described (Moore et al. 2004;Deet al. 2010). For the detec- Fig. 1 Flow diagram for screening for lactic acid bacteria resistant to gastric acids and bile salts. The isolate WU-P19 was tested for survival tion of 16S rDNA sequences, the primers UFUL (5′-GCCT under the gastrointestinal tract conditions as outlined in this figure AACACATGCAAGTCGA-3′) and 1500R (5′-TTCA GCATTGTTCCATTGG-3′) were used for amplification. When needed, 10 mL of this cell suspension was transferred to The polymerase chain reaction (PCR) was run under the fol- 90 mL of the MRS broth either with or without 6 mM of the lowing conditions: initial activation at 94 °C for 5 min; 30 cy- desired bile salt (i.e. 2.93 g GCA/L of MRS medium; 3.23 g cles of the denaturation step (94 °C for 30 s each), the anneal- TCA/L of MRS medium; 2.83 g GDCA/L of MRS medium; ing step (55 °C for 30 s each) and the extension step (72 °C for and 3.13 g TDCA/L of MRS medium). The cells were grown 2 min each); and a final treatment (72 °C, 5 min). The for 24 h in the MRS broth without the bile salt and for 36 h in amplicon size was 1436 bp. The PCR primers used for DNA the broth containing the bile salt. When needed, the cells were sequencing were the following: UFUL, 350F (5′-TACG harvested by centrifugation and washed twice with PBS, pH 7. GGAGGCAGCAG-3′), 785F (5′-GGATTAGATACCCT 0. Generally, the cells were suspended in PBS, pH 7.0, and GGTAGTC-3′) and 1099F (5′-GCAACGAGCGCAACCC- adjusted to the desired OD , unless stated otherwise. 600 3′). A portion of the sequences were compared with the se- quences in the GenBank® public database (www.ncbi.nlm. nih.gov/BLAST/). Hexadecane The cells were grown in liquid media and washed as described Assay of the bile salt hydrolase enzyme above. The cell pellet was suspended in PBS (pH 4.0 and of L. plantarum WU-P19 pH 7.0 in separate experiments) to an optical density of 0.25 ± 0.05 measured at 600 nm. Next, 1 mL of n- The bile salt hydrolase activity of L. plantarum WU-P19 was hexadecane was added to 3 mL of the suspended cells and assayed by using a cell-free extract of the cells grown in the mixed for 3 min. The cell suspensions were incubated at 37 °C MRS medium with and without the bile salts GCA and for 1 h. A 200-μL portion of the aqueous part of the cell GDCA. The enzyme activity measured for the cells grown 82 Ann Microbiol (2018) 68:79–91 without the bile salts was taken as the control measurement. In These capsules were carefully sealed to minimise air within separate experiments, both the primary bile salts (GCA, TCA) the capsules. The capsules were then combusted at 950 °C. and secondary bile salts (GDCA, TDCA) were used as sub- The combustion gases were detected by a thermal conductiv- strates for the enzymatic assay. ity detector to determine the carbon and nitrogen contents of Bile salt hydrolase activity in a cell extract was assayed the dry cells. using a modification of a previously published method (Tanaka et al. 2000; Liong and Shah 2005). Thus, the washed Cell morphology of L. plantarum WU-P19 cell pellet from 20 mL of culture broth (OD = 1.0) was suspended in 2 mL of 0.1 M sodium phosphate buffer, Lactobacillus plantarum WU-P19 was grown in a 250-mL pH 7.0. This suspension was sonicated (Vibra-cell, Sonics Duran bottle that contained 250 mL of the MRS medium with Materials, Inc., USA, www.sonics.com) at an amplitude of and without 6 mM of bile salts GCA and GDCA. The culture 42% for 3 min while being held in an ice bath. The broth was sampled 48 h after inoculation. The cells were sonicated suspension was centrifuged to remove the cell washed twice with PBS, pH 7, and recovered by centrifuga- debris. The supernatant was used in determining the bile salt tion. The cell pellet was suspended in 0.1 M sodium phos- hydrolase activity using a two-step procedure. phate buffer, pH 7.2, and thin smeared on a glass slide In the first step, 100 μL of a bile salt substrate (20 mM (0.5 × 0.5 cm). The sample was primary fixed in 2.5% (w/v) aqueous solution) was added to 100 μL of the cell-free extract glutaraldehyde for 2 h and washed three times with 0.1 M containing the enzymes and incubated at 37 °C for 30 min. To sodium phosphate buffer, pH 7.2. Afterwards, the sample this mixture, 50 μL of 15% (w/v) trichloroacetic acid was was secondary fixed with 1% (w/v) osmium tetroxide for added to stop the enzyme reaction. The mixture was then 30 min and washed three times with 