Inhibitory potential of lactobacilli against <Emphasis Type="Italic">Escherichia coli</Emphasis> internalization by HT 29 cells

A quantitative fluorescent in situ hybridization method was employed to evaluate the competitive inhibitory effect of three Lactobacillus strains (Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus plantarum) against Escherichia coli internalization in a model system of HT 29 cells. Furthermore, aggregation and adhesion abilities of the Lactobacillus strains were examined. All lactobacilli were able to attach to the HT 29 cells and aggregate with pathogens; however, the adhesion and aggregation degree was strain-dependent. L. reuteri possessed a high capacity of adhesion (6.80 ± 0.63; log CFU ± SEM per well), whereas lower capacities were expressed by L. gasseri (4.52 ± 0.55) and L. plantarum (4.90 ± 0.98). Additionally, L. reuteri showed the rapid or normal ability to aggregate with selected E. coli in comparison with remaining two lactobacilli, which showed only slow or negative aggregative reaction. Internalization of E. coli into the cell lines was markedly suppressed by L. reuteri, while L. gasseri and L. plantarum caused only a minimum anti-invasion effect. The fact that L. reuteri in our experiments showed an outstanding potential for adhering to the colon epithelial cell line, compared with the rest strains, suggested that one of the possible mechanisms of preventing pathogen adhesion and invasion is simple competitions at certain receptors and capability to block receptor binding sites, or that an avid interaction between L. reuteri and the host cell might be modulating intracellular events responsible for the E. coli internalization. Moreover, L. reuteri exhibited a strong ability to aggregate with E. coli, which could be another limiting factor of pathogen invasion.     

Adhesion of the probiotic strains <Emphasis Type="Italic">Enterococcus mundtii</Emphasis> ST4SA and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> 423 to Caco-2 cells under conditions simulating the intestinal tract,and in the presence of antibiotics and anti-inflammatory medicaments

Adhesion of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 (human carcinoma epithelial) cells was visualized by fluorescent staining. Both strains showed good adhesion compared to L. casei MB1, L. casei Shirota, L. johnsonii La1 and L. rhamnosus GG. No correlation was found between hydrophobicity, aggregation and adhesion to Caco-2 cells. Presence of antibiotics and anti-inflammatory medicaments reduced adhesion of bacterial strains to Caco-2 cells. Proteins sensitive to pepsin, trypsin and pronase are involved in the adhesion of E. mundtii ST4SA and L. plantarum 423 to Caco-2 cells. Adhesion of Listeria monocytogenes ScottA to Caco-2 cells was not prevented by E. mundtii ST4SA and L. plantarum 423. Cell-free culture supernatants of strains ST4SA and 423, containing the antimicrobial peptides plantaricin 423 and peptide ST4SA, prevented the invasion of L. monocytogenes ScottA into Caco-2 cells.     

Expression of fluorescent proteins in Lactobacillus rhamnosus to study host–microbe and microbe–microbe interactions

Probiotic Lactobacillus strains are widely used to benefit human and animal health, although the exact mechanisms behind their interactions with the host and the microbiota are largely unknown. Fluorescent tagging of live probiotic cells is an important tool to unravel their modes of action. In this study, the implementation of different heterologously expressed fluorescent proteins for the labelling of the model probiotic strains Lactobacillus rhamnosusGG (gastrointestinal) and Lactobacillus rhamnosusGR‐1 (vaginal) was explored. Heterologous expression of mTagBFP2 and mCherry resulted in long‐lasting fluorescence of L. rhamnosusGG and GR‐1 cells, using the nisin‐controlled expression (NICE) system. These novel fluorescent strains were then used to study in vitro aspects of their microbe–microbe and microbe–host interactions. Lactobacillus rhamnosusGG and L. rhamnosusGR‐1 expressing mTagBFP2 and mCherry could be visualized in mixed‐species biofilms, where they inhibited biofilm formation by Salmonella Typhimurium–gfpmut3 expressing the green fluorescent protein. Likewise, fluorescent L. rhamnosusGG and L. rhamnosusGR‐1 were implemented for the visualization of their adhesion patterns to intestinal epithelial cell cultures. The fluorescent L. rhamnosus strains developed in this study can therefore serve as novel tools for the study of probiotic interactions with their environment.     

