Display of heterologous protein on the surface of Lactobacillus plantarum by using the CspI anchor protein

The C-terminus of the putative cell surface protein CspI which contains one putative LPxTG motif region and a signal peptides fragment were amplified from L. plantarum CICC6024, and the green fluorescent protein gene gfp was amplified from the plasmid pACGFP. The three genes were ligated and the fusion gene was named SgfpL. The fusion gene SgfpL was then cloned into shuttle expression vector pMG36e and transformed into L. plantarum. SDS-PAGE identified that the fusion protein was expressed and the band of fusion protein was observed at the predicated molecular size. Fluorescence assay, western blot against GFP antibody, protease accessibility and SDS sensitivity assays were performed to determine that the GFP was successfully displayed on the surfaces of L. plantarum cells and the maximum display capacity of the GFP fusion protein was ca. 65 μg?ml^?1. The fermentation condition experiments determined that the amounts of GFP fusion protein were increased at a higher temperature and reached the peak at 2.5 h. Then, the β-galactosidase from Bifidobacterium bifidum was functionally displayed on the surface of L. plantarum cells via CspI to demonstrate the applicability of the CspI-mediated surface display system.     

Enhancing the stability of xylanase from Cellulomonas fimi by cell-surface display on Escherichia coli

Aims: The cell‐surface display of Cex, which encodes xylanase and exoglucanase from Cellulomonas fimi, was constructed on Escherichia coli using PgsA as the anchor protein. Characterization of the cell‐surface display of Cex was performed. Methods and Results: PgsA was fused to the N‐terminus of Cex and six histidines were utilized as spacers between the targeting and anchor proteins. Successful cell‐surface display of Cex was demonstrated by Western blot and immunofluorescence analyses on E. coli C41 (DE3). According to the time‐course analysis, the xylanase activity of Cex was achieved at 49 U g^?1 dry cell weight after 12 h culture at 37°C. The optimal temperature and pH ranges of the cell‐surface displayed protein with whole‐cell were broader than the corresponding ranges of the purified form. Further determination of thermostability indicated that the half‐life of cell‐surface displayed Cex was 1·6 times longer than that of purified Cex at 60°C. Conclusions: We have successfully developed the cell‐surface display of xylanase on E. coli. The cell‐surface display can enhance the stability of xylanase against changes in temperature and has the potential of becoming a whole‐cell biocatalyst for industrial applications, such as biobleaching of paper and production of renewable energy. Significance and Impact of the Study: The results demonstrated that the cell‐surface display of xylanase embedded in the cell membrane is more stable than that of the purified enzyme. Thus, to improve the stability of heterologous proteins production, cell‐surface display using the PgsA anchor protein as a tool can be considered in E. coli.     

Display of heterologous proteins on the surface of Lactococcus lactis using the H and W domain of PrtB from Lactobacillus delburueckii subsp. bulgaricus as an anchoring matrix

Aims: The aim of this study was to develop a cell‐surface display system for foreign antigens on the surface of a Lactococcus lactis strain using an H and W domain of PrtB from Lactobacillus delburueckii subsp. bulgaricus as an anchoring matrix. Methods and Results: To construct a cell‐surface display pACL1 vector, a derivative of pSECE1 vector, we amplified the H and W domain of the cell‐surface proteinase Prt B from Lact. bulgaricus using specific primers and then cloned it into a site downstream of the secretion signal sequence in the pSECE1 vector. The new system, designed for cell‐surface display of recombinant proteins on L. lactis, was evaluated by the expression and display of the FliC protein of Salmonella enterica serovar Enteritidis as a reporter gene (pALC1:FliC). The expression of the FliC protein by the transformed cells was analysed by Western blot analysis, and the localization of FliC on the cell surface was confirmed by immunofluorescence microscopy and flow cytometry analysis. A specific band corresponding in size (approx. 110 kDa) to FliC plus the anchor residues was detected by anti‐FliC antibody in the cell extract of L. lactis H61 harbouring pALC1:FliC, but not L. lactis H61 harbouring pALC1. In addition, flow cytometry and immunofluorescence microscopy revealed FliC‐specific positive signals and a significant increase of fluorescence, respectively, in cells harbouring pALC1:FliC compared with that in control cells harbouring the parental pALC1 plasmid. These findings demonstrated that FliC was successfully displayed on the cell surface by the anchor domain of PrtB. Conclusions: A pALC1 vector using the H and W domain of PrtB from Lact. bulgaricus as an anchoring matrix can be used to successfully display the FliC protein on the surface of L. lactis. Significance and Impact of the Study: This novel way of displaying heterologous proteins on the cell surface of L. lactis using the PrtB anchor domain should prove useful for the delivery of antigens and other proteins.     