0.1 M sodium phosphate centrifuged to remove the precipitate. Each bile salt was tested buffer, pH 7.2. The fixed cells were dehydrated sequentially separately with a given cell extract. with 30, 50, 70, 90, 100% ethanol. The cells were further dried In the second step, 200 μL of the supernatant from the and coated with gold in a sputter coater. The sample was previous step was diluted with 800 μL of distilled water and observed using a scanning electron microscope (Merlin mixed with 1 mL of ninhydrin reagent. This solution was Compact, Zeiss; www.zeiss.com). boiled for 14 min in a test tube and then cooled to room temperature in tap water. Antibiotic susceptibility of L. plantarum WU-P19 The concentrations of the amino acids released after the above treatment were determined by measuring the optical Antibiotic susceptibility of L. plantarum WU-P19 was tested density at 570 nm (OD ) against a blank prepared the same by agar diffusion method described by Vijayakumar et al. way as explained in step 2 above for the supernatant, but with (2015). Analyses were done in duplicate. Petri dishes (diam- the supernatant replaced with 200 μL of distilled water. The eter = 90 mm) containing solidified MRS agar (25 mL per OD measurement was compared to a calibration curve pre- dish) were used. The agar layer thickness in the petri dishes pared using glycine or taurine solutions of a precisely known was around 4 mm. The antibiotic discs were placed on the concentration treated the same way as explained in step 2 surface of MRS agar plates that had been swabbed with the above. One unit of bile salt hydrolase activity (U) was defined bacterial suspension (1.5 × 10 cells/mL grown in MRS broth as the amount of enzyme that liberated 1 μmol of the amino for 24 h at 37 °C). Plates were incubated for 24 h at 37 °C and acid from the substrate (GCA, TCA, GDCA and TDCA) per the diameters of inhibition zones around the discs were mea- min. The protein concentration in the cell-free extract was sured. The bacterium was classified as sensitive (S), interme- determined using the Bradford method (Bradford 1976). The diate (I) or resistant (R) according to the diameters of inhibi- standard curve was prepared with bovine serum albumin. tion zones in accordance with the Clinical and Laboratory Standards Institute (CLSI 2016). Carbon and nitrogen measurements of L. plantarum WU-P19 β-Galactosidase activity of L. plantarum WU-P19 The freshly harvested washed L. plantarum WU-P19 cells β-Galactosidase activity was determined by the method of from the culture broth were frozen at − 80 °C for 1 h and then Miller (1972) as modified by Vinderola and Reinheimer freeze-dried (FDU-2100 freeze dryer; EYELA, Japan, www. (2003). The strain WU-P19 was inoculated in MRS medium eyelaworld.com). Carbon and nitrogen contents in the freeze- supplemented with 2% (w/v) lactose instead of glucose and dried cells were determined by using a CN elemental analyzer grown for 24 h at 37 °C. The cells were harvested by centrifu- (LECO CHN-600; TruSpec, CN, USA). In brief, the freeze- gation (2907 × g, 10 min), washed twice and suspended in Z dried biomass powder (0.2 g) was pressed into tin capsules. buffer. The optical density of the suspension was measured at The pressing eliminated any air pockets from the sample. 600 nm. The cells in suspension (1 mL) were then Ann Microbiol (2018) 68:79–91 83 permeabilised by vortex mixing (7 min) with 50 μLofa tolu- in eight samples. These counts were generally consistent with ene–acetone mixture (toluene:acetone ratio of 1:9 v/v). The the earlier reports. For example, in Vietnamese fermented veg- resulting suspension of permeabilised cells (100 μL) was etables (mustard, beet, eggplant), the reported counts have 8 9 added to 900 μL of Z buffer containing 200 μL of 4 mg/mL been in the range of 10 to 10 CFU/g (Nguyen et al. 2013). o-nitrophenyl-β-D-galactopyranoside (ONPG). The mixture Similarly, in fermented vegetables of the Eastern Himalayas was incubated at 37 °C for 15 min. The reaction was then (gundruk, sinki and khaipi) the microbial counts have ranged 7 8 stopped by adding 0.5 mL of 1 M Na CO and the optical from 10 to 10 CFU/g (Tamang et al. 2005). 