Lactobacillus plantarum AS1 binds to cultured human intestinal cell line HT-29 and inhibits cell attachment by enterovirulent bacterium Vibrio parahaemolyticus

Aim: Lactobacillus plantarum AS1 was incubated with HT‐29 adenocarcinoma cell line to assess its adhesion potency and examined for its inhibitory effect on the cell attachment by an enterovirulent bacterium Vibrio parahaemolyticus. Methods and Results: Lactobacillus plantarum AS1 attached efficiently to HT‐29 cells as revealed by scanning electron microscopy and bacterial adhesion assay. Lactobacillus plantarum AS1 significantly reduced V. parahaemolyticus attached to HT‐29 cells by competition, exclusion and displacement mode. Lactobacillus plantarum AS1 seems to adhere to human intestinal cells via mechanisms that involve different combinations of carbohydrate and protein factors on the bacteria and eukaryotic cell surface. Conclusion: Strain Lact. plantarum AS1 inhibits the cell attachment of a pathogen V. parahaemolyticus by steric hindrance mechanism. Also, antibacterial factors such as bacteriocins, lactic acid and exopolysaccharides could be involved. Significance and Impact of the Study: The ability to inhibit the adhesion of V. parahaemolyticus to intestinal cell line warrants further investigation to explore the use of probiotic strain Lact. plantarum AS1 in the management of gastroenteritis caused by V. parahaemolyticus.     

The proteins (12 and 15 kDa) isolated from heat‐killed Lactobacillus plantarum L67 induces apoptosis in HT‐29 cells

A number of scientific studies have revealed that Lactobacillus strains have beneficial bioactivities in the gastrointestinal tract. In this study, the production of intracellular reactive oxygen species (ROS) and the amounts of intracellular calcium, protein kinase C activity, cytochrome c, Bid, Bcl‐2, Bax and the apoptosis‐mediated proteins [caspase‐8, caspase‐3 and poly ADP ribose polymerase (PARP)] were evaluated to understand the induction of programmed cell death in HT‐29 cells by Lactobacillus plantarum L67. The results obtained from this study indicated that the relative intensities of the apoptotic‐related factors (intracellular ROS and intracellular calcium) and of apoptotic signals (Bax and t‐Bid) increased with increasing concentrations of the membrane proteins isolated from heat‐killed L. plantarum L67, whereas the relative intensities of cytochrome c, Bcl‐2, caspase‐8, caspase‐3 and PARP decreased. This study determines whether proteins (12 and 15 kDa) isolated from heat‐killed L. plantarum L67 induce programmed cell death in HT‐29 cells. Proteins isolated from L. plantarum L67 can stimulate the apoptotic signals and then consequently induce programmed cell death in HT‐29 cells. The results in this study suggest that the proteins isolated from L. plantarum L67 could be used as an antitumoural agent in probiotics and as a component of supplements or health foods. Copyright © 2015 John Wiley & Sons, Ltd.     

Antimicrobial susceptibility of microflora from ovine cheese

Strains identified in ovine cheese and bryndza by matrix-assisted laser desorption/ionization time-of-flight analysis belonged to ten species of non-enterococcal lactic acid bacteria and included Lactobacillus casei/Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus brevis, Lactococcus lactis, Pediococcus pentosaceus and Pediococcus acidilactici. The susceptibility toward antibiotics was determined in lactobacilli, lactococci and pediococci and also in Escherichia coli for comparison. Analysis of L. fermentum and pediococci revealed the presence of non-wild-type epidemiological cut-offs in streptomycin, clindamycin or gentamicin. E. coli were resistant to ampicillin, tetracycline, enrofloxacin and florfenicol. No extended spectrum β-lactamases were detected.     

Probiotic Lactobacilli Interfere with <Emphasis Type="Italic">Streptococcus mutans</Emphasis> Biofilm Formation In Vitro

