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Identification and analysis of the function of surface layer proteins from three Lactobacillus strains

Identification and analysis of the function of surface layer proteins from three Lactobacillus... To identify and investigate the role of surface layer proteins (SLPs) on the probiotic properties of Lactobacillus strains, SLPs were extracted from Lactobacillus bulgaricus fb04, L. rhamnosus fb06, L. gasseri fb07, and L. acidophilus NCFM by 5 mol/L lithium chloride. The molecular masses of the four SLPs were approximately 45–47 kDa as analyzed by SDS-PAGE. Hydrophobic amino acids were the main components of the four SLPs. The secondary structure content of the four SLPs showed extensive variability among different strains. After the SLPs were removed from the cell surface, the autoaggregation ability, coaggregation ability, and gastrointestinal tolerability of the four lactobacilli were significantly reduced as compared with the intact cells (P < 0.05). When exposed to bile salt stress, L. rhamnosus fb06, L. gasseri fb07, and L. acidophilus NCFM expressed more SLPs as determined by Bradford method. In conclusion, the four lactobacilli all possessed functional SLPs, which had positive contributions to the probiotic properties of the four Lactobacillus strains. This research could reveal the biological contributions of SLPs from Lactobacillus strains and offer a theoretical basis for the application of lactobacilli and their SLPs in food and pharmaceutical industries. . . . . Keywords Surface layer proteins Lactobacillus Amino acid composition Secondary structure Probiotic function Introduction intestinal tissues are reported to be relevant to the cell-surface components of bacteria, such as lipoteichoic acids, extracellular Lactobacilli have been identified as main members of probiotic polysaccharides, and cell surface proteins (Jakava-Viljanen and strains. They could maintain the stability of gastrointestinal tract Palva 2007). and prevent pathogen infections, when ingested in sufficient Bacterial surface layers, usually found outside the cell wall number (Jakava-Viljanen and Palva 2007;Martínezetal. of Eu- and Archae-bacteria, are composed of proteins, which 2012). Due to their health-promoting functions, lactobacilli could self-assemble into a sub-lattice array structure (Smit and have been widely used in food and pharmaceutical industries. Pouwels 2002). Surface layers are attached to cell wall The mechanisms of their probiotic activities have been attribut- through noncovalent interactions, including hydrogen bond, ed to the mucosal barrier function, modulation of the immune ionic bond, and hydrophobic interactions, and can be routinely response, coaggregation with pathogens, competitive exclu- extracted by chaotropic denaturants or high concentration dis- sion, and displacement of pathogens (Beganović et al. 2011; sociating agents (Navarre and Schneewind 1999). Many spe- Lee et al. 2003). The interactions between lactobacilli and cies of lactobacillus have been demonstrated to possess sur- face layer proteins (SLPs) and show the molecular masses ranging from 25 to 71 kDa, accounting for 10–15% of the total cellular protein (Sára and Sleytr 2000;Chenetal. * Rong-Rong Lu 2007). SLPs are highly basic proteins, with calculated pI lurr@jiangnan.edu.cn values ranging from 9.35 to 10.40. School of Food Science and Technology, Province Key Laboratory SLPs may have various biological functions. Many reports of Transformation and Utilization of Cereal Resource, Henan have revealed that SLPs are the key components for lactobacilli University of Technology, Zhengzhou, Henan Province 450001, to play their probiotic role in the host. Autoaggregation and China coaggregation abilities are the important surface properties of School of Food Science and Technology, Jiangnan University, 1800 lactobacilli, which could contribute a lot to the colonization Lihu Avenue, Wuxi, Jiangsu 214122, China 208 Ann Microbiol (2018) 68:207–216 and antibacterial function of lactobacilli in the gastrointestinal L. rhamnosus fb06, and L. bulgaricus fb04 were isolated from tract (Collado et al. 2008). It has been reported that the SLPs of fermented food. The lactobacilli were grown under aerobic con- Lactobacillus crispatus, L. acidophilus,and L. helveticus have a ditions in De Man-Rogosa-Sharpe (MRS) broth (Oxoid) over- positive contribution to the autoaggregation and coaggregation night for 18 h at 37 °C in a shaker (DKY-II, Duke automation ability of their strains (Kos et al. 2003; Chen et al. 2007; equipment Co., Ltd., Shanghai, China) at 180 rpm. E. coli Beganović et al. 2011). The SLPs of L. acidophilus M92 and ATCC 43893 and S. typhimurium CMCC 50013 were grown L. acidophilus ATCC 4356 could help the strains to adapt to in Luria-Bertani (LB) medium overnight for 18 h at 37 °C in a adverse living conditions (Frece et al. 2005; Khaleghi et al. shaker. 2010). Many studies have implicated the involvement of some SLPs from lactobacilli in adhesion to epithelial cells or extracel- Transmission electron microscopy lular matrix proteins (Lorca et al. 2002; Leeuw et al. 2006;Sun et al. 2012;Mengetal. 2014). The SLPs of L. acidophilus, Lactobacilli cells were collected by centrifugation (5000×g, L. helveticus,and L. plantarum were reported to be involved 10 min, 4 °C) and washed twice with phosphate buffer solu- in pathogen inhibition (Meng et al. 2014). tion (PBS; pH 7.2). For untreated samples, the washed cells Due to the great diversity of SLPs, many kinds of SLPs were suspended in PBS. For LiCl-treated samples, the cells have not been clearly investigated. Most of the reports about were suspended in 5 mol/L lithium chloride, and all the sam- SLPs were focused on the L. acidophilus, L. helveticus, ples were agitated for 30 min at room temperature. L. crispatus,and L. brevis strains. Some strains, which were TEM was performed according to Gerbino et al. (2011). researched in our manuscript, such as L. gasseri, L. rhamnosus, Cells were suspended in glutaraldehyde solution and fixed for and L. bulgaricus, have been widely used as probiotics in food 2 h. Fixed cells were stained with phosphotungstic acid and industry. But the characterization and function of their SLPs examined through an H-7650 TEM (Hitachi, Tokyo, Japan) at were lacking of report. In this study, the SLPs were extracted an operating voltage of 80 kV. from L. gasseri fb07, L. rhamnosus fb06, and L. bulgaricus fb04. L. acidophilus NCFM was set as a reference strain, as its Preparation of SLPs and SDS-PAGE analysis SLPs were verified in previous studies (Johnson et al. 2013; Meng et al. 2015). The physicochemical properties of the four SLPs of the four lactobacilli were extracted according to SLPs and their possible contributions to the probiotic proper- Meng et al. (2014). Lactobacilli cells were washed twice with ties of Lactobacillus strains were studied. This research could PBS and suspended in 5 mol/L lithium chloride (LiCl). After reveal the biological contribution of these SLPs to the probiotic treatment for 30 min at room temperature, SLPs were collect- properties of lactobacilli and advance our understanding about ed by centrifugation (12,000×g, 4 °C, 15 min) and dialyzed by the probiotic mechanism. distilled water at 4 °C, then lyophilized (Labconco Corp., Kansas City, MO, USA). The SLPs were mixed with 2× loading buffer, boiled for Materials and methods 5 min, and analyzed by SDS-PAGE using 12% polyacryl- amide gels. Protein bands were made visible by staining with Chemicals Comassie brilliant blue. SLPs were purified according to Meng et al. (2015). Low molecular weight marker from 14.4 to 97.4 kDa was Briefly, the lyophilized SLPs were dissolved in 8 mol/L purchased from the China National Medicines Corporation urea–Tris–hydrochloric acid solution and purified by chroma- (Shanghai, China). Pepsin (1:10,000) and trypsin (1:250) were tography on a cation-exchange column. purchased from the Bio Basic Company (Bio Basic Inc., Canada). The other chemicals were common reagent of ana- Amino acid content analysis of SLPs lytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). The amino acid content of SLPs was identified using the method described by Lu et al. (2010). Briefly, SLP samples Bacterial strains and growth conditions were digested with 6 M HCl under atmospheric nitrogen at 110 °C for 24 h. Then, they were filtered with two filter mem- L. acidophilus NCFM, L. gasseri fb07, L. rhamnosus fb06, branes. The filtrate was centrifuged at 3000g for 30 min, and L. bulgaricus fb04, Escherichia coli ATCC 43893 (O124:NM, the clear supernatant, containing mainly amino acids, was causing infectious diarrhea), and Salmonella typhimurium derivatized by O-phthaldialdehyde (OPA) and 9- CMCC 50013 (O4,5:Hi:2, causing acute gastroenteritis) were fluorenylmethoxycarbonyl chlorine (FMOC-Cl), followed provided by the Research Center of Food Biotechnology, by RP-HPLC analysis in an Agilent (Agilent Technologies, Jiangnan University, Jiangsu, China. L. gasseri fb07, Palo Alto, CA, USA) assembly system, using a Zorbax 80A Ann Microbiol (2018) 68:207–216 209 C column (4.6 × 180 mm), running at 0.5 mL/min. Survival of lactobacilli in simulated gastric and small Methionine and cysteine were determined separately as their intestinal juice oxidation products, according to the performic acid procedure, prior to hydrolysis in 6 M HCl. The results were processed This experiment was carried out according to Frece et al. (2005) with the aid of ChemStation for LC 3D software (Agilent with some modifications. Simulated gastric juice was prepared −1 Technologies, Palo Alto, CA, USA). by dissolving pepsin into sterile NaCl solution (5.0 g L ). The −1 final concentrationofpepsinwas 3.0g L ,and thepHofthe solution was adjusted to 2.0 with HCl. Simulated small intesti- Secondary structure analysis by circular dichroism nal juice was prepared by dissolving trypsin and bile salts into −1 sterile NaCl solution (5.0 g L ). The final concentration of −1 Circular dichroism analysis was performed according to the trypsinandbilesalts was1.0and1.5gL . The pH of the method described by Meng et al. (2014) using a MOS-450/ solution was adjusted to 8.0 with 0.1 mol/L NaOH. AF-CD spectrometer (Bio-Logic, Grenoble, France). SLPs Lactobacilli cells were collected by centrifugation were placed in a 1 mm path length quartz cuvette at the (5000×g, 10 min, 4 °C) and washed twice with phosphate- −1 concentration of 0.1–0.2mgmL . The scanning wave- buffered saline (PBS, pH 7.2). The washed cells were length was set as 190–250 nm, and the scanning rate was suspended in simulated gastric or small intestinal juice to −1 8 set as 100 nm min . A buffer sample with no SLPs was 5×10 CFU/mL and incubated for 4 h at 37 °C. The changes subtracted from all spectra to account for any background in total viable counts were monitored every 1 h by the pour signal. plate method, using MRS agar incubated at 37 °C for 48 h. SLP expression under bile salt stress conditions Autoaggregation assays For studying the effect of bile salt stress, the experiment was Autoaggregation assays were performed according to Chen performed as described by Khaleghi et al. (2010) with some et al. (2007) with some modifications. Lactobacilli cells were modifications. MRS broth containing 0, 0.1, 0.2, 0.5, or −1 collected by centrifugation (5000×g,10min,4 °C)and 1.0 g L bile was prepared. Inoculum of 2% (v/v)of washed twice with PBS (pH 7.2). The washed cells were lactobacilli was used in the samples. All the samples were suspended in PBS to approximately 10 CFU/mL. The absor- incubated at 37 °C for 18 h in a shaker. After incubation, bance of the bacteria suspension was measured at 600 nm lactobacilli cells were washed twice with phosphate-buffered (A ). Bacteria suspension of 3 mL was stirred 10 s. After 4-h saline (PBS, pH 7.2). The OD was adjusted to 0.5 by PBS. 0 600 incubation at room temperature, the absorbance was measured Lactobacilli cells collected from 100 mL of the prepared sus- at 600 nm (A ). The percentage of autoaggregation was cal- pension were used to extract SLPs according to Meng et al. culated as (1–A /A ) × 100. (2014). The concentration of the SLPs was determined by 4 0 Bradford method (Bradford 1976). Briefly, 100-μg/mL bovine serum albumin solution was prepared as protein standard so- Coaggregation assays lution. Coomassie® Brilliant Blue G-250(100 mg) was dis- solved in 50 mL 95% ethanol. To this solution, 100 mL 85% The coaggregation of lactobacilli with E. coli ATCC 43893 or (w/V) phosphoric acid was added. The resulting solution was S. typhimurium CMCC 50013 was determined according to then diluted to a final volume of 1 L by distilled water. Three- the method of Kos et al. (2003). The method for preparing cell milliliter G-250 solution was added into 0–1.0-mL protein suspensions of lactobacilli, E. coli ATCC 43893, and standard solution, respectively, to establish a standard curve S. typhimurium CMCC 50013 was the same as that for equation. The absorbance was measured at 595 nm. Three- autoaggregation. Equal volumes (2 mL) of lactobacilli and milliliter G-250 solution was added into 100-μL SLP solutions pathogen suspensions were mixed together by vortexing for (PBS, pH 7.2). After reaction, OD was determined. SLP 10 s. Four milliliters of each bacterial suspension was set up as concentrations were calculated by the standard curve equation. control groups at the same time. The absorbance of the sus- pensions at 600 nm was determined after 4-h incubation at Statistical analysis room temperature. The coaggregation percentage was calcu- lated as (1 − A /[(A + A )/2]) × 100, where A represents the All of the experiments were conducted at least in triplicate. M L P M absorbance of the mixture after 4-h incubation, A represents The data were analyzed with a one-way ANOVA that set the absorbance of lactobacilli suspension, A represents the significance at P < 0.05. SPSS 18.0 (SPSS Inc., Chicago, absorbance of pathogen suspension (E. coli ATCC 43893 or USA) for Windows was used in the statistical analysis. The S. typhimurium CMCC 50013). results were presented as means ± standard errors. 210 Ann Microbiol (2018) 68:207–216 Fig. 1 Transmission electron microscopy of the cell surface structures of the four lactobacilli. a L. bulgaricus fb04; b L. rhamnosus fb06; c L. gasseri fb07; d L. acidophilus NCFM. 1- Untreated lactobacilli; 2- Lactobacilli treated by 5 mol/L LiCl. Arrows: the surface layer Ann Microbiol (2018) 68:207–216 211 shown in Fig. 1, the untreated lactobacilli were covered with a layer of extracellular structure. After treatment by LiCl, the surface layers were removed. In order to identify the SLPs, the LiCl-extracts were analyzed in the following SDS-PAGE experiment. In each LiCl-extracts, one major band was found (lanes 1 to 4, Fig. 2). The SLP of L. acidophilus NCFM (lane 1) was set as a reference protein, because its molecular mass had been reported as 46 kDa (Johnson et al. 2013). SDS-PAGE analysis identified the presence of SLPs as extracted from L. gasseri fb07, L. rhamnosus fb06, and L. bulgaricus fb04 strains. The molecular masses were approximately 45–47 kDa. Fig. 2 SDS-PAGE profiles of SLPs extracted from lactobacilli by 5 mol/L LiCl. Line 1, L. acidophilus NCFM; line 2, L. gasseri fb07; line 3, L. rhamnosus fb06; line 4, L. bulgaricus fb04 Amino acid composition of SLPs Results The amino acid compositions of the four extracted SLPs are shown in Table 1. The hydrophobic amino acids in the four Identify the presence of SLPs from lactobacilli SLPs accounted for about 35–45%, indicating that the confor- mation of the SLPs might be maintained by the strong hydro- In this work, SLPs were extracted by 5 mol/L LiCl. To ob- phobic interactions. The content of basic amino acids in the serve the surface structure of the four lactobacilli, both untreat- four SLPs was all higher than that of the acid amino acids. It ed and LiCl-treated samples were examined by TEM. As could be speculated that the isoelectric points of these proteins −1 Table 1 Amino acid composition of the SLPs (mg g protein) Amino acid L. bulgaricus fb04 L. rhamnosus fb06 L. gasseri fb07 L. acidophilus NCFM Asp 7.16 ± 0.43 6.02 ± 0.21 10.31 ± 0.56 10.20 ± 0.47 Glu 7.48 ± 0.55 7.21 ± 0.32 8.03 ± 0.38 4.36 ± 0.24 Ser 6.61 ± 0.44 7.50 ± 0.48 2.10 ± 0.27 6.75 ± 0.22 His 4.35 ± 0.50 2.62 ± 0.14 2.01 ± 0.49 4.88 ± 0.84 Gly 3.21 ± 0.66 6.22 ± 0.37 5.81 ± 0.41 3.17 ± 0.43 Thr 5.96 ± 0.65 6.78 ± 0.88 5.46 ± 0.49 11.79 ± 1.82 Arg 6.19 ± 0.34 4.52 ± 0.59 10.91 ± 0.61 5.52 ± 0.75 Ala 9.73 ± 0.93 11.7 ± 1.44 10.82 ± 0.98 10.62 ± 1.01 Tyr 2.42 ± 0.28 4.97 ± 0.73 1.03 ± 0.13 7.64 ± 0.44 Cys-s 0.10 ± 0.02 0.06 ± 0.01 0.21 ± 0.03 0.00 Val 9.38 ± 0.48 9.21 ± 0.86 8.13 ± 0.45 11.81 ± 0.77 Met 1.03 ± 0.15 1.92 ± 0.20 0.07 ± 0.01 0.55 ± 0.02 Phe 5.89 ± 0.14 5.07 ± 0.29 5.41 ± 0.41 2.73 ± 0.20 Ile 7.81 ± 0.71 4.37 ± 0.78 5.55 ± 0.84 3.85 ± 0.33 Leu 7.60 ± 0.82 8.11 ± 0.69 9.80 ± 0.94 3.85 ± 0.25 Lys 11.03 ± 1.33 11.07 ± 1.56 9.47 ± 0.83 10.18 ± 0.97 Pro 3.64 ± 0.31 2.21 ± 0.19 5.20 ± 0.72 1.90 ± 0.05 a d d d d Trp n.d n.d n.d n.d Hydrophobic amino acid 45.08 ± 2.38 42.69 ± 2.79 44.98 ± 2.05 35.31 ± 1.84 Basic amino acids 21.57 ± 1.33 18.21 ± 1.52 22.39 ± 1.71 20.58 ± 1.78 Acid amino acids 14.64 ± 1.35 13.23 ± 1.12 18.34 ± 1.27 14.56 ± 1.17 Hydrophobic amino acids Acid amino acids Basic amino acids Not determined 212 Ann Microbiol (2018) 68:207–216 Table 2 Secondary structure Strains α-helix (%) β-sheet (%) β-turn (%) random coil (%) content of the SLPs L. bulgaricus fb04 44.2 ± 1.09 3.2 ± 0.41 29.3 ± 1.88 27.1 ± 1.64 L. rhamnosus fb06 20.5 ± 1.47 24.4 ± 1.26 19.2 ± 0.53 36.5 ± 2.73 L. gasseri fb07 23.9 ± 1.58 22.4 ± 1.44 19.2 ± 0.71 36.1 ± 1.97 L. acidophilus NCFM 33.7 ± 1.12 13.6 ± 0.46 24.6 ± 0.81 32.3 ± 1.37 were alkaline, which was in line with the characteristic of Effects of SLPs on the survival of lactobacilli Lactobacillus SLPs. in simulated gastrointestinal conditions After incubation in the simulated gastric juice for 4 h Secondary structure analysis of SLPs (Table 4), the survival of the untreated lactobacilli was about 82–95%, but the survival of the four LiCl-treated lactobacilli The proportion of different secondary structures is shown in significantly (P < 0.05) decreased to 