2 3 density was measured at 420 nm and 560 nm. The β- Screening of LAB from fermented herbs samples was per- galactosidase activity was expressed in Miller units as follows: formed by sequentially exposing the cells to a PBS solution (pH 2.5), an artificial gastric enzyme solution and an artificial β−galactosidase activityðÞ Miller units small intestinal enzyme solution (Fig. 1). This screening se- quence mimicked the environment any ingested cells would OD −ðÞ 1:75 OD 420 560 ¼ 1000 ð2Þ encounter in the human gastrointestinal tract. Any microor- t v OD ganisms surviving this exposure are likely to be able to persist In Eq. (2), OD is the absorbance of o-nitrophenol released, t and colonize the human gut, the characteristics required of a is the reaction time (min), v is the volume of culture used (mL), potential probiotic LAB. The number of cells surviving after OD is the optical density of the suspension of permeabilised each step of the sequential exposure are shown Table 1. cells and OD is the optical density of the suspension of 600 Exposing the microbes to the simulated environment of the whole cells used in the assay. gastrointestinal tract generally resulted in a 1–2 log reduction in counts (Table 1). Antimicrobial activity of L. plantarum WU-P19 Approximately 4–5 colonies from each fermented herb sample were selected such that they had all of the following Antimicrobial activity of L. plantarum WU-P19 was deter- properties: an ability to survive for 3 h at pH 2.5 and body mined using agar spot method (Hernandez et al. 2005; temperature (37 °C); resistant to porcine gastric mucosa pep- Pithva et al. 2014). An overnight culture of the strain WU- sin and pancreatin enzymes; and resistant to oxgall (Fig. 1). P19 was centrifuged (2907 × g, 10 min), the cells were resus- Therefore, these isolates were potentially able to survive in the pended in 0.85% (w/v) NaCl and the OD measurement was conditions of the gastrointestinal tract. The 19 selected isolates adjusted to 0.132 [1.5 × 10 colony-forming units (CFU)/mL] were gram-positive; of rod or coccus morphology; non-spore- using 0.85% NaCl. A 2 μLsample of this cellsuspension was forming; and catalase-negative. spotted on MRS agar plates and incubated anaerobically at 37 °C for 24 h. After incubation, the indicator strain (OD = 0.5) was mixed with 10 mL of brain heart infusion Table 1 Microbial counts in the enriched MRS broth samples of the (BHI) soft agar (0.7%, w/v agar) and overlaid on the previ- fermented herbs and suspended WU-P19 cells at various stages of expo- ously spotted MRS agar. The plate was incubated at 37 °C for sure to the gastric conditions 24 h and the diameter of the inhibition zone around the spot a b c d Sample M M M M S A AGM ASM was measured. (log CFU/mL) Statistical analyses Fermented herbs Starfruit 7.43 ± 0.02 6.14 ± 0.03 5.96 ± 0.05 5.18 ± 0.03 The BSH activities and hydrophobicity data are presented as Roselle 7.73 ± 0.05 7.46 ± 0.01 6.76 ± 0.03 6.44 ± 0.01 mean values (± standard deviation) of three independent rep- Gac 8.21 ± 0.01 7.47 ± 0.02 6.54 ± 0.06 6.14 ± 0.08 licates. The data were analysed by one-way analysis of vari- Indian trumpet 7.97 ± 0.04 7.26 ± 0.02 6.15 ± 0.05 5.87 ± 0.03 ance (ANOVA) and Tukey’s test. A value of p < 0.05 was Cell suspension taken as a significant difference. WU-P19 8.97 ± 0.02 8.77 ± 0.04 7.62 ± 0.02 6.76 ± 0.03 Microbial count in a sample (fermented herb) after enrichment in MRS medium Results and discussion Microbial count in a sample after exposure to PBS, pH 2.5, for 3 h Microbial count in a sample after exposure to the artificial gastric en- Screening of LAB from fermented herb samples zyme solution for 3 h Microbial count in a sample after exposure to the artificial small intes- The microbial counts of the fermented herb samples (M ,Fig. tine solution for 4 h 1) were in the range of 7.43 ± 0.02 to 8.21 ± 0.01 log CFU/mL The values are averages ± standard deviation of duplicate determinations (Table 1). For each herb, the microbial counts were measured 84 Ann Microbiol (2018) 68:79–91 Once an ingested bacterium has passed through the acid to the host tissue and cause infection compared to pathogens environment of the stomach, it enters the small intestine, with a lower surface hydrophobicity (Doyle 2000; Collado where it mixes with bile. The concentration of bile in human et al. 2008). The cell surface hydrophobicity is therefore one small intestinal tract can range from less than 1 mM to 40 mM of the several criteria used in selecting probiotic bacteria (Islam et al. 2011). Human bile contains nearly 70% w/w (Bautista-Gallego et al. 2013). This work assessed the hydro- GCA