In clinical studies, probiotic bacteria have decreased the counts of salivary mutans streptococci (MS). We compared the effects of probiotic Lactobacillus strains on the biofilm formation of Streptococcus mutans. The bacterial strains used included four S. mutans strains (reference strains NCTC 10449 and Ingbritt and clinical isolates 2366 and 195) and probiotic strains Lactobacillus rhamnosus GG, L. plantarum 299v, and L. reuteri strains PTA 5289 and SD2112. The ability of MS to adhere and grow on a glass surface, reflecting biofilm formation, was studied in the presence of the lactobacilli (LB). The effect of LB culture supernatants on the viability of the MS was studied as well. All of the LB inhibited the biofilm formation of the clinical isolates of MS (P < 0.001). The biofilm formation of the reference strains of MS was also inhibited by the LB, but L. plantarum and L. reuteri PTA 5289 showed a weaker inhibition when compared to L. reuteri SD2112 and L. rhamnosus GG. Viable S. mutans cells could be detected in the biofilms and culture media only when the experiments were performed with the L. reuteri strains. The L. reuteri strains were less efficient in killing the MS also in the tests performed with the culture supernatants. The pHs of the supernatants of L. reuteri were higher compared to those of L. rhamnosus GG and L. plantarum; P < 0.001. In conclusion, our results demonstrated that four commonly used probiotics interfered with S. mutans biofilm formation in vitro, and that the antimicrobial activity against S. mutans was pH-dependent.     

In Vitro Characterization of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strains with Inhibitory Activity on Enteropathogens for Use as Potential Animal Probiotics

The present study evaluates the probiotic properties of three Lactobacillus plantarum strains MJM60319, MJM60298, and MJM60399 possessing antimicrobial activity against animal enteric pathogens. The three strains did not show bioamine production, mucinolytic and hemolytic activity and were susceptible to common antibiotics. The L. plantarum strains survived well in the simulated orogastrointestinal transit condition and showed adherence to Caco-2 cells in vitro. The L. plantarum strains showed strong antimicrobial activity against enterotoxigenic Escherichia coli, Shiga toxin-producing E. coli, Salmonella enterica subsp. enterica serovar Typhimurium, Choleraesuis and Gallinarum compared to the commercial probiotic strain Lactobacillus rhamnosus GG. The mechanism of antimicrobial activity of the L. plantarum strains appeared to be by the production of lactic acid. Furthermore, the L. plantarum strains tolerated freeze-drying and maintained higher viability in the presence of cryoprotectants than without cryoprotectants. Finally, the three L. plantarum strains tolerated NaCl up to 8% and maintained >60% growth. These characteristics of the three L. plantarum strains indicate that they could be applied as animal probiotic after appropriate in vivo studies.     

The effect of cell surface components on adhesion ability of Lactobacillus rhamnosus

The aim of this study was to analyze the cell envelope components and surface properties of two phenotypes of Lactobacillus rhamnosus isolated from the human gastrointestinal tract. The ability of the bacteria to adhere to human intestinal cells and to aggregate with other bacteria was determined. L. rhamnosus strains E/N and PEN differed with regard to the presence of exopolysaccharides (EPS) and specific surface proteins. Transmission electron microscopy showed differences in the structure of the outer cell surface of the strains tested. Bacterial surface properties were analyzed by Fourier transform infrared spectroscopy, fatty acid methyl esters and hydrophobicity assays. Aggregation capacity and adhesion of the tested strains to the human colon adenocarcinoma cell line HT29 was determined. The results indicated a high adhesion and aggregation ability of L. rhamnosus PEN, which possessed specific surface proteins, had a unique fatty acid content, and did not synthesize EPS. Adherence of L. rhamnosus was dependent on specific interactions and was promoted by surface proteins (42–114 kDa) and specific fatty acids. Polysaccharides likely hindered bacterial adhesion and aggregation by masking protein receptors. This study provides information on the cell envelope constituents of lactobacilli that influence bacterial aggregation and adhesion to intestinal cells. This knowledge will help to understand better their specific contribution in commensal–host interactions and adaptation to this ecological niche.     

Influence of orally administered Lactobacillus GG on respiratory immune response in a murine model of diet‐induced obesity

Mice with diet‐induced obesity were fed with Lactobacillus rhamnosus GG (LGG) suspended in saline or saline alone (control mice). Pulmonary mRNA expression of IFN‐γ; IFN‐α receptor 1; CD247 antigen; killer cell lectin‐like receptor subfamily K, member 1; TNF‐α; IL‐12 receptor β1 and IL‐2 receptor β, and the proportion of Lactobacillales in feces were significantly greater in the LGG group than in the control mice (P < 0.05 and P < 0.01, respectively). These results suggest that LGG alters the respiratory immunity of obese subjects through having a potent impact on intestinal immunity.     