Display of both N- and C-terminal target fusion proteins on the Aspergillus oryzae cell surface using a chitin-binding module

A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein.     

Construction of an <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> cell-surface display system using a putative GPI-anchored protein

A novel cell-surface display system was constructed in Aspergillus oryzae. Each of the five genes encoding the putative cell-wall-localized protein from the A. oryzae genome was cloned and these cell-surface anchor functions were examined by fusion to the C-terminal of the green fluorescent protein (GFP). Using the MP1 and CWP proteins as anchor proteins, GFP signals were strongly observed on the cell surface of recombinant A. oryzae. When these proteins were used as anchor proteins for cell-surface display of β-glucosidase from A. oryzae, enzyme activity was detected on the cell surface. In particular, β-glucosidase activity of recombinant A. oryzae using MP1, a putative glycosylphosphatidylinositol (GPI) anchor protein was higher than CWP. Based on these results, it was concluded that the MP1 protein can act as a GPI-anchor protein in A. oryzae, and the proposed cell-surface display system using MP1 allows for the display of heterogeneous and endogenous proteins.     

Kinetic characterization of galacto‐oligosaccharide (GOS) synthesis by three commercially important β‐galactosidases

Many β‐galactosidases show large differences in galacto‐oligosaccharide (GOS) production and lactose hydrolysis. In this study, a kinetic model is developed in which the effect of lactose, glucose, galactose, and oligosaccharides on the oNPG converting activity of various β‐galactosidases is quantified. The use of oNPG as a competing substrate to lactose yields more information than can be obtained by examining only the conversion of lactose itself. The reaction rate with lactose or oligosaccharides as substrate relative to that with water as acceptor is much higher for the β‐galactosidase of Bacillus circulans than the β‐galactosidases of Aspergillus oryzae and Kluyveromyces lactis. In addition, the β‐galactosidase of B.circulans has a high reaction rate with galactose as acceptor, in contrast to those of A. oryzae and K. lactis. The latter two are strongly inhibited by galactose. These differences explain why β‐galactosidase of B. circulans gives higher yields in GOS production than other β‐galactosidases. Many of the reaction rate constants for the β‐galactosidase isoforms of B. circulans increase with increasing molecular weight of the isoform. This indicates that the largest isoform β‐gal‐A is most active in GOS production. However, its hydrolysis rate is also much higher than that of the other isoforms, which results in a faster hydrolysis of oligosaccharides as well. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:38–47, 2014     

Antagonistic activity of two potential probiotic bacteria from fish intestines and investigation of their effects on growth performance and immune response in rainbow trout (Oncorhynchus mykiss)

The probiotic activity of two bacterial strains, Lactobacillus casei and Lactobacillus plantarum, isolated from common carp intestines was studied using antagonistic tests in vitro against Yersinia ruckeri. Randomly assigned to triplicate groups were 450 rainbow trout (mean weight, 20 ± 3 g) fed three different diets: a commercial feed, or the same feed incorporated into either 5 × 10⁷ CFU g^?1 of L. casei or L. plantarum. After a 30‐day feeding trial, 30 fish in each group were challenged with Yersinia ruckeri by intraperitoneal injection. Growth parameters were significantly increased in both treatment groups. Immune parameters such as lysozyme activity, alternative complement activity and total immunoglobulin level were significantly higher in the L. casei group than in fish fed the control diet, while no significant differences were revealed between the L. plantarum and control groups. Mortality rates of fish fed L. casei and L. plantarum were lower than in fish fed the control diet after challenging with Y. ruckeri.     

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.     

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.     

Immobilization of Lactococcus lactis to cellulosic material by cellulose‐binding domain of Cellvibrio japonicus

Aims: Immobilization of whole cells can be used to accumulate cells in a bioreactor and thus increase the cell density and potentially productivity, also. Cellulose is an excellent matrix for immobilization purposes because it does not require chemical modifications and is commercially available in many different forms at low price. The aim of this study was to construct a Lactococcus lactis strain capable of immobilizing to a cellulosic matrix. Methods and Results: In this study, the Usp45 signal sequence fused with the cellulose‐binding domain (CBD) (112 amino acids) of XylA enzyme from Cellvibrio japonicus was fused with PrtP or AcmA anchors derived from L. lactis. A successful surface display of L. lactis cells expressing these fusion proteins under the P45 promoter was achieved and detected by whole‐cell ELISA. A rapid filter paper assay was developed to study the cellulose‐binding capability of these recombinant strains. As a result, an efficient immobilization to filter paper was demonstrated for the L. lactis cells expressing the CBD‐fusion protein. The highest immobilization (92%) was measured for the strain expressing the CBD in fusion with the 344 amino acid PrtP anchor. Conclusions: The result from the binding tests indicated that a new phenotype for L. lactis with cellulose‐binding capability was achieved with both PrtP (LPXTG type anchor) and AcmA (LysM type anchor) fusions with CBD. Significance and Impact of the Study: We demonstrated that an efficient immobilization of recombinant L. lactis cells to cellulosic matrix is possible. This is a step forward in developing efficient immobilization systems for lactococcal strains for industrial‐scale fermentations.     