and glycochenodeoxycholic acid (GCDCA) (Holm phobicity characteristics of the isolate as one of the properties et al. 2013). Three other bile salts, GDCA, TCA and determining the potential for adhesion to the intestinal walls. taurochenodeoxycholic acid (TCDCA) - together make The eight isolates that withstood the four bile salts were around 23% w/w of the bile (Holm et al. 2013). In addition, tested for surface hydrophobicity at pH 4 and 7, the pH ex- around 2% w/w of TDCA is present (Holm et al. 2013). tremes encountered in the greater part of the human gastroin- Bacteria resistant to bile salts are likely to have a higher sur- testinal tract (Cotter and Hill 2003; Abuhelwa et al. 2016). In a vival rate during passage through the duodenum (Begley et al. dispersion of a hydrophobic liquid such as n-hexadecane in 2005). In view of this, the 19 isolates were further tested for water, the hydrophobic cells attach to the surface of the hy- tolerance to the four bile salts individually (GCA, GDCA, drophobic droplets and this lowers the cell concentration in TCA or TDCA). The results showed that eight isolates the aqueous phase relative to the initial concentration. The (WU-P05, WU-P06, WU-P07, WU-P08, WU-P11, WU- fraction of the cells removed by adsorption to the hydrophobic P13, WU-P14 and WU-P19) grew in the MRS medium sup- dispersed phase is, therefore, a measure of the surface hydro- plemented with the four bile salts. Thus, this group was most phobicity of the cells. likely to afford isolates capable of surviving in the human The hydrophobicity measured at pH 4 and 7 is shown in gastrointestinal tract. Among eight isolates, the fermented Fig. 2 for the relevant isolates. The hydrophobicity of a given herb samples of H. sabdariffa and M. cochinchinensis yielded isolate depended on the pH of the aqueous phase. For all three isolates each. isolates, the hydrophobicity at neutral pH was lower than the The colonies of eight isolates grown in the MRS medium hydrophobicity at the acidic pH (Fig. 2). The pH dependence supplemented with one of the four bile salts exhibited two of hydrophobicity is linked to the effect of pH on the ioniza- distinct characteristics. One group (Group A) of the isolates tion of the functional groups at the surface of a cell. Changes (WU-P07 and WU-P19) produced opaque white colonies and in ionization affect the surface charge density and therefore the caused a precipitate to form in all of the MRS media supple- hydrophobicity. The increased hydrophobicity at acidic pH mented with the bile salts. A second set (Group B) of the implied a lower surface charge density under acidic condi- isolates (WU-P05, WU-P06, WU-P08, WU-P11, WU-P13 tions. This would occur if the predominant functional group and WU-P14) formed visibly opaque white colonies and on the cell surface was −COOH, for example. Under acidic conditions such a functional group would be uncharged. The caused precipitation in the MRS supplemented with TCA and TDCA. The Group B isolates also produced translucent colonies in the MRS medium supplemented separately with pH 7 GDCA. Precipitation in media supplemented with the bile pH 4 salts is associated with the BSH-mediated formation of the 40 a free bile acids that are poorly soluble in water in a low pH environment (Begley et al. 2006). LAB produce both the BSH and the acids that lower the culture pH. d d Selectionof LAB based onhydrophobicity atpH 4 bc 20 c and 7 To colonize the human gut, a bacterium must not only survive the conditions encountered during transit through the gastro- intestinal tract, but must also adhere to the intestinal walls or it will be removed with the intestinal flow. Bacterial character- istics known to influence adhesion to surfaces are hydropho- bicity, the zeta potential, the motility, and the ability to pro- Bacterial isolate duce extracellular adhesive substances such as polysaccha- Fig. 2 Hydrophobicity of lactic acid bacteria in n-hexadecane. All rides, proteins and biosurfactants (Roosjen et al. 2006). isolates had been grown in the MRS medium free of bile salts. The Many pathogenic bacteria are known to attach to the host washed cells were suspended in PBS, pH 4 or pH 7, before the tissue prior to multiplying and causing infection. Pathogens measurements. The different letters above the bars for a given pH value indicate a statistically significant difference (p <0.05) with a high surface hydrophobicity