An In Vitro Study on Bacterial Growth Interactions and Intestinal Epithelial Cell Adhesion Characteristics of Probiotic Combinations

The aims of this study were to examine long-term growth interactions of five probiotic strains (Lactobacillus casei 01, Lactobacillus plantarum HA8, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC 55730 and Bifidobacterium lactis Bb12) either alone or in combination with Propionibacterium jensenii 702 in a co-culture system and to determine their adhesion ability to human colon adenocarcinoma cell line Caco-2. Growth patterns of probiotic Lactobacillus strains were not considerably affected by the presence of P. jensenii 702, whereas lactobacilli exerted a strong antagonistic action against P. jensenii 702. In the co-culture of Bif. lactis Bb12 and P. jensenii 702, a significant synergistic influence on growth of both bacteria was observed (P < 0.05). The results of adhesion assay showed that when probiotic strains were tested in combination, there was evidence of an associated effect on percentage adherence. However, in most cases these differences were not statistically significant (P < 0.05). Adhesion percentage of Lb. casei 01 and Lb. rhamnosus GG both decreased significantly in the presence of P. jensenii 702 compared to their adhesion levels when alone (P < 0.05). These results show that the survival and percentage adhesion of some probiotic strains may be influenced by the presence of other strains and this should be considered when formulating in the probiotic products.     

Strain-Dependent Augmentation of Tight-Junction Barrier Function in Human Primary Epidermal Keratinocytes by Lactobacillus and Bifidobacterium Lysates

In this study, we investigated whether probiotic lysates can modify the tight-junction function of human primary keratinocytes. The keratinocytes were grown on cell culture inserts and treated with lysates from Bifidobacterium longum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus fermentum, or Lactobacillus rhamnosus GG. With the exception of L. fermentum (which decreased cell viability), all strains markedly enhanced tight-junction barrier function within 24 h, as assessed by measurements of transepithelial electrical resistance (TEER). However, B. longum and L. rhamnosus GG were the most efficacious, producing dose-dependent increases in resistance that were maintained for 4 days. These increases in TEER correlated with elevated expression of tight-junction protein components. Neutralization of Toll-like receptor 2 abolished both the increase in TEER and expression of tight-junction proteins induced by B. longum, but not L. rhamnosus GG. These data suggest that some bacterial strains increase tight-junction function via modulation of protein components but the different pathways involved may vary depending on the bacterial strain.     

Survival of freeze-dried Lactobacillus plantarum and Lactobacillus rhamnosus during storage in the presence of protectants

No significant differences were observed in the viability of Lactobacillus plantarum and Lactobacillus rhamnosus cells during freeze-drying in the presence or absence of inositol, sorbitol, fructose, trehalose, monosodium glutamate and propyl gallate. However, survival was higher during storage when drying took place in the presence of these compounds. Sorbitol produced more significant effects than the other compounds toward maintaining viability of freeze-dried L. plantarum and L. rhamnosus.     

Isolation and characterization of probiotic properties of Lactobacilli isolated from rat fecal microbiota

The objective of the present study was to characterize lactobacilli isolates from the feces of male Wistar rats. Various physiological features of the candidate probiotic isolates were preliminarily investigated, including tolerance to simulated gastric juice and bile salts, antimicrobial activity, antibiotic susceptibility and in vitro aggregation. Based on their morphological and biochemical characteristics, four potential probiotic isolates (CS2, CS3, CS4, and CS7) were screened. The isolates showed good tolerance to stimulated gastric juice and bile salts. CS4 and CS7 exhibited strong antibacterial activities against the pathogens tested as assessed in neutral pH culture supernatants. All lactobacilli isolates were susceptible to all the tested antibiotics, except vancomycin. Moreover, the isolate CS4 and CS7 were found to possess stronger cell surface traits such as hydrophobicity, auto‐aggregation and co‐aggregation capacity. In addition, CS4 and CS7 had greater β‐galactosidase activities than the others. Biochemical tests and 16S rRNA gene sequencing confirmed that CS2, CS3, CS4 and CS7 are Lactobacillus intestinalis PJ2, L. sakei PJ3, L. helveticus PJ4, and L. plantarum PJ7, respectively. Based on the obtained results, L. helveticus PJ4 and L. plantarum PJ7 are ideal in vitro probiotic candidates and require further in vivo evaluation.     