45–66%. Similarly, after Table 2. Among the four SLPs, the SLP of L. bulgaricus fb04 treatment with small intestinal juice for 4 h (Table 5), the showed the most α-helix content (44.2%) and the least β- viability of the four LiCl-treated lactobacilli decreased by sheet content (3.2%). The L. acidophilus NCFM SLP also 22–34% as compared with the untreated lactobacilli. These showed a high content of α- helix (33.7%) and a low propor- results confirmed the protective role of SLPs. tion of β-sheet (13.6%). The SLP of L. rhamnosus fb06 was the only one that had more β-sheet than α-helix. These results Effects of bile salt stress on the expression of SLPs suggested that SLPs from different species showed a low level of similarity on the content of secondary structures. The four tested strains were subjected to bile salt stress during their growth. Then, their SLPs were extracted and analyzed by Bradford method. When under bile salt stress, L. rhamnosus Effects of SLPs on the autoaggregation fb06, L. gasseri fb07, and L. acidophilus NCFM expressed and coaggregation of lactobacilli more SLPs than in the control group (without bile salt). The SLP concentration significantly augmented (P <0.05) when The autoaggregation of the four lactobacilli decreased signif- the bile concentration was increased as determined by icantly (P < 0.05) due to the loss of the SLPs (Table 3), sug- Bradford method (Fig. 3). gesting that the SLPs had positive contributions to the autoaggregation ability of the four lactobacilli. Similarly, after LiCl treatment, the coaggregation of the four lactobacilli with E. coli ATCC 43893 (4–8% reduction) or S. typhimurium Discussion CMCC 50013 (5–8% reduction) was significantly reduced (P < 0.05). These results indicated that the SLPs could posi- LiCl was widely used to extracted bacterial surface layer in tively affect the coaggregation of the four strains. many previous reports (Chen et al. 2007; Zhang et al. 2010; Table 3 Effects of the SLPs on the autoaggregation and coaggregation of lactobacilli Strains Autoaggregation (%) E. coli ATCC 43893 coaggregation (%) S. typhimurium CMCC 50013 coaggregation (%) Untreated LiCl-treated Untreated LiCl-treated Untreated LiCl-treated Ba Ab Ba Cb Ba Bb L. bulgaricus fb04 27.97 ± 0.53 21.75 ± 1.60 20.57 ± 0.90 12.84 ± 0.46 20.27 ± 0.60 11.87 ± 1.59 Ca Bb Ba Bb Ba Bb L. rhamnosus fb06 21.41 ± 0.82 13.56 ± 1.27 21.02 ± 1.75 16.20 ± 2.04 20.90 ± 0.82 12.93 ± 0.95 Ca Bb Ca Cb Ba Bb L. gasseri fb07 20.04 ± 0.73 13.39 ± 0.64 16.83 ± 0.50 12.75 ± 0.20 18.73 ± 1.14 13.37 ± 0.46 Aa Ab Aa Ab Aa Ab L. acidophilus NCFM 35.58 ± 1.64 21.71 ± 0.60 28.10 ± 2.22 23.98 ± 0.93 24.87 ± 0.38 16.70 ± 0.98 a, b represent significant difference between the untreated and LiCl-treated groups (P <0.05) A, B, C indicate the significant difference among different strains (P <0.05) The table presents mean numbers ± standard deviation Ann Microbiol (2018) 68:207–216 213 Table 4 Effects of the SLPs on the survival of lactobacilli in simulated gastric juice Strains Groups 0 h (log) 1 h (log) 2 h (log) 3 h (log) 4 h (log) Survival (%) a a b bc c A L. bulgaricus fb04 Untreated 7.38 ± 0.27 7.18 ± 0.04 6.80 ± 0.05 6.57 ± 0.18 6.37 ± 0.03 86.31 a b c d e B LiCl-treated 6.86 ± 0.06 6.31 ± 0.09 5.41 ± 0.06 4.86 ± 0.12 3.86 ± 0.14 56.27 a b c d e A L. rhamnosus fb06 Untreated 8.45 ± 0.02 7.90 ± 0.10 7.62 ± 0.14 7.41 ± 0.03 7.10 ± 0.11 84.02 a b c d e B LiCl-treated 7.90 ± 0.08 7.18 ± 0.07 6.00 ± 0.11 5.41 ± 0.03 5.21 ± 0.02 65.95 a b c d e A L. gasseri fb07 Untreated 8.38 ± 0.03 8.15 ± 0.11 7.82 ± 0.12 7.37 ± 0.06 6.90 ± 0.07 82.34 a b c d e B LiCl-treated 7.99 ± 0.07 7.21 ± 0.07 6.38 ± 0.04 4.92 ± 0.12 3.65 ± 0.06 45.68 a a ab b c A L. acidophilus NCFM Untreated 9.30 ± 0.10 9.26 ± 0.06 9.13 ± 0.09 9.02 ± 0.08 8.85 ± 0.11 95.16 a b c d e B LiCl-treated 8.42 ± 0.05 8.07 ± 0.10 7.08 ± 0.10 6.72 ± 0.14 5.59 ± 0.06 66.38 a, b, c, d, e represent significant difference in different time of treatments (P < 0.05) A, B represent significant difference between the untreated and LiCl-treated groups (P <0.05) The table presents mean numbers ± standard deviation Beganović et al. 2011). After LiCl treatment, the surface layer typical feature of Lactobacillus SLPs, they usually have a high of L. crispatus K313 and K243 was lost as determined by content of hydrophobic amino acid residues (Sleytr 1997). TEM (Sun et al. 2012). This phenomenon was similar with The hydrophobic feature of the cell surface of bacteria has our results (Fig. 1). The surface layers of the four tested strains been implicated in the attachment to their host tissue (Zhang were stripped because of the LiCl treatment. et al. 2010). In addition, sulfur-containing amino acids were The masses of Lactobacillus SLPs were reported ranging often reported no more than 2% in Lactobacillus SLPs and the from 25 to 71 kDa (Åvall-Jääskeläinen and Palva 2005). lysine content was usually higher, at about 10% (Boot et al. Zhang et al. (2010) had isolated L. rhamnosus J10-L from 1993; Sleytr 1997; Åvall-Jääskeläinen and Palva 2005). The traditional Chinese fermentation food and the molecular mass amino acid compositions of the four extracted SLPs were of its SLP was revealed as 45 kDa. This was similar with the consistent with the common characteristics of Lactobacillus molecular weight of L. rhamnosus fb06. Previous studies re- SLPs. ported that the molecular masses of the SLPs from L. gasseri Protein secondary structures refer to the regularly repeating VPI 11759 and L. gasseri ATCC 19992 were 29.8 and conformation in polypeptide chains, mainly including α-he- 26.3 kDa (Ventura et al. 2002). By comparison, the molecular lix, β-sheet, β-turn, and random coil. Circular dichroism was weight of L. gasseri fb07 SLP was larger than that of the two often used to analyze the secondary structure of proteins. reported strains. Meng et al. (2014) reported that the SLPs of L. acidophilus The amino acid composition of Lactobacillus SLPs resem- fb116 and L. acidophilus fb214 had about 34% α-helix, 12% bles in most parts the composition of other bacterial SLPs, but β-sheet, 24% β-turn, and 32% random coil, which was similar some Lactobacillus-specific features can also be found. As a with the secondary structure content of L. acidophilus NCFM Table 5 Effects of the SLPs on the survival of lactobacilli in simulated intestinal juice Strains Groups 0 h (log) 1 h (log) 2 h (log) 3 h (log) 4 h (log) Survival (%) a b c d e A L. bulgaricus fb04 Untreated 7.19 ± 0.14 6.91 ± 0.07 6.14 ± 0.05 5.71 ± 0.11 4.92 ± 0.06 68.42 a b c d e B LiCl-treated 6.51 ± 0.07 6.05 ± 0.05 5.26 ± 0.11 4.20 ± 0.22 2.82 ± 0.07 43.31 a a b c c A L. rhamnosus fb06 Untreated 8.26 ± 0.21 8.19 ± 0.08 7.84 ± 0.12 7.41 ± 0.07 7.23 ± 0.03 87.53 a b c d e B LiCl-treated 8.09 ± 0.04 7.82 ± 0.16 7.27 ± 0.04 6.33 ± 0.08 5.30 ± 0.17 65.51 a b c d e A L. gasseri fb07 Untreated 7.85 ± 0.12 7.32 ± 0.07 6.94 ± 0.07 6.12 ± 0.03 5.57 ± 0.08 70.96 a b c d e B LiCl-treated 7.20 ± 0.03 5.97 ± 0.11 5.31 ± 0.03 3.18 ± 0.14 2.66 ± 0.09 36.94 a b c d e A L. acidophilus NCFM Untreated 8.74 ± 0 .08 7.98 ± 0.04 7.27 ± 0.01 6.96 ± 0.08 6.66 ± 0.14 76.20 a b c d e B LiCl-treated 8.36 ± 0.08 7.70 ± 0.15 6.91 ± 0.10 5.62 ± 0.12 4.19 ± 0.09 50.12 a, b, c, d, e represent significant difference in different time of treatments (P < 0.05) A, B represent significant difference between the untreated and LiCl-treated groups (P <0.05) The table presents mean numbers ± standard deviation 214 Ann Microbiol (2018) 68:207–216 Fig. 3 SLP concentrations under different level of bile stress determined by Bradford method SLP determined in our experiment. These findings indicated strains was likely to be reduced after the SLPs were removed. that the secondary structure content of SLPs may be related to Moreover, among the four tested strains, L. acidophilus NCFM the genetic similarity of the strains. In addition, Mobili et al. exhibited the highest autoaggregation and L. gasseri fb07 the (2009) found that the thermal denaturation temperature of lowest one. Interestingly, L. acidophilus NCFM and L. gasseri SLPs was associated with their secondary structure content. fb07 also showed the highest and the lowest coaggregation More β-sheet could make the protein more stable. Therefore, ability, respectively. This finding was in agreement with we could infer that the SLP of L. bulgaricus may have the Collado et al. (2008) who suggested that coaggregation abili- lowest thermal denaturation temperature in the four extracted ties were related to autoaggregation properties. SLPs. Ventura et al. (2002) reported two kinds of surface When lactobacilli were ingested in a sufficient number, they proteins from L. gasseri. The two proteins were suggested to would exert their beneficial functions effectively. Therefore, the possess a content of β-sheet from 26.1 to 28.5% by secondary key point in enabling lactobacilli to play their role is to maintain structure prediction, which was a little higher than our results the viability during their transit through the gastrointestinal tract of L. gasseri (β-sheet 22.4%). of the host. L. acidophilus was capable of displaying adaptive Bacterial aggregation is of considerable