tend to adhere more easily Hydrophobicity (%) WU-P05 WU-P06 WU-P07 WU-P08 WU-P11 WU-P13 WU-P14 WU-P19 Ann Microbiol (2018) 68:79–91 85 Table 2 Specific activity of BSH enzyme in cell-free extracts of the of some LAB were reported to be much higher compared to isolate WU-P19 grown in different media the values shown in Fig. 2. For example, the hydrophobicity has ranged: from 28% to 50% for L. paracasei;from 74%to Medium Specific activity (U/mg)** 94% for L. johnsonii; and from 23% to 88% for L. acidophilus GCA* TCA* GDCA* TDCA* (Schillinger et al. 2005). In the present study, the hydropho- bicity never exceeded about 40% (Fig. 2) and for a majority of b a b a MRS 4.04 ± 0.18 3.81 ± 0.33 4.63 ± 0.65 3.42 ± 0.47 the isolates it was less than 25% (Fig. 2). A hydrophobicity a a b a MRS-GCA 5.41 ± 0.04 3.74 ± 0.21 4.28 ± 0.55 3.58 ± 0.44 value of 31% has been previously reported for L. plantarum b a a a MRS-GDCA 4.10 ± 0.50 3.30 ± 0.27 7.38 ± 0.55 3.23 ± 0.16 (Zago et al. 2011). Many bacterial pathogens are known to attach to epithelial Results are expressed as the means ± standard deviations of three independent replicates. The different superscript letters within a column cells to initiate infection. Therefore, a comparison of the hy- indicate a statistically significant difference (p <0.05) drophobicity of such bacteria with the values found for the *Substrate used for measuring the BSH activity isolates in the present work (Fig. 2) is of potential interest. **Specific activity (U/mg) was defined as the amount of enzyme that For the food-borne pathogen Listeria monocytogenes, the hy- liberated 1 μmol of amino acid from a substrate (i.e. GCA, TCA, drophobicity values have ranged from about 10% to 39% GDCA and TDCA) per min per milligram of protein (Skovager et al. 2013). For Escherichia coli 35150, Escherichia coli 8 and Bacillus cereus, the hydrophobicity pH-dependent hydrophobicity changes were generally consis- values have ranged from 4% to 39% (Popovici et al. 2014). tent with the literature. For example, the surface charge of In the present study, the hydrophobicity value of the isolate LAB has been reported to depend on pH (Poortinga et al. WU-P06 (Fig. 2) was comparable to the hydrophobicity value 2002). The hydrophobicity of L. lactis subsp. lactis biovar. reported for the pathogen Salmonella typhimurium diacetylactis strain LLD18 was reported to increase with a ATCC14028 (Nguyen et al. 2014). decrease in pH (Ly et al. 2008), as was the case for most of the isolates in the present study (Fig. 2). For different bacteria, the hydrophobicity and its depen- Identification of the isolate WU-P19 dence on the pH was different (Fig. 2). This was likely be- cause of the strain-dependent differences in the surface density The isolate WU-P19 having a high hydrophobicity at both of the ionizable functional groups and the specific types of the pH 4 and pH 7 (Fig. 2) was selected for further evaluation of functional groups present. For example, at pH 3, L. plantarum potential probiotic properties. This isolate was identified as C4 was found to have a hydrophobicity of 16%, whereas L. plantarum WU-P19 based in the 16S rDNA sequence. L. rhamnosus GG had a much higher hydrophobicity of (This sequence was 100% homologous with the 16S rDNA 74% (Bergillos-Meca et al. 2015). of Lactobacillus plantarum strain Ni1323. The GenBank® The cell wall of LAB consists mainly of peptidoglycans, accession number for the reference 16S rDNA gene sequence (lipo)teichoic acids, proteins and polysaccharides (N- is AB598972.1.) L. plantarum have been isolated from vari- acetylglucosamine and N-acetyl-muramic acid units) ous other fermented foods and also from the human gastroin- (Delcour et al. 1999). Differences in the relative proportions testinal tract (Kaushik et al. 2009; Iqbal et al. 2010). of these surface components in different strains affect the mag- Lactobacillus plantarum is accepted as probiotic in many nitude of the pH effect on the surface ionization. In general, jurisdictions. For example, Health Canada (http://www.hc-sc. isolates rich in surface proteins and (lipo)teichoic acids tend to gc.ca/fn-an/label-etiquet/claims-reclam/probiotics_claims- be more hydrophobic compared to isolates having a high pro- allegations_probiotiques-eng.php#fnb1)accepts L. plantarum portion of the hydrophilic polysaccharides (Schär-Zammaretti and several other species of the genus Lactobacillus as and