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.     

Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface

An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of α-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface, selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including nutrient concentrations in the media, through the emission of fluorescence. Received: 23 August 1999 / Received revision: 16 November 1999 / Accepted: 29 November 1999     

The activity of beta-galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate

Aims: Kluyveromyces lactis was cultured in cheese whey permeate on both batch and continuous mode to investigate the effect of time course and growth rate on β‐galactosidase activity, lactose consumption, ethanol production and protein profiles of the cells. Methods and Results: Cheese whey was the substrate to grow K. lactis as a batch or continuous culture. In order to precise the specific growth rate for maximum β‐galactosidase activity a continuous culture was performed at five dilution (growth) rates ranging from 0·06, 0·09, 0·12, 0·18 to 0·24 h^?1. The kinetics of lactose consumption and ethanol production were also evaluated. On both batch and continuous culture a respirofermentative metabolism was detected. The growth stage for maximum β‐gal activity was found to be at the transition between late exponential and entrance of stationary growth phase of batch cultures. Fractionating that transition stage in several growth rates at continuous culture a maximum β‐galactosidase activity at 0·24 h^?1 was observed. Following that stage β‐gal activity undergoes a decline which does not correlate to the density of its corresponding protein band on the gel prepared from the same samples. Conclusion: The maximum β‐galactosidase activity per unit of cell mass was found to be 341·18 mmol ONP min^?1 g^?1 at a dilution rate of 0·24 h^?1. Significance and Impact of the Study: The physiology of K. lactis growing in cheese whey permeate can proven useful to optimize the conversion of that substrate in biomass rich in β‐gal or in ethanol fuel. In addition to increasing the native enzyme the conditions established here can be set to increase yields of recombinant protein production based on the LAC4 promoter in K. lactis host.     

Surface display of respiratory syncytial virus glycoproteins in Lactococcus lactis NZ9000

Aims: A system for displaying heterologous respiratory syncytial virus (RSV) glycoproteins on the surface of Lactococcus lactis NZ9000 was developed. Methods and Results: Fusion of the USP45 signal peptide and the cA (C terminus of the peptidoglycan‐binding) domains of AcmA, a major autolysin from L. lactis, to the N‐ and C‐terminal of the target proteins, respectively, was carried out. The target protein was the major immunogenic domain of either the F (40·17‐kDa) or G (11·49‐kDa) glycoprotein domains of the RSV. Whole‐cell ELISA readings obtained after 24 h of induction showed an increase in protein expression as the cA domain repeats increased, for the G glycoprotein of RSV. On the other hand, the F glycoprotein indicated decreasing expression levels as the number of cA domain repeats increased. The difference in the expression levels of the F and G domains may be attributed to the different sizes of the antigenic domains. Conclusions: The size and properties of the target proteins are vital in determining the amount of antigenic domains being displayed on the surface of live cells. Significance and Impact of the Study: The system demonstrated here can aid in the utilization of the generally regarded as safe (GRAS) bacteria L. lactis, as a vaccine delivery vehicle to surface display the antigenic proteins of RSV.     

AGROBACTERIUM‐MEDIATED TRANSFORMATION IN THE GREEN ALGA HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE,VOLVOCALES)1

The first successful Agrobacterium‐mediated transformation of the green alga Haematococcus pluvialis Flot. using the binary vectors hosting the genes coding for GUS (β‐glucuronidase), GFP (green fluorescent protein), and hpt (hygromycin phosphotransferase) is reported here. Colonies resistant to hygromycin at 10 mg · L^?1 expressed β‐glucuronidase. The greenish yellow fluorescence of GFP was observed when the hygromycin‐resistant cells were viewed with a fluorescent microscope. PCR was used to successfully amplify fragments of the hpt (407 bp) and GUS (515 bp) genes from transformed cells, while Southern blots indicated the integration of the hygromycin gene into the genome of H. pluvialis. SEM indicated that the cell wall of H. pluvialis was altered on infection with Agrobacterium. The transformation achieved here by Agrobacterium does not need treatment with acetosyringone or the wounding of cells. A robust transformation method for this alga would pave the way for manipulation of many important pathways relevant to the food, pharmaceutical, and nutraceutical industries.     