Quantitative Approach in the Study of Adhesion of Lactic Acid Bacteria to Intestinal Cells and Their Competition with Enterobacteria

To describe the phenomena of bacterial adhesion to intestinal cells and the competition for adhesion between bacteria, mathematical equations based on a simple dissociation process involving a finite number of bacterial receptors on intestinal cell surface were developed. The equations allow the estimation of the maximum number of Lactobacillus sp. and Escherichia coli cells that can adhere to Caco-2 cells and intestinal mucus; they also characterize the affinity of the bacteria to Caco-2 cells and intestinal and fecal mucus and the theoretical adhesion ratio of two bacteria present in a mixed suspension. The competition for adhesion between Lactobacillus rhamnosus GG and E. coli TG1 appeared to follow the proposed kinetics, whereas the competition between Lactobacillus casei Shirota and E. coli TG1 may involve multiple adhesion sites or a soluble factor in the culture medium of the former. The displacement of the adhered Lactobacillus by E. coli TG1 seemed to be a rapid process, whereas the displacement of E. coli TG1 by the Lactobacillus took more than an hour.     

Lactobacillus rhamnosus GR-1 Ameliorates Escherichia coli-Induced Inflammation and Cell Damage via Attenuation of ASC-Independent NLRP3 Inflammasome Activation

Escherichia coli is a major environmental pathogen causing bovine mastitis, which leads to mammary tissue damage and cell death. We explored the effects of the probiotic Lactobacillus rhamnosus GR-1 on ameliorating E. coli-induced inflammation and cell damage in primary bovine mammary epithelial cells (BMECs). Increased Toll-like receptor 4 (TLR4), NOD1, and NOD2 mRNA expression was observed following E. coli challenge, but this increase was attenuated by L. rhamnosus GR-1 pretreatment. Immunofluorescence and Western blot analyses revealed that L. rhamnosus GR-1 pretreatment decreased the E. coli-induced increases in the expression of the NOD-like receptor family member pyrin domain-containing protein 3 (NLRP3) and the serine protease caspase 1. However, expression of the adaptor protein apoptosis-associated speck-like protein (ASC, encoded by the Pycard gene) was decreased during E. coli infection, even with L. rhamnosus GR-1 pretreatment. Pretreatment with L. rhamnosus GR-1 counteracted the E. coli-induced increases in interleukin-1β (IL-1β), -6, -8, and -18 and tumor necrosis factor alpha mRNA expression but upregulated IL-10 mRNA expression. Our data indicate that L. rhamnosus GR-1 reduces the adhesion of E. coli to BMECs, subsequently ameliorating E. coli-induced disruption of cellular morphology and ultrastructure and limiting detrimental inflammatory responses, partly via promoting TLR2 and NOD1 synergism and attenuating ASC-independent NLRP3 inflammasome activation. Although the residual pathogenic activity of L. rhamnosus, the dosage regimen, and the means of probiotic supplementation in cattle remain undefined, our data enhance our understanding of the mechanism of action of this candidate probiotic, allowing for development of specific probiotic-based therapies and strategies for preventing pathogenic infection of the bovine mammary gland.     

Functional properties of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains isolated from dairy products

Twenty-four acid- and bile-tolerant lactobacilli isolates from dairy products were identified and further in vitro characterized for the presence of functional traits potentially useful for probiotic applications, which included desirable and undesirable traits, such as biofilm formation, ability to inhibit intestinal pathogens, antibiotic susceptibility, and enzyme activity. The majority of examined strains were susceptible to certain antimicrobial agents (streptomycin, gentamicin, clindamycin, erythromycin, tetracycline, quinupristin–dalfopristin), except for three strains of Lactobacillus rhamnosus with minimal inhibitory concentration levels for streptomycin higher than the microbiological breakpoints (≥32 μg/mL), which are considered as resistant. Undesirable traits such as α-chymotrypsin or N-acetyl-β-glucosaminidase activities were not detected, but low β-glucuronidase, and moderate and high β-glucosidase activities were recorded in nine strains, which were eliminated from further examination together with three isolates showing unsuitable antibiotic resistance. Of the remaining 12 isolates, 4 (Lactobacillus fermentum 202, Lactobacillus gallinarum 7001, L. rhamnosus 183, and Lactobacillus plantarum L2-1) manifested an outstanding potential to inhibit selected intestinal pathogens in an agar spot test, including Escherichia coli and Salmonella spp., and simultaneously demonstrated strong biofilm-forming capacity. In conclusion, the results of our in vitro experiments showed that the above four strains had a potential probiotic value and met the criteria to be identified as a possible probiotic microorganism, with the necessity of verification through well-designed in vivo experimental, clinical, and technological studies before the strains can be used as probiotics or as starter probiotic cultures.     