Ubbink 2003). In earlier work, the hydrophobicity values probiotics contributing to a Bhealthy gut flora^ when Table 3 Hydrophobicity and Medium Hydrophobicity Carbon (%) Nitrogen (%) carbon and nitrogen contents of the isolate WU-P19 grown in Hexadecane (%) Contact angle (°) different media MRS 32.0 ± 0.5 37.37 ± 0.32 13.3 ± 0.2 2.9 ± 0.0 MRS-GCA 26.1 ± 0.9 25.73 ± 1.03 20.8 ± 2.5 2.7 ± 0.1 MRS-TCA 25.2 ± 1.7 22.85 ± 0.50 20.9 ± 0.3 2.9 ± 0.1 MRS-GDCA 14.3 ± 1.0 17.20 ± 0.57 26.3 ± 0.3 0.7 ± 0.0 MRS-TDCA 20.8 ± 0.5 23.55 ± 0.49 34.2 ± 0.4 2.1 ± 0.1 86 Ann Microbiol (2018) 68:79–91 Fig. 3 Contact angles of the isolate WU-P19. The spread of a distilled water drop on a lawn of the isolate WU-P19. The isolate had been grown in the MRS medium: free of bile salts (a); and with the bile salt GDCA (b) delivered in food at a level of 1 × 10 CFU per serving, so long Thus, excretion of the deconjugated bile salts in the faeces is as no strain-specific claims are made. increased through the action of BSH producing microorgan- isms. To compensate for the loss, the liver must produce more bile salts (Begley et al. 2006), and this consumes some of the Bile salt hydrolase and β-galactosidase activities cholesterol that would otherwise appear in the bloodstream. of L. plantarum WU-P19 A probiotic microorganism possessing β-galactosidase ac- tivity is potentially useful as it can impart an ability to digest The specific activities of bile salt hydrolase of L. plantarum lactose in cases of low lactose tolerance. Furthermore, β- WU-P19 grown in the control medium (i.e. MRS without the galactosidase in a microorganism also imparts the ability to bile salts) were lower than the corresponding specific activi- make prebiotic galacto-oligosaccharides (Iqbal et al. 2010). β- ties in the MRS media supplemented with the bile salt galactosidase activity of L. plantarum WU-P19 was 747 (Table 2). Thus, the presence of the substrate in the culture Miller units. This was comparable to other reported data, al- medium induced the production of the enzyme. though L. plantarum strains can differ greatly in their β- Many strains of L. plantarum are known to produce BSH galactosidase activities (Zago et al. 2011). For example, for (Kumar et al. 2012, 2014), but the specific activity differs strains isolated from cheeses, the strain Lp996 had an activity from strain to strain (Kumar et al. 2012, 2014). BSH activities of 1062.14 Miller units, but the activity of strain Lp994 was of 138.05 U/mg-protein (GCA as substrate), 65.01 U/mg-pro- only 32.17 Miller units (Zago et al. 2011). Lactobacillus tein (GDCA as substrate), 70.54 U/mg-protein (TCA as sub- plantarum MCC2156 was reported to have the same level of strate) and 89.41 U/mg-protein (TDCA as substrate) have β-galactosidase activity as the strain Lp996 (Gobinath and been reported in L. plantarum NCDO82 from the National Prapulla 2014). Collection of Dairy Organisms, Australia (Kumar et al. 2012, 2014). Similarly, the BSH activities of L. plantarum Lp21 isolated from human faeces were 157.01 U/mg-protein Effects of bile salts on the hydrophobicity (GCA as substrate), 67.66 U/mg-protein (GDCA as substrate), of L. plantarum WU-P19 89.92 U/mg-protein (TCA as substrate) and 98.02 U/mg-pro- tein (TDCA as substrate) (Kumar et al. 2012, 2014). In com- The effects of bile salts on hydrophobicity of L. plantarum parison with these data, the activities for L. plantarum WU- WU-P19 were assessed. Thus, the bacterium was grown in the P19 in the present work were generally higher (Table 2), as the MRS medium without the bile salts (control experiment) and activities measured for the strains NCDO82 and Lp21 had in the same medium supplemented separately with 6 mM of been defined as the amount of enzyme that liberated 1 nmol one of the four bile salts (GCA, TCA, GDCA and TDCA) of the amino acid from the substrate per min (Kumar et al. (exposed experiments). Hydrophobicity tests used both the 2012, 2014). n-hexadecane method and the water contact angle method. In principle, intestinal colonization by a probiotic microor- The results are shown in Table 3 and Fig. 3. ganism possessing BSH activity has the potential to lower The contact angle of a water droplet on a surface decreases blood cholesterol in vivo. Bile salts are produced in the liver as the surface becomes more hydrophilic. The contact angle through conjugation of a cholesterol-derived steroidal moiety measurements