importance in human response to various stress conditions (Kim et al. 2001). From gut, where probiotics are to be active (Jankovic et al. 2003). our data (Table 4), it can be found that the four tested Autoaggregation was reported to be correlated with adhesion, lactobacilli had good tolerance to gastric juice before LiCl treat- which was known as a prerequisite for colonization (Collado ment. Therefore, they may play their probiotic functions effec- et al. 2008). Coaggregation is a part of competitive exclusion tively in the host. But the loss of their SLPs led to the viability mechanism which is coupled with the antimicrobial activity of reduction. These results demonstrated that the protective effects the probiotic strain and enables a decrease of the pathogenic load of the SLPs could ensure the amount of the lactobacilli to in- during infections (Beganović et al. 2011). Compared with the teract with the gastrointestinal tract. In previous reports, the non-coaggregating strains, the coaggregating ones were easier to removal of SLPs from L. acidophilus M92 reduced their via- colonize in intestine and inhibit pathogen infection (Golowczyc bility in simulated gastric and small intestinal juices. et al. 2007). Furthermore, it was approved that SLPs of L. acidophilus As reported by other researchers, the autoaggregation and M92 was not sensitive to pepsin and pancreatin which could the adhesion ability of L. crispatus ZJ001, L. acidophilus M92, suggest possible protective role in the gastrointestinal tract and L. helveticus M92 strains were significantly decreased af- (Frece et al. 2005). ter the removal of their SLPs (Kos et al. 2003;Chenetal. 2007; L. acidophilus LA1-1, L. crispatus ZJ001, and L. casei Beganović et al. 2011). In our study, the autoaggregation abil- Zhang were reported to be able to adapt to salt, heat, and bile ity of the four tested strains was also impaired after the lost of stress conditions (Kim et al. 2001;Chen etal. 2007; Guo et al. the SLPs. Beganović et al. (2011) demonstrated that the 2009). Lactobacillus SLPs have shown the abilities to help the coaggregation of L. helveticus M92 with S. typhimurium FP1 growth of lactobacilli in harsh environments (Boot et al. 1993; was negatively affected after the removal of SLPs. The Kos et al. 2003; Åvall-Jääskeläinen and Palva 2005; Frece coaggregation of L. kefir with Saccharomyces lipolytica was et al. 2005). Khaleghi et al. (2010) reported that the SLP inhibited due to the loss of SLPs (Golowczyc et al. 2009). expression of L. acidophilus ATCC 4356 was increased in These outcomes were consistent with our results. All the find- the stress condition to be a protective sheath, which was con- sistent with our results. Nevertheless, the bile salt stress did ings indicated that the antimicrobial activity of Lactobacillus Ann Microbiol (2018) 68:207–216 215 Collado MC, Meriluoto J, Salminen S (2008) Adhesion and aggregation not affect the production of SLPs from L. bulgaricus fb04. properties of probiotic and pathogen strains. Eur Food Res Technol These results suggested that SLPs from different strains might 226:1065–1073 have different characteristics. Frece J, Kos B, Svetec I-K, Zgaga Z, MršaV, Šušković J (2005) Lactobacillus SLPs may have great potential in industrial Importance of S-layer proteins in probiotic activity of Lactobacillus acidophilus M92. J Appl Microbiol 98:285–292 applications due to their protective effects in hostile environ- Gerbino E, Mobili P, Tymczyszyn E, Fausto R, Gómez-Zavaglia A (2011) ments. Hollmann et al. (2007) reported a kind of liposome FTIR spectroscopy structural analysis of the interaction between coated with Lactobacillus SLPs. The SLPs enhanced the sta- Lactobacillus kefir S-layers and metal ions. J Mol Struct 987:186–192 bility of the liposome after its exposure to bile salts, pancreatic Golowczyc M, Mobili P, Garrote G, Abraham A, De Antoni G (2007) Protective action of Lactobacillus kefir carrying S-layer protein extract, pH change, and thermal shock, which provided an against Salmonella enterica serovar Enteritidis. Int J Food application of Lactobacillus SLPs in pharmaceutical industry. Microbiol 118:264–273 More applications may be explored in the future research. Golowczyc MA, Mobili P, Garrote GL, de los Angeles Serradell M, Abraham AG, De Antoni GL (2009) Interaction between Lactobacillus kefir and Saccharomyces lipolytica isolated from kefir grains: evidence for lectin-like activity of bacterial surface proteins. Conclusions JDairy Res 76:111–116 Guo Z, Wang J, Yan L, Chen W, Liu X-M, Zhang H-P (2009) In vitro comparison of probiotic properties of Lactobacillus casei Zhang, a In this study, the presence of SLPs from L. bulgaricus fb04, potential new probiotic, with selected probiotic strains. LWT-Food L. rhamnosus fb06, L. gasseri fb07, and L. acidophilus NCFM Sci Technol 42:1640–1646 was verified and their physicochemical properties were charac- Hollmann A, Delfederico L, Glikmann G, De Antoni G, Semorile L, Disalvo E (2007) Characterization of liposomes coated with S-layer terized. In addition, the biological functions of the four SLPs proteins from lactobacilli. Biochim Biophys Acta 1768:393–400 were confirmed. The results indicated that the SLPs could pos- Jakava-Viljanen M, Palva A (2007) Isolation of surface (S) layer protein itively affect the aggregation ability and gastrointestinal tolera- carrying Lactobacillus species from porcine intestine and faeces and bility of the four lactobacilli. Under adverse conditions, characterization of their adhesion properties to different host tissues. Vet Microbiol 124:264–273 L. rhamnosus fb06, L. gasseri fb07, and L. acidophilus Jankovic I, Ventura M, Meylan V, Rouvet M, Elli M, Zink R (2003) NCFM were shown to express more SLP. These observations Contribution of aggregation-promoting factor to maintenance of cell provided a theoretical basis for the practical application of SLPs shape in Lactobacillus gasseri 4B2. J Bacteriol 185:3288–3296 and the lactobacilli in food and pharmaceutical industries. Johnson B, Selle K, O’Flaherty S, Goh YJ, Klaenhammer T (2013) Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM. Microbiology 159:2269–2282 Acknowledgements This study was supported by the grant from the Khaleghi M, Kermanshahi RK, Yaghoobi M, Zarkesh-Esfahani S, National Natural Science Foundation of China (No. 31471696, Baghizadeh A (2010) Assessment of bile salt effects on s-layer pro- 31701542) and Province Key Laboratory of Transformation and duction, slp gene expression and some physicochemical properties Utilization of Cereal Resource (PL2016009). of Lactobacillus acidophilus ATCC 4356. J Microbiol Biotechnol 20:749–756 Compliance with ethical standards Kim WS, Perl L, Park JH, Tandianus JE, Dunn NW (2001) Assessment of stress response of the probiotic Lactobacillus acidophilus. Curr Conflict of interest The authors declare that they have no conflict of Microbiol 43:346–350 interest. Kos B, Šušković J, Vuković S, Šimpraga M, Frece J, Matošić S(2003) Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J Appl Microbiol 94:981–987 Lee Y-K, Puong K-Y, Ouwehand AC, Salminen S (2003) Displacement of bacterial pathogens from mucus and Caco-2 cell surface by References lactobacilli. J Med Microbiol 52:925–930 Leeuw E, Li X, Lu W (2006) Binding characteristics of the Lactobacillus Åvall-Jääskeläinen S, Palva A (2005) Lactobacillus surface layers and brevis ATCC 8287 surface layer to extracellular matrix proteins. their applications. FEMS Microbiol Rev 29:511–529 FEMS Microbiol Lett 260:210–215 Beganović J, Frece J, Kos B, Leboš Pavunc A, Habjanič K, Šušković J Lorca G, Torino MI, Font de Valdez G, Ljungh Å (2002) Lactobacilli (2011) Functionality of the S-layer protein from the probiotic strain express cell surface proteins which mediate binding of immobilized Lactobacillus helveticus M92. Antonie Van Leeuwenhoek 100:43–53 collagen and fibronectin. FEMS Microbiol Lett 206:31–37 Boot HJ, Kolen C, Van Noort JM, Pouwels PH (1993) S-layer protein of Lu R-R, Qian P, Sun Z, Zhou X-H, Chen T-P, He J-F, Zhang H, Wu J Lactobacillus acidophilus ATCC 4356: purification, expression in (2010) Hempseed protein derived antioxidative peptides: purifica- Escherichia coli, and nucleotide sequence of the corresponding tion, identification and protection from hydrogen peroxide-induced gene. J Bacteriol 175:6089–6096 apoptosis in PC12 cells. Food Chem 123:1210–1218 Bradford MM (1976) A rapid and sensitive method for the quantitation of Martínez MG, Acosta MP, Candurra NA, Ruzal SM (2012) S-layer pro- microgram quantities of protein utilizing the principle of protein-dye teins of Lactobacillus acidophilus inhibits JUNV infection. binding. Anal Biochem 72:248–254 Biochem Biophys Res Commun 422:590–595 Meng J, Zhu X, Gao S-M, Zhang Q-X, Sun Z, Lu R-R (2014) Chen X, Xu J, Shuai J, Chen J, Zhang Z, Fang W (2007) The S-layer proteins of Lactobacillus crispatus strain ZJ001 is responsible for Characterization of surface layer proteins and its role in probiotic properties of three Lactobacillus strains. Int J Biol Macromol 65: competitive exclusion against Escherichia coli O157: H7 and Salmonella