Heterologous protein secretion by Lactobacillus plantarum using homologous signal peptides

Aims: To test seven selected putative signal peptides from Lactobacillus plantarum WCFS1 in terms of their ability to drive secretion of two model proteins in Lact. plantarum, and to compare the functionality of these signal peptides with that of well‐known heterologous signal peptides (Usp45, M6). Methods and Results: Signal peptide functionality was assessed using a series of modular derivatives of the pSIP vectors for peptide pheromone‐controlled high‐level gene expression in lactobacilli. Several of the constructs with homologous signal peptides yielded similar or higher reporter protein activities than constructs with heterologous signal peptides. Two of the homologous signal peptides (Lp_0373 and Lp_0600) appeared as especially promising candidates for directing secretion, as they were among the best performing with both reporter proteins. Conclusions: We have identified homologous signal peptides for high‐level secretion of heterologous proteins in Lact. plantarum. With the model proteins, some of these performed better than commonly used heterologous signal peptides. Significance and Impact of the Study: The homologous signal peptides tested out, in this study, could be useful in food‐grade systems for secretion of interesting proteins in Lact. plantarum. The constructed modular secretion vectors are easily accessible for rapid signal peptide screening.     

Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj‐Apis362 for high gamma‐aminobutyric acid production

Gamma‐aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full‐length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA‐production, the GAD gene was cloned into pMG36e‐LbGAD, and then expressed in Lactobacillus plantarum Taj‐Apis362 cells. The overexpression was confirmed by SDS‐PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone‐back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA‐rich products.     

Basic characterization and partial purification of β-glucosidase from cell-free extracts of Oenococcus oeni ST81

Aims: This study was designed to characterize a β‐glucosidase of Oenococcus oeni ST81, a strain isolated from a Spanish wine of the origin appellation Ribeira Sacra. Methods and Results: The β‐glucosidase of O. oeni ST81 seems to have a periplasmic localization into the cells. This activity was strongly inhibited by gluconic acid, partially inhibited by glucose and not inhibited by fructose, lactate, malate, mannitol or sorbitol. Ethanol increased the activity of this enzyme up to 147%. Among the several metal ions assayed, only Fe²⁺ (10 mmol l^?1) and Cu²⁺ (5 mmol l^?1) exhibited a partial inhibitory effect (40%). This enzyme was partially purified using a combination of ammonium sulfate precipitation and chromatographic methods. The single peak because of β‐glucosidase in all chromatographic columns indicates the presence of a single enzyme with an estimated molecular mass of 140 kDa. The calculated K_m and V_max values for 4‐nitrophenyl‐β‐d ‐glucopyranoside were 0·38 mmol l^?1 and 5·21 nmol min^?1, respectively. The enzyme was stable at pH 5·0 with a value of t_1/2 = 50 days for the crude extract. Conclusions: The β‐glucosidase of O. oeni ST81 is substantially different from those characterized from other wine‐related lactic acid bacteria (LAB), such as Lactobacillus plantarum and Lactobacillus brevis; however, it appears to be closely related to a β‐glucosidase from O. oeni ATCC BAA‐1163 cloned into Escherichia coli. The periplasmic localization of the enzyme together with its high tolerance to ethanol and fructose, the low inhibitory effect of some wine‐related compounds on the enzymatic activity and long‐term stability of the enzyme could be of interest for winemaking. Significance and Impact of the Study: Information regarding a β‐glucosidase from O. oeni ST81 is presented. Although the release of aroma compounds by LAB has been demonstrated, little information exists concerning the responsible enzymes. To our knowledge, this study contains the first characterization of a native β‐glucosidase purified from crude extracts of O. oeni ST81.     

The effects of a vegetable‐derived probiotic lactic acid bacterium on the immune response

The objective of this study was to investigate the probiotic properties of the fermented vegetable derived lactic acid bacterium, L. plantarum. L. plantarum 10hk2 showed antibacterial activity against pathogenic bacteria and immunomodulating effects on murine macrophage cell lines. RAW 264.7 cells stimulated with viable cells of this probiotic strain increased the amounts of pro‐inflammatory mediators such as IL‐1β, IL‐6 and TNF‐α, as well as the anti‐inflammatory mediator, IL‐10. ICR mice fed with viable cells of L. plantarum 10hk2 had reduced numbers of enteric Salmonella and Shigella species in comparison to controls from 2 weeks after supplementation, and this effect was observed for up to 4 weeks. The findings of this study suggest that this specific lactic acid bacterial strain, which is derived from vegetable fermentation, holds great promise for use in probiotics and as a food additive since it can reduce the number of some pathogenic bacteria through production of lactic acids.