shown in Table 3 fully concurred with the hy- with amino acids. A substantial fraction of the bile salts re- drophobicity measurements using hexadecane. The contact leased into the duodenum is reabsorbed in the gastrointestinal angle of a water droplet on a lawn of bacterial cells reduced tract. BSH cleaves the peptide linkage of the bile salts to relative to the control, if the cells were grown in media sup- remove the amino acid moiety from the steroidal part. This plemented with bile salts (Table 3,Fig. 3). The bile salt GDCA deconjugated steroidal substance is poorly soluble in water was the most effective in reducing the droplet contact angle and is less readily reabsorbed than the counterpart bile salt. (Fig. 3, Table 3) and hydrophobicity (Table 3). Ann Microbiol (2018) 68:79–91 87 That is, they coat the hydrophobic oil droplets to make them more hydrophilic, or water-soluble. A similar mech- anism might be operating in reducing the hydrophobicity of the cells. Bile salts attach to hydrophobic surfaces through the hydrophobic regions in their molecules. Thus, the hydrophobic surface becomes coated with bile salts such that the hydrophilic part of the molecule faces outwards towards the aqueous phase. The surface, there- fore, becomes less hydrophobic. A water droplet in contact with a hydrophilic surface spreads more, or better wets the surface, compared to a droplet in contact with a hydrophobic surface. A lawn of L. plantarum WU-P19 grown in the control medium was 200 nm Mag = 20.00 K X EHT = 1.00 kV WD = 3.6 mm clearly more hydrophobic (Fig. 3a) compared to a lawn of the same bacterium grown in the medium supplemented with GDCA (Fig. 3b). In keeping with these results, bile salts were reported to reduce the cell hydrophobicity of L. delbrueckii subsp. lactic 200 (Burns et al. 2011)al- though supplementation with bile has been reported to make L. brevis KB290 cells more hydrophobic relative to the control (Kimoto-Nira et al. 2015). Carbon and nitrogen contents of L. plantarum WU-P19 In view of the above results, the cells grown in the media supplemented with the bile salts were less hydrophobic compared to the cells grown in the medium without the 300 nm Mag = 20.00 K X EHT = 1.00 kV WD = 3.8 mm bile salts (Table 3). This suggested that the presence of the bile salts in the growth medium potentially modified the chemical composition of the bacterial cell. As carbon (C) and nitrogen (N) are among the main constituents of a microbial cell, these elements were measured in the bio- mass of L. plantarum WU-P19 grown in the MRS with and without the bile salts. The results are shown in Table 3. On exposure to bile salts, the C- and N- contents of the biomass changed. The magnitude of this change depended on the specific bile salt added to the growth medium. Scanning electron microscopy (SEM) of L. plantarum WU-P19 300 nm Mag = 20.00 K X EHT = 1.00 kV WD = 4.7 mm SEM images of L. plantarum WU-P19 grown in the MRS Fig. 4 Scanning electron microscopy images of the isolate WU-P19 medium with and without GCA and GDCA are shown in grown in the MRS medium: free of bile salts (a); supplemented with 6 mM GCA (b); and supplemented with 6 mM GDCA (c). All cultures Fig. 4. Presence of GCA (Fig. 4b) and GDCA (Fig. 4c) were incubated anaerobically at 37 °C for 48 h affected cell morphology relative to control (Fig. 4a). Cells grown with the bile salts were clearly surrounded by an external translucent shroud. The volume of the Molecules of bile salts contain both hydrophilic (water- shroud was much larger for the cells grown with GDCA soluble) and non-polar hydrophobic regions. In human (Fig. 4c) compared to cells grown with GCA (Fig. 4b). intestine, the bile salts act as emulsifiers of fats and oils. Therod-likecells withintheshroudhadaroughsurface 88 Ann Microbiol (2018) 68:79–91 Table 4 Antibiotic susceptibility of the isolate WU-P19 using the disc Table 5 Antimicrobial spectrum of the isolate WU-P19 against indicat- diffusion method ed bacterial pathogens a,b Antibiotic Disc Zone of Classification Indicator pathogen Zone of potency (μg) inhibition (mm) inhibition (mm) Amikacin 30 15 I Bacillus cereus 32 ± 1 Ampicillin 10 37 S b Enterococcus faecalis 24 ± 1 Bacitracin 10 19 S Escherichia coli O157:H7 27 ± 1 Cefotaxime 30 44 S Cefoxitin 30 15 S Listeria monocytogenes ATCC 7644 34 ± 1 Ceftriaxone 30 44 S Salmonella enteritidis DMST 15676 22 ± 1 Ciprofloxacin 5 0 R Salmonella