typhimurium. Int J Food Microbiol 115:307–312 110–114 216 Ann Microbiol (2018) 68:207–216 Meng J, Gao S-M, Zhang Q-X, Lu R-R (2015) Murein hydrolase activity Sun Z, Huang L, Kong J, Hu S, Zhang X, Kong W (2012) In vitro evaluation of Lactobacillus crispatus K313 and K243: high- of surface layer proteins from Lactobacillus acidophilus against Escherichia coli. Int J Biol Macromol 79:527–532 adhesion activity and anti-inflammatory effect on Salmonella Mobili P, Londero A, Maria TMR, Eusébio MES, De Antoni GL, Fausto braenderup infected intestinal epithelial cell. Vet Microbiol 159: R, Gómez-Zavaglia A (2009) Characterization of S-layer proteins of 212–220 Lactobacillus by FTIR spectroscopy and differential scanning calo- Ventura M, Jankovic I, Walker DC, Pridmore RD, Zink R (2002) rimetry. Vib Spectrosc 50:68–77 Identification and characterization of novel surface proteins in Navarre WW, Schneewind O (1999) Surface proteins of gram-positive Lactobacillus johnsonii and Lactobacillus gasseri. Appl Environ bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol 68:6172–6181 Microbiol Mol Biol Rev 63:174–229 Zhang Y-C, Zhang L-W, Tuo Y-F, Guo C-F, Yi H-X, Li J-Y, Han X, Du M Sára M, Sleytr UB (2000) S-layer proteins. J Bacteriol 182:859–868 (2010) Inhibition of Shigella sonnei adherence to HT-29 cells by Sleytr UB (1997) Basic and applied S-layer research: an overview. FEMS lactobacilli from Chinese fermented food and preliminary character- Microbiol Rev 20:5–12 ization of S-layer protein involvement. Res Microbiol 161:667–672 Smit E, Pouwels PH (2002) One repeat of the cell wall binding domain is sufficient for anchoring the Lactobacillus acidophilus surface layer protein. J Bacteriol 184:4617–4619 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Identification and analysis of the function of surface layer proteins from three Lactobacillus strains

Annals of Microbiology , Volume 68 (4) – Mar 17, 2018

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Springer Journals
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Copyright © 2018 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
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1590-4261
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1869-2044
DOI
10.1007/s13213-018-1335-1
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Abstract

To identify and investigate the role of surface layer proteins (SLPs) on the probiotic properties of Lactobacillus strains, SLPs were extracted from Lactobacillus bulgaricus fb04, L. rhamnosus fb06, L. gasseri fb07, and L. acidophilus NCFM by 5 mol/L lithium chloride. The molecular masses of the four SLPs were approximately 45–47 kDa as analyzed by SDS-PAGE. Hydrophobic amino acids were the main components of the four SLPs. The secondary structure content of the four SLPs showed extensive variability among different strains. After the SLPs were removed from the cell surface, the autoaggregation ability, coaggregation ability, and gastrointestinal tolerability of the four lactobacilli were significantly reduced as compared with the intact cells (P < 0.05). When exposed to bile salt stress, L. rhamnosus fb06, L. gasseri fb07, and L. acidophilus NCFM expressed more SLPs as determined by Bradford method. In conclusion, the four lactobacilli all possessed functional SLPs, which had positive contributions to the probiotic properties of the four Lactobacillus strains. This research could reveal the biological contributions of SLPs from Lactobacillus strains and offer a theoretical basis for the application of lactobacilli and their SLPs in food and pharmaceutical industries. . . . . Keywords Surface layer proteins Lactobacillus Amino acid composition Secondary structure Probiotic function Introduction intestinal tissues are reported to be relevant to the cell-surface components of bacteria, such as lipoteichoic acids, extracellular Lactobacilli have been identified as main members of probiotic polysaccharides, and cell surface proteins (Jakava-Viljanen and strains. They could maintain the stability of gastrointestinal tract Palva 2007). and prevent pathogen infections, when ingested in sufficient Bacterial surface layers, usually found outside the cell wall number (Jakava-Viljanen and Palva 2007;Martínezetal. of Eu- and Archae-bacteria, are composed of proteins, which 2012). Due to their health-promoting functions, lactobacilli could self-assemble into a sub-lattice array structure (Smit and have been widely used in food and pharmaceutical industries. Pouwels 2002). Surface layers are attached to cell wall The mechanisms of their probiotic activities have been attribut- through noncovalent interactions, including hydrogen bond, ed to the mucosal barrier function, modulation of the immune ionic bond, and hydrophobic interactions, and can be routinely response, coaggregation with pathogens, competitive exclu- extracted by chaotropic denaturants or high concentration dis- sion, and displacement of pathogens (Beganović et al. 2011; sociating agents (Navarre and Schneewind 1999). Many spe- Lee et al. 2003). The interactions between lactobacilli and cies of lactobacillus have been demonstrated to possess sur- face layer proteins (SLPs) and show the molecular masses ranging from 25 to 71 kDa, accounting for 10–15% of the total cellular protein (Sára and Sleytr 2000;Chenetal. * Rong-Rong Lu 2007). SLPs are highly basic proteins, with calculated pI lurr@jiangnan.edu.cn values ranging from 9.35 to 10.40. School of Food Science and Technology, Province Key Laboratory SLPs may have various biological functions. Many reports of Transformation and Utilization of Cereal Resource, Henan have revealed that SLPs are the key components for lactobacilli University of Technology, Zhengzhou, Henan Province 450001, to play their probiotic role in the host. Autoaggregation and China coaggregation abilities are the important surface properties of School of Food Science and Technology, Jiangnan University, 1800 lactobacilli, which could contribute a lot to the colonization Lihu Avenue, Wuxi, Jiangsu 214122, China 208 Ann Microbiol (2018) 68:207–216 and antibacterial function of lactobacilli in the gastrointestinal L. rhamnosus fb06, and L. bulgaricus fb04 were isolated from tract (Collado et al. 2008). It has been reported that the SLPs of fermented food. The lactobacilli were grown under aerobic con- Lactobacillus crispatus, L. acidophilus,and L. helveticus have a ditions in De Man-Rogosa-Sharpe (MRS) broth (Oxoid) over- positive contribution to the autoaggregation and coaggregation night for 18 h at 37 °C in a shaker (DKY-II, Duke automation ability of their strains (Kos et al. 2003; Chen et al. 2007; equipment Co., Ltd., Shanghai, China) at 180 rpm. E. coli Beganović et al. 2011). The SLPs of L. acidophilus M92 and ATCC 43893 and S. typhimurium CMCC 50013 were grown L. acidophilus ATCC 4356 could help the strains to adapt to in Luria-Bertani (LB) medium overnight for 18 h at 37 °C in a adverse living conditions (Frece et al. 2005; Khaleghi et al. shaker. 2010). Many studies have implicated the involvement of some SLPs from lactobacilli in adhesion to epithelial cells or extracel- Transmission electron microscopy lular matrix proteins (Lorca et al. 2002; Leeuw et al. 2006;Sun et al. 2012;Mengetal. 2014). The SLPs of L. acidophilus, Lactobacilli cells were collected by centrifugation (5000×g, L. helveticus,and L. plantarum were reported to be involved 10 min, 4 °C) and washed twice with phosphate buffer solu- in pathogen inhibition (Meng et al. 2014). tion (PBS; pH 7.2). For untreated samples, the washed cells Due to the great diversity of SLPs, many kinds of SLPs were suspended in PBS. For LiCl-treated samples, the cells have not been clearly investigated. Most of the reports about were suspended in 5 mol/L lithium chloride, and all the sam- SLPs were focused on the L. acidophilus, L. helveticus, ples were agitated for 30 min at room temperature. L. crispatus,and L. brevis strains. Some strains, which were TEM was performed according to Gerbino et al. (2011). researched in our manuscript, such as L. gasseri, L. rhamnosus, Cells were suspended in glutaraldehyde solution and fixed for and L. bulgaricus, have been widely used as probiotics in food 2 h. Fixed cells were stained with phosphotungstic acid and industry. But the characterization and function of their SLPs examined through an H-7650 TEM (Hitachi, Tokyo, Japan) at were lacking of report. In this study, the SLPs were extracted an operating voltage of 80 kV. from L. gasseri fb07, L. rhamnosus fb06, and L. bulgaricus fb04. L. acidophilus NCFM was set as a reference strain, as its Preparation of SLPs and SDS-PAGE analysis SLPs were verified in previous studies (Johnson et al. 2013; Meng et al. 2015). The physicochemical properties of the four SLPs of the four lactobacilli were extracted according to SLPs and their possible contributions to the probiotic proper- Meng et al. (2014). Lactobacilli cells were washed twice with ties of Lactobacillus strains were studied. This research could PBS and suspended in 5 mol/L lithium chloride (LiCl). After reveal the biological contribution of these SLPs to the probiotic treatment