Modulation of the inflammation–coagulation interaction during pneumococcal pneumonia by immunobiotic Lactobacillus rhamnosus CRL1505: Role of Toll‐like receptor 2

The present study evaluated the effect of nasally given Lactobacillus rhamnosus CRL1505 on the immunocoagulative response during pneumococcal infection in immunocompetent mice. In addition, we aimed to gain insight into the mechanism involved in the immunomodulatory effect of the L. rhamnosus CRL1505 strain by evaluating the role of TLR2. Results showed that nasally given L. rhamnosus CRL1505 effectively regulates inflammation and hemostatic alterations during the pneumococcal infection. Immunobiotic treatment significantly reduced permeability of the bronchoalveolar–capillary barrier, and general cytotoxicity, decreasing lung tissue damage. The CRL1505 strain improved the production of TNF‐α, IFN‐γ, and IL‐10 after pneumococcal challenge. In addition, increased TM and TF expressions were found in lungs of L. rhamnosus CRL1505‐treated mice. Moreover, we demonstrated, for the first time, that the TLR2 signaling pathway has a role in the induction of IFN‐γ and IL‐10 and in the reduction of TF. The results also allow us to speculate that a PRR, other than TLR2, may mediate the immunobiotic activity of L. rhamnosus CRL1505 and could explain changes in TNF‐α and TM.     

Characterization of Biosynthesized Silver Nanoparticles Using Lactobacillus rhamnosus GG and its In Vitro Assessment Against Colorectal Cancer Cells

Silver nanoparticles are the most desirable nanoparticles broadly used in diverse fields. This study intends to investigate the anticancer properties of synthesized silver/Lactobacillus rhamnosus GG nanoparticles (Ag-LNPs) as a reducing and stabilizing agent in the synthesis process. To prepare silver/Lactobacillus rhamnosus GG nanoparticles, 1 mg/ml cell lysate of Lactobacillus rhamnosus GG and 1 mM silver nitrate solution were mixed and incubated for 72 h. XRD, FTIR, and TEM methods were used for nanoparticle characterization. MTT assay and annexin/PI staining were employed to analyze the toxicity and apoptotic cells levels of Ag-LNPs, respectively. TEM showed that these nanoparticles are spherical shaped about 233 nm in size. FTIR spectroscopy demonstrated that Ag-LNPs were functionalized with biomolecules. XRD pattern showed high purity and face-centered crystal structure of Ag-LNPs. MTT assay revealed that the percentages of HT-29 live cells significantly reduced in the high concentration of Ag-LNPs. Annexin/PI staining showed that these nanoparticles could lead HT-29 cells to apoptosis. This study showed the new Ag-LNP-synthesizing method using Lactobacillus rhamnosus GG as a cost-effective and efficient approach. Also, it showed that these nanoparticles can be considered as a potential active agent for biomedical applications and drug delivery due to their anticancer activities.

     

Cloning,expression and functional validation of a β-fructofuranosidase from Lactobacillus plantarum

Fructooligosaccharides (FOS) are prebiotics that selectively stimulate the growth and activity of lactobacilli and bifidobacteria. These strains metabolize FOS with endogenous β-fructofuranosidase. In this study, a β-fructofuranosidase gene from Lactobacillus plantarum ST-III designated sacA was cloned into Escherichia coli, and the properties of the recombinant protein (SacA) were examined. The sacA gene encodes a peptide of 501 amino acids with a predicted molecular weight of 56.7 kDa. Sequence alignment revealed the presence of three highly conserved motifs, NDPNG, RDP and EC, indicating that the enzyme belongs to glycoside hydrolase family 32. The predicted three-dimensional structure of the SacA enzyme was similar to β-fructofuranosidases of bifidobacteria, such that it contained a five-blade β-propeller module and a β-sandwich domain with one additional N-terminal α-helix. The optimal reaction temperature and pH of the enzyme were 37 °C and 6.0, respectively. Substrate hydrolysis and kinetic parameters demonstrated that β-fructofuranosidase from L. plantarum ST-III liberated fructosyl residues from the non-reducing terminus of fructans, such as sucrose, FOS, levan or inulin, and FOS was the preferred substrate. The expression of the sacA gene in a non-FOS-fermenting strain, Lactobacillus rhamnosus GG, enabled the recombinant strain to metabolize FOS and sucrose.