serovar Typhi DMST 02842 27 ± 1 Chloramphenicol 30 32 S Clindamycin 2 16 I Staphylococcus aureus ATCC 25923 17 ± 1 Erythromycin 15 26 S Gentamicin 10 0 R The results are expressed as the means ± standard deviations of three Kanamycin 30 0 R independent replicates Nalidixic acid 30 0 R The indicator strains Bacillus cereus and Enterococcus faecalis were Penicillin G 10 29 S obtained from Thasala Hospital (Nakhon Si Thammarat, Thailand). The Streptomycin 10 15 S other strains were obtained from the American Type Culture Collection Vancomycin 30 0 R (ATCC), USA, and the Department of Medical Sciences Culture Tetracycline 30 28 S Collection (DMST), Thailand Tobramycin 10 0 R Trimethoprim 5 24 S discussed earlier with reference to Table 3,the translucent R: resistant, I: intermediate, S: susceptible substances surrounding the cells grown in MRS supple- Clinical and Laboratory Standards Institute (CLSI 2016) mented with GDCA reduced the surface hydrophobicity to 14.3, or about 45% of the control (MRS medium, when grown with GCA (Fig. 4b) but the surface was Table 3). The envelope around the cells (Fig. 4b, c) likely smooth when the cells were grown with GDCA. consisted of exopolysaccharides. In certain intestinal bac- teria, bile salts have been found to induce the production These differences in cell morphology were attributed to differences in toxicity (i.e. bactericidal effect) of the bile of exopolysaccharides (Ruas-Madiedo et al. 2009), and these have the potential to alter the hydrophobicity. salts added to the medium. The cells within the shroud had a rough surface when grown with the toxic GCA. As Production of an extracellular layer of polysaccharides Fig. 5 Representative photographs of: (a) antibiotic susceptibility of isolate WU-P19 measured by the disc diffusion method, (b) inhibition zones on lawns of the specified bacterial pathogens, produced by the isolate WU-P19 using the agar spot method. In b, inhibition zones are shown for the pathogens Bacillus cereus, Salmonella enteritidis DMST 15676 and Staphylococcus aureus ATCC 25923 Ann Microbiol (2018) 68:79–91 89 around the cell wall may be a protective response (Ruas- TCA, GDCA and TDCA as substrates and possessed β- Madiedo et al. 2009) against the bactericidal bile salts. galactosidase activity. Presence of these bile salts in the growth medium affected the carbon and nitrogen contents in Antibiotic susceptibility and antimicrobial properties cells, altered the cell morphology and reduced the cell hydro- of L. plantarum WU-P19 phobicity. WU-P19 inhibited growth of both gram negative and gram positive bacteria at different levels. WU-P19 was The bacterium WU-P19 was found to be resistant to gentami- resistant to several antibiotics. Passaging the bacterium cin, kanamycin, vancomycin, ciprofloxacin, tobramycin and through the conditions of the gastrointestinal tract in vitro nalidixic acid, but it was susceptible to ampicillin, bacitracin, caused an approximate 2 log reduction in cell counts. In view cefotaxime, cefoxitin, ceftriaxone, chloramphenicol, erythro- of its capabilities in vitro, a probiotic assessment of the isolate mycin, penicillin G, streptomycin, tetracycline and trimetho- WU-P19 is planned in vivo. prim (Table 4,Fig. 5a). This resistance and susceptibility pat- Acknowledgements This research was funded by the Royal Golden tern generally agreed with data reported previously (Abriouel Jubilee (RGJ) PhD Program (PHD/0258/2553 code 6.Q.WL/53/A.1) et al. 2015). Resistance to vancomycin of L. plantarum, and the Higher Education Research Promotion and National Research L. paracasei, L. salivarius and L. rhamnosus is an intrinsic University Project of Thailand, Office of the Higher Education resistance that is a consequence of structural components of Commission. their peptidoglycan (Blandino et al. 2008). This intrinsic re- Compliance with ethical standards sistance to vancomycin is not transferred to vancomycin- susceptible enterococci (Klein et al. 1998; Klein et al. 2000). Conflict of interest The authors declare that they have no competing Although the strain WU-P19 was not resistant to penicillin interest. G and streptomycin, some LAB isolated from fermented foods are known to have developed resistance to penicillin G (Belletti et al. 2009; Lapsiri et al. 2011). 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Journal
Annals of Microbiology – Springer Journals
Published: Dec 22, 2017