for 30 min at room temperature, SLPs were collect- properties of lactobacilli and advance our understanding about ed by centrifugation (12,000×g, 4 °C, 15 min) and dialyzed by the probiotic mechanism. distilled water at 4 °C, then lyophilized (Labconco Corp., Kansas City, MO, USA). The SLPs were mixed with 2× loading buffer, boiled for Materials and methods 5 min, and analyzed by SDS-PAGE using 12% polyacryl- amide gels. Protein bands were made visible by staining with Chemicals Comassie brilliant blue. SLPs were purified according to Meng et al. (2015). Low molecular weight marker from 14.4 to 97.4 kDa was Briefly, the lyophilized SLPs were dissolved in 8 mol/L purchased from the China National Medicines Corporation urea–Tris–hydrochloric acid solution and purified by chroma- (Shanghai, China). Pepsin (1:10,000) and trypsin (1:250) were tography on a cation-exchange column. purchased from the Bio Basic Company (Bio Basic Inc., Canada). The other chemicals were common reagent of ana- Amino acid content analysis of SLPs lytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). The amino acid content of SLPs was identified using the method described by Lu et al. (2010). Briefly, SLP samples Bacterial strains and growth conditions were digested with 6 M HCl under atmospheric nitrogen at 110 °C for 24 h. Then, they were filtered with two filter mem- L. acidophilus NCFM, L. gasseri fb07, L. rhamnosus fb06, branes. The filtrate was centrifuged at 3000g for 30 min, and L. bulgaricus fb04, Escherichia coli ATCC 43893 (O124:NM, the clear supernatant, containing mainly amino acids, was causing infectious diarrhea), and Salmonella typhimurium derivatized by O-phthaldialdehyde (OPA) and 9- CMCC 50013 (O4,5:Hi:2, causing acute gastroenteritis) were fluorenylmethoxycarbonyl chlorine (FMOC-Cl), followed provided by the Research Center of Food Biotechnology, by RP-HPLC analysis in an Agilent (Agilent Technologies, Jiangnan University, Jiangsu, China. L. gasseri fb07, Palo Alto, CA, USA) assembly system, using a Zorbax 80A Ann Microbiol (2018) 68:207–216 209 C column (4.6 × 180 mm), running at 0.5 mL/min. Survival of lactobacilli in simulated gastric and small Methionine and cysteine were determined separately as their intestinal juice oxidation products, according to the performic acid procedure, prior to hydrolysis in 6 M HCl. The results were processed This experiment was carried out according to Frece et al. (2005) with the aid of ChemStation for LC 3D software (Agilent with some modifications. Simulated gastric juice was prepared −1 Technologies, Palo Alto, CA, USA). by dissolving pepsin into sterile NaCl solution (5.0 g L ). The −1 final concentrationofpepsinwas 3.0g L ,and thepHofthe solution was adjusted to 2.0 with HCl. Simulated small intesti- Secondary structure analysis by circular dichroism nal juice was prepared by dissolving trypsin and bile salts into −1 sterile NaCl solution (5.0 g L ). The final concentration of −1 Circular dichroism analysis was performed according to the trypsinandbilesalts was1.0and1.5gL . The pH of the method described by Meng et al. (2014) using a MOS-450/ solution was adjusted to 8.0 with 0.1 mol/L NaOH. AF-CD spectrometer (Bio-Logic, Grenoble, France). SLPs Lactobacilli cells were collected by centrifugation were placed in a 1 mm path length quartz cuvette at the (5000×g, 10 min, 4 °C) and washed twice with phosphate- −1 concentration of 0.1–0.2mgmL . The scanning wave- buffered saline (PBS, pH 7.2). The washed cells were length was set as 190–250 nm, and the scanning rate was suspended in simulated gastric or small intestinal juice to −1 8 set as 100 nm min . A buffer sample with no SLPs was 5×10 CFU/mL and incubated for 4 h at 37 °C. The changes subtracted from all spectra to account for any background in total viable counts were monitored every 1 h by the pour signal. plate method, using MRS agar incubated at 37 °C for 48 h. SLP expression under bile salt stress conditions Autoaggregation assays For studying the effect of bile salt stress, the experiment was Autoaggregation assays were performed according to Chen performed as described by Khaleghi et al. (2010) with some et al. (2007) with some modifications. Lactobacilli cells were modifications. MRS broth containing 0, 0.1, 0.2, 0.5, or −1 collected by centrifugation (5000×g,10min,4 °C)and 1.0 g L bile was prepared. Inoculum of 2% (v/v)of washed twice with PBS (pH 7.2). The washed cells were lactobacilli was used in the samples. All the samples were suspended in PBS to approximately 10 CFU/mL. The absor- incubated at 37 °C for 18 h in a shaker. After incubation, bance of the bacteria suspension was measured at 600 nm lactobacilli cells were washed twice with phosphate-buffered (A ). Bacteria suspension of 3 mL was stirred 10 s. After 4-h saline (PBS, pH 7.2). The OD was adjusted to 0.5 by PBS. 0 600 incubation at room temperature, the absorbance was measured Lactobacilli cells collected from 100 mL of the prepared sus- at 600 nm (A ). The percentage of autoaggregation was cal- pension were used to extract SLPs according to Meng et al. culated as (1–A /A ) × 100. (2014). The concentration of the SLPs was determined by 4 0 Bradford method (Bradford 1976). Briefly, 100-μg/mL bovine serum albumin solution was prepared as protein standard so- Coaggregation assays lution. Coomassie® Brilliant Blue G-250(100 mg) was dis- solved in 50 mL 95% ethanol. To this solution, 100 mL 85% The coaggregation of lactobacilli with E. coli ATCC 43893 or (w/V) phosphoric acid was added. The resulting solution was S. typhimurium CMCC 50013 was determined according to then diluted to a final volume of 1 L by distilled water. Three- the method of Kos et al. (2003). The method for preparing cell milliliter G-250 solution was added into 0–1.0-mL protein suspensions of lactobacilli, E. coli ATCC 43893, and standard solution, respectively, to establish a standard curve S. typhimurium CMCC 50013 was the same as that for equation. The absorbance was measured at 595 nm. Three- autoaggregation. Equal volumes (2 mL) of lactobacilli and milliliter G-250 solution was added into 100-μL SLP solutions pathogen suspensions were mixed together by vortexing for (PBS, pH 7.2). After reaction, OD was determined. SLP 10 s. Four milliliters of each bacterial suspension was set up as concentrations were calculated by the standard curve equation. control groups at the same time. The absorbance of the sus- pensions at 600 nm was determined after 4-h incubation at Statistical analysis room temperature. The coaggregation percentage was calcu- lated as (1 − A /[(A + A )/2]) × 100, where A represents the All of the experiments were conducted at least in triplicate. M L P M absorbance of the mixture after 4-h incubation, A represents The data were analyzed with a one-way ANOVA that set the absorbance of lactobacilli suspension, A represents the significance at P < 0.05. SPSS 18.0 (SPSS Inc., Chicago, absorbance of pathogen suspension (E. coli ATCC 43893 or USA) for Windows was used in the statistical analysis. The S. typhimurium CMCC 50013). results were presented as means ± standard errors. 210 Ann Microbiol (2018) 68:207–216 Fig. 1 Transmission electron microscopy of the cell surface structures of the four lactobacilli. a L. bulgaricus fb04; b L. rhamnosus fb06; c L. gasseri fb07; d L. acidophilus NCFM. 1- Untreated lactobacilli; 2- Lactobacilli treated by 5 mol/L LiCl. Arrows: the surface layer Ann Microbiol (2018) 68:207–216 211 shown in Fig. 1, the untreated lactobacilli were covered with a layer of extracellular structure. After treatment by LiCl, the surface layers were removed. In order to identify the SLPs, the LiCl-extracts were analyzed in the following SDS-PAGE experiment. In each LiCl-extracts, one major band was found (lanes 1 to 4, Fig. 2). The SLP of L. acidophilus NCFM (lane 1) was set as a reference protein, because its molecular mass had been reported as 46 kDa (Johnson et al. 2013). SDS-PAGE analysis identified the presence of SLPs as extracted from L. gasseri fb07, L. rhamnosus fb06, and L. bulgaricus fb04 strains. The molecular masses were approximately 45–47 kDa. Fig. 2 SDS-PAGE profiles of SLPs extracted from lactobacilli by 5 mol/L LiCl. Line 1, L. acidophilus NCFM; line 2, L. gasseri fb07; line 3, L. rhamnosus fb06; line 4, L. bulgaricus fb04 Amino acid composition of SLPs Results The amino acid compositions of the four extracted SLPs are shown in Table 1. The hydrophobic amino acids in the four Identify the presence of SLPs from lactobacilli SLPs accounted for about 35–45%, indicating that the confor- mation of the SLPs might be maintained by the strong hydro- In this work, SLPs were extracted by 5 mol/L LiCl. To ob- phobic interactions. The content of basic amino acids in the serve the surface structure of the four lactobacilli, both untreat- four SLPs was all higher than that of the acid amino acids. It ed and LiCl-treated samples were examined by TEM. As could be speculated that the isoelectric points of these proteins −1 Table 1 Amino acid composition of the SLPs (mg g protein) Amino acid L. bulgaricus fb04 L. rhamnosus fb06 L. gasseri fb07 L. acidophilus NCFM Asp 7.16 ± 0.43 6.02 ± 0.21 10.31 ± 0.56 10.20 ± 0.47 Glu 7.48 ± 0.55 7.21 ± 0.32 8.03 ± 0.38 4.36 ± 0.24 Ser 6.61 ± 0.44 7.50 ± 0.48 2.10 ± 0.27 6.75 ± 0.22 His 4.35 ± 0.50 2.62 ± 0.14 2.01 ± 0.49 4.88 ± 0.84 Gly 3.21 ± 0.66 6.22 ± 0.37 5.81 ± 0.41 3.17 ± 0.43 Thr 5.96 ± 0.65 6.78 ± 0.88 5.46 ± 0.49 11.79 ± 1.82 Arg 6.19 ± 0.34 4.52 ± 0.59 10.91 ± 0.61 5.52 ± 0.75 Ala 9.73 ± 0.93 11.7 ± 1.44 10.82 ± 0.98 10.62 ± 1.01 Tyr 2.42 ± 0.28 4.97 ± 0.73 1.03 ± 0.13 7.64 ± 0.44 Cys-s 0.10 ± 0.02 0.06 ± 0.01 0.21 ± 0.03 0.00 Val 9.38 ± 0.48 9.21 ± 0.86 8.13 ± 0.45 11.81 ± 0.77 Met 1.03 ± 0.15 1.92 ± 0.20 0.07 ± 0.01 0.55 ± 0.02 Phe 5.89 ± 0.14 5.07 ± 0.29 5.41 ± 0.41 2.73 ± 0.20 Ile 7.81 ± 0.71 4.37 ± 0.78 5.55 ± 0.84 3.85 ± 0.33 Leu 7.60 ± 0.82 8.11 ± 0.69 9.80 ± 0.94 3.85 ± 0.25 Lys 11.03 ± 1.33 11.07 ± 1.56 9.47 ± 0.83 10.18 ± 0.97 Pro 3.64 ± 0.31 2.21 ± 0.19 5.20 ± 0.72 1.90 ± 0.05 a d d d d Trp n.d n.d n.d n.d Hydrophobic amino acid 45.08 ± 2.38 42.69 ± 2.79 44.98 ± 2.05 35.31 ± 1.84 Basic amino acids 21.57 ± 1.33 18.21 ± 1.52 22.39 ± 1.71 20.58 ± 1.78 Acid amino acids 14.64 ± 1.35 13.23 ± 1.12 18.34 ± 1.27 14.56 ± 1.17 Hydrophobic amino acids Acid amino acids Basic amino acids Not determined 212 Ann Microbiol (2018) 68:207–216 Table 2 Secondary structure Strains α-helix (%) β-sheet (%) β-turn (%) random coil (%) content of the SLPs L. bulgaricus fb04 44.2 ± 1.09 3.2 ± 0.41 29.3 ± 1.88 27.1 ± 1.64 L. rhamnosus fb06 20.5 ± 1.47 24.4 ± 1.26 19.2 ± 0.53 36.5 ± 2.73 L. gasseri fb07 23.9 ± 1.58 22.4 ± 1.44 19.2 ± 0.71 36.1 ± 1.97 L. acidophilus NCFM 33.7 ± 1.12 13.6 ± 0.46 24.6 ± 0.81 32.3 ± 1.37 were alkaline, which was in line with the characteristic of Effects of SLPs on the survival of lactobacilli Lactobacillus SLPs. in simulated gastrointestinal conditions After incubation in the simulated gastric juice for 4 h Secondary structure analysis of SLPs (Table 4), the survival of the untreated lactobacilli was about 82–95%, but the survival of the four LiCl-treated lactobacilli The proportion of different secondary structures is shown in significantly (P < 0.05) decreased to 45–66%. Similarly, after Table 2. Among the four SLPs, the SLP of L. bulgaricus fb04 treatment with small intestinal juice for 4 h (Table 5), the showed the most α-helix content (44.2%) and the least β- viability of the four LiCl-treated lactobacilli decreased by sheet content (3.2%). The L. acidophilus NCFM SLP also 22–34% as compared with the untreated lactobacilli. These showed a high content of α- helix (33.7%) and a low propor- results confirmed the protective role of SLPs. tion of β-sheet (13.6%). The SLP of L. rhamnosus fb06 was the only one that had more β-sheet than α-helix. These results Effects of bile salt stress on the expression of SLPs suggested that SLPs from different species showed a low level of similarity on the content of secondary structures. The four tested strains were subjected to bile salt stress during their growth. Then, their SLPs were extracted and analyzed by Bradford method. When under bile salt stress, L. rhamnosus Effects of SLPs on the autoaggregation fb06, L. gasseri fb07, and L. acidophilus NCFM expressed and coaggregation of lactobacilli more SLPs than in the control group (without bile salt). The SLP concentration significantly augmented (P <0.05) when The autoaggregation of the four lactobacilli decreased signif- the bile concentration was increased as determined by icantly (P < 0.05) due to the loss of the SLPs (Table 3), sug- Bradford method (Fig. 3). gesting that the SLPs had positive contributions to the autoaggregation ability of the four lactobacilli. Similarly, after LiCl treatment, the coaggregation of the four lactobacilli with E. coli ATCC 43893 (4–8% reduction) or S. typhimurium Discussion CMCC 50013 (5–8% reduction) was significantly reduced (P < 0.05). These results indicated that the SLPs could posi- LiCl was widely used to extracted bacterial surface layer in tively affect the coaggregation of the four strains. many previous reports (Chen et al. 2007; Zhang et al. 2010; Table 3 Effects of the SLPs on the autoaggregation and coaggregation of lactobacilli Strains Autoaggregation (%) E. coli ATCC 43893 coaggregation (%) S. typhimurium CMCC 50013 coaggregation (%) Untreated LiCl-treated Untreated LiCl-treated Untreated LiCl-treated Ba Ab Ba Cb Ba Bb L. bulgaricus fb04 27.97 ± 0.53 21.75 ± 1.60 20.57 ± 0.90 12.84 ± 0.46 20.27 ± 0.60 11.87 ± 1.59 Ca Bb Ba Bb Ba Bb L. rhamnosus fb06 21.41 ± 0.82 13.56 ± 1.27 21.02 ± 1.75 16.20 ± 2.04 20.90 ± 0.82 12.93 ± 0.95 Ca Bb Ca Cb Ba Bb L. gasseri fb07 20.04 ± 0.73 13.39 ± 0.64 16.83 ± 0.50 12.75 ± 0.20 18.73 ± 1.14 13.37 ± 0.46 Aa Ab Aa Ab Aa Ab L. acidophilus NCFM 35.58 ± 1.64 21.71 ± 0.60 28.10 ± 2.22 23.98 ± 0.93 24.87 ± 0.38 16.70 ± 0.98 a, b represent significant difference between the untreated and LiCl-treated groups (P <0.05) A, B, C indicate the significant difference among different strains (P <0.05) The table presents mean numbers ± standard deviation Ann Microbiol (2018) 68:207–216 213 Table 4 Effects of the SLPs on the survival of lactobacilli in simulated gastric juice Strains Groups 0 h (log) 1 h (log) 2 h (log) 3 h (log) 4 h (log) Survival (%) a a b bc c A L. bulgaricus fb04 Untreated 7.38 ± 0.27 7.18 ± 0.04 6.80 ± 0.05 6.57 ± 0.18 6.37 ± 0.03 86.31 a b c d e B LiCl-treated 6.86 ± 0.06 6.31 ± 0.09 5.41 ± 0.06 4.86 ± 0.12 3.86 ± 0.14 56.27 a b c d e A L. rhamnosus fb06 Untreated 8.45 ± 0.02 7.90 ± 0.10 7.62 ± 0.14 7.41 ± 0.03 7.10 ± 0.11 84.02 a b c d e B LiCl-treated 7.90 ± 0.08 7.18 ± 0.07 6.00 ± 0.11 5.41 ± 0.03 5.21 ± 0.02 65.95 a b c d e A L. gasseri fb07 Untreated 8.38 ± 0.03 8.15 ± 0.11 7.82 ± 0.12 7.37 ± 0.06 6.90 ± 0.07 82.34 a b c d e B LiCl-treated 7.99 ± 0.07 7.21 ± 0.07 6.38 ± 0.04 4.92 ± 0.12 3.65 ± 0.06 45.68 a a ab b c A L. acidophilus NCFM Untreated 9.30 ± 0.10 9.26 ± 0.06 9.13 ± 0.09 9.02 ± 0.08 8.85 ± 0.11 95.16 a b c d e B LiCl-treated 8.42 ± 0.05 8.07 ± 0.10 7.08 ± 0.10 6.72 ± 0.14 5.59 ± 0.06 66.38 a, b, c, d, e represent significant difference in different time of treatments (P < 0.05) A, B represent significant difference between the untreated and LiCl-treated groups (P <0.05) The table presents mean numbers ± standard deviation Beganović et al. 2011). After LiCl treatment, the surface layer typical feature of Lactobacillus SLPs, they usually have a high of L. crispatus K313 and K243 was lost as determined by content of hydrophobic amino acid residues (Sleytr 1997). TEM (Sun et al. 2012). This phenomenon was similar with The hydrophobic feature of the cell surface of bacteria has our results (Fig. 1). The surface layers of the four tested strains been implicated in the attachment to their host tissue (Zhang were stripped because of the LiCl treatment. et al. 2010). In addition, sulfur-containing amino acids were The masses of Lactobacillus SLPs were reported ranging often reported no more than 2% in Lactobacillus SLPs and the from 25 to 71 kDa (Åvall-Jääskeläinen and Palva 2005). lysine content was usually higher, at about 10% (Boot et al. Zhang et al. (2010) had isolated L. rhamnosus J10-L from 1993; Sleytr 1997; Åvall-Jääskeläinen and Palva 2005). The traditional Chinese fermentation food and the molecular mass amino acid compositions of the four extracted SLPs were of its SLP was revealed as 45 kDa. This was similar with the consistent with the common characteristics of Lactobacillus molecular weight of L. rhamnosus fb06. Previous studies re- SLPs. ported that the molecular masses of the SLPs from L. gasseri Protein secondary structures refer to the regularly repeating VPI 11759 and L. gasseri ATCC 19992 were 29.8 and conformation in polypeptide chains, mainly including α-he- 26.3 kDa (Ventura et al. 2002). By comparison, the molecular lix, β-sheet, β-turn, and random coil. Circular dichroism was weight of L. gasseri fb07 SLP was larger than that of the two often used to analyze the secondary structure of proteins. reported strains. Meng et al. (2014) reported that the SLPs of L. acidophilus The amino acid composition of Lactobacillus SLPs resem- fb116 and L. acidophilus fb214 had about 34% α-helix, 12% bles in most parts the composition of other bacterial SLPs, but β-sheet, 24% β-turn, and 32% random coil, which was similar some Lactobacillus-specific features can also be found. As a with the secondary structure content of L. acidophilus NCFM Table 5 Effects of the SLPs on the survival of lactobacilli in simulated intestinal juice Strains Groups 0 h (log) 1 h (log) 2 h (log) 3 h (log) 4 h (log) Survival (%) a b c d e A L. bulgaricus fb04 Untreated 7.19 ± 0.14 6.91 ± 0.07 6.14 ± 0.05 5.71 ± 0.11 4.92 ± 0.06 68.42 a b c d e B LiCl-treated 6.51 ± 0.07 6.05 ± 0.05 5.26 ± 0.11 4.20 ± 0.22 2.82 ± 0.07 43.31 a a b c c A L. rhamnosus fb06 Untreated 8.26 ± 0.21 8.19 ± 0.08 7.84 ± 0.12 7.41 ± 0.07 7.23 ± 0.03 87.53 a b c d e B LiCl-treated 8.09 ± 0.04 7.82 ± 0.16 7.27 ± 0.04 6.33 ± 0.08 5.30 ± 0.17 65.51 a b c d e A L. gasseri fb07 Untreated 7.85 ± 0.12 7.32 ± 0.07 6.94 ± 0.07 6.12 ± 0.03 5.57 ± 0.08 70.96 a b c d e B LiCl-treated 7.20 ± 0.03 5.97 ± 0.11 5.31 ± 0.03 3.18 ± 0.14 2.66 ± 0.09 36.94 a b c d e A L. acidophilus NCFM Untreated 8.74 ± 0 .08 7.98 ± 0.04 7.27 ± 0.01 6.96 ± 0.08 6.66 ± 0.14 76.20 a b c d e B LiCl-treated 8.36 ± 0.08 7.70 ± 0.15 6.91 ± 0.10 5.62 ± 0.12 4.19 ± 0.09 50.12 a, b, c, d, e represent significant difference in different time of treatments (P < 0.05) A, B represent significant difference between the untreated and LiCl-treated groups (P <0.05) The table presents mean numbers ± standard deviation 214 Ann Microbiol (2018) 68:207–216 Fig. 3 SLP concentrations under different level of bile stress determined by Bradford method SLP determined in our experiment. These findings indicated strains was likely to be reduced after the SLPs were removed. that the secondary structure content of SLPs may be related to Moreover, among the four tested strains, L. acidophilus NCFM the genetic similarity of the strains. In addition, Mobili et al. exhibited the highest autoaggregation and L. gasseri fb07 the (2009) found that the thermal denaturation temperature of lowest one. Interestingly, L. acidophilus NCFM and L. gasseri SLPs was associated with their secondary structure content. fb07 also showed the highest and the lowest coaggregation More β-sheet could make the protein more stable. Therefore, ability, respectively. This finding was in agreement with we could infer that the SLP of L. bulgaricus may have the Collado et al. (2008) who suggested that coaggregation abili- lowest thermal denaturation temperature in the four extracted ties were related to autoaggregation properties. SLPs. Ventura et al. (2002) reported two kinds of surface When lactobacilli were ingested in a sufficient number, they proteins from L. gasseri. The two proteins were suggested to would exert their beneficial functions effectively. Therefore, the possess a content of β-sheet from 26.1 to 28.5% by secondary key point in enabling lactobacilli to play their role is to maintain structure prediction, which was a little higher than our results the viability during their transit through the gastrointestinal tract of L. gasseri (β-sheet 22.4%). of the host. L. acidophilus was capable of displaying adaptive Bacterial aggregation is of considerable importance in human response to various stress conditions (Kim et al. 2001). From gut, where probiotics are to be active (Jankovic et al. 2003). our data (Table 4), it can be found that the four tested Autoaggregation was reported to be correlated with adhesion, lactobacilli had good tolerance to gastric juice before LiCl treat- which was known as a prerequisite for colonization (Collado ment. Therefore, they may play their probiotic functions effec- et al. 2008). Coaggregation is a part of competitive exclusion tively in the host. But the loss of their SLPs led to the viability mechanism which is coupled with the antimicrobial activity of reduction. These results demonstrated that the protective effects the probiotic strain and enables a decrease of the pathogenic load of the SLPs could ensure the amount of the lactobacilli to in- during infections (Beganović et al. 2011). Compared with the teract with the gastrointestinal tract. In previous reports, the non-coaggregating strains, the coaggregating ones were easier to removal of SLPs from L. acidophilus M92 reduced their via- colonize in intestine and inhibit pathogen infection (Golowczyc bility in simulated gastric and small intestinal juices. et al. 2007). Furthermore, it was approved that SLPs of L. acidophilus As reported by other researchers, the autoaggregation and M92 was not sensitive to pepsin and pancreatin which could the adhesion ability of L. crispatus ZJ001, L. acidophilus M92, suggest possible protective role in the gastrointestinal tract and L. helveticus M92 strains were significantly decreased af- (Frece et al. 2005). ter the removal of their SLPs (Kos et al. 2003;Chenetal. 2007; L. acidophilus LA1-1, L. crispatus ZJ001, and L. casei Beganović et al. 2011). In our study, the autoaggregation abil- Zhang were reported to be able to adapt to salt, heat, and bile ity of the four tested strains was also impaired after the lost of stress conditions (Kim et al. 2001;Chen etal. 2007; Guo et al. the SLPs. Beganović et al. (2011) demonstrated that the 2009). Lactobacillus SLPs have shown the abilities to help the coaggregation of L. helveticus M92 with S. typhimurium FP1 growth of lactobacilli in harsh environments (Boot et al. 1993; was negatively affected after the removal of SLPs. The Kos et al. 2003; Åvall-Jääskeläinen and Palva 2005; Frece coaggregation of L. kefir with Saccharomyces lipolytica was et al. 2005). Khaleghi et al. (2010) reported that the SLP inhibited due to the loss of SLPs (Golowczyc et al. 2009). expression of L. acidophilus ATCC 4356 was increased in These outcomes were consistent with our results. All the find- the stress condition to be a protective sheath, which was con- sistent with our results. Nevertheless, the bile salt stress did ings indicated that the antimicrobial activity of Lactobacillus Ann Microbiol (2018) 68:207–216 215 Collado MC, Meriluoto J, Salminen S (2008) Adhesion and aggregation not affect the production of SLPs from L. bulgaricus fb04. properties of probiotic and pathogen strains. Eur Food Res Technol These results suggested that SLPs from different strains might 226:1065–1073 have different characteristics. Frece J, Kos B, Svetec I-K, Zgaga Z, MršaV, Šušković J (2005) Lactobacillus SLPs may have great potential in industrial Importance of S-layer proteins in probiotic activity of Lactobacillus acidophilus M92. J Appl Microbiol 98:285–292 applications due to their protective effects in hostile environ- Gerbino E, Mobili P, Tymczyszyn E, Fausto R, Gómez-Zavaglia A (2011) ments. Hollmann et al. (2007) reported a kind of liposome FTIR spectroscopy structural analysis of the interaction between coated with Lactobacillus SLPs. The SLPs enhanced the sta- Lactobacillus kefir S-layers and metal ions. J Mol Struct 987:186–192 bility of the liposome after its exposure to bile salts, pancreatic Golowczyc M, Mobili P, Garrote G, Abraham A, De Antoni G (2007) Protective action of Lactobacillus kefir carrying S-layer protein extract, pH change, and thermal shock, which provided an against Salmonella enterica serovar Enteritidis. Int J Food application of Lactobacillus SLPs in pharmaceutical industry. Microbiol 118:264–273 More applications may be explored in the future research. Golowczyc MA, Mobili P, Garrote GL, de los Angeles Serradell M, Abraham AG, De Antoni GL (2009) Interaction between Lactobacillus kefir and Saccharomyces lipolytica isolated from kefir grains: evidence for lectin-like activity of bacterial surface proteins. Conclusions JDairy Res 76:111–116 Guo Z, Wang J, Yan L, Chen W, Liu X-M, Zhang H-P (2009) In vitro comparison of probiotic properties of Lactobacillus casei Zhang, a In this study, the presence of SLPs from L. bulgaricus fb04, potential new probiotic, with selected probiotic strains. LWT-Food L. rhamnosus fb06, L. gasseri fb07, and L. acidophilus NCFM Sci Technol 42:1640–1646 was verified and their physicochemical properties were charac- Hollmann A, Delfederico L, Glikmann G, De Antoni G, Semorile L, Disalvo E (2007) Characterization of liposomes coated with S-layer terized. In addition, the biological functions of the four SLPs proteins from lactobacilli. Biochim Biophys Acta 1768:393–400 were confirmed. The results indicated that the SLPs could pos- Jakava-Viljanen M, Palva A (2007) Isolation of surface (S) layer protein itively affect the aggregation ability and gastrointestinal tolera- carrying Lactobacillus species from porcine intestine and faeces and bility of the four lactobacilli. Under adverse conditions, characterization of their adhesion properties to different host tissues. Vet Microbiol 124:264–273 L. rhamnosus fb06, L. gasseri fb07, and L. acidophilus Jankovic I, Ventura M, Meylan V, Rouvet M, Elli M, Zink R (2003) NCFM were shown to express more SLP. These observations Contribution of aggregation-promoting factor to maintenance of cell provided a theoretical basis for the practical application of SLPs shape in Lactobacillus gasseri 4B2. J Bacteriol 185:3288–3296 and the lactobacilli in food and pharmaceutical industries. Johnson B, Selle K, O’Flaherty S, Goh YJ, Klaenhammer T (2013) Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM. Microbiology 159:2269–2282 Acknowledgements This study was supported by the grant from the Khaleghi M, Kermanshahi RK, Yaghoobi M, Zarkesh-Esfahani S, National Natural Science Foundation of China (No. 31471696, Baghizadeh A (2010) Assessment of bile salt effects on s-layer pro- 31701542) and Province Key Laboratory of Transformation and duction, slp gene expression and some physicochemical properties Utilization of Cereal Resource (PL2016009). of Lactobacillus acidophilus ATCC 4356. J Microbiol Biotechnol 20:749–756 Compliance with ethical standards Kim WS, Perl L, Park JH, Tandianus JE, Dunn NW (2001) Assessment of stress response of the probiotic Lactobacillus acidophilus. Curr Conflict of interest The authors declare that they have no conflict of Microbiol 43:346–350 interest. 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Journal

Annals of MicrobiologySpringer Journals

Published: Mar 17, 2018

References