Isolation, technological characterization and in vitro probiotic evaluation of Lactococcus strains from traditional Turkish skin bag Tulum cheeses Abstract Purpose The present study was undertaken to evaluate in vitro prerequisite probiotic and technological characteristics of ten Lactococcus strains isolated from traditional goat skin bags of Tulum cheeses from the Central Taurus mountain range in Turkey. Methods All isolates were identified based on the nucleotide sequences of the 16S rRNA gene. Eight isolates belonged to Lactococcus lactis and two belonged to Lactococcus garvieae. Probiotic potential was determined from resistance to acid and bile salt, resistance to gastric and pancreatic juices, resistance to antibiotic, auto-aggregation, co-aggregation, diacetyl, hydrogen peroxide and exopolysaccharide productions. Technological properties were verified by alcohol, NaCl and hydrogen peroxide resistance and temperature tests. Results L. lactis NTH7 displayed high growth at all alcohol concentrations while L. lactis NTH4 grew very well even at NaCl concentrations of 10%. All strains showed to some extent resistance to acid and bile. Five strains exhibited desirable survival in gastric juice (pH 2.0), while three strains survived in pancreatic juice (pH 8.0). All Lactococcus isolates were sensitive to ampicillin, chloramphenicol, erythromycin, vancomycin, kanamycin, gentamycin and tetracycline. Also, only L. lactis NTH7 from among the isolates showed resistance against penicillin. L. lactis NTH10 and L. lactis NTH7 had higher auto-aggregation values in comparison with all other strains. All the strains demonstrated a co-aggregation ability against model food pathogens, particularly, L. lactis NTH10 which showed a superior ability with L. monocytogenes. All the ten strains produced H2O2 and exopolysaccharide (EPS); however, diacetyl production was detected for only four strains including L. lactis NTH10. Conclusion These results demonstrate that the L. lactis NTH10 isolate could be regarded as a favorable probiotic candidate for future in vivo studies.

Analysis of the influence of cyclo (L-phenylalanine-L-proline) on the proteome of Staphylococcus aureus using iTRAQ Abstract Purpose Cyclo (L-phenylalanine-L-proline) (cFP) is an extracellular quorum sensing (QS) signal molecule that coordinates communication between Gram-negative bacteria. Some studies have also reported QS in Gram-positive bacteria. However, the effect of cFP on Gram-positive bacteria remains unknown. Therefore, an isobaric tags for relative and absolute quantitation (iTRAQ) proteomic experiment were designed to elucidate whether cFP influences protein expression in Staphylococcus aureus (S. aureus). Methods The iTRAQ proteomics method was used to analyze untreated (control) and S. aureus treated with cFP for 12 h. Samples were then processed by liquid-phase tandem mass spectrometry (LC-MS/MS) and analyzed using bioinformatics tools. Results The results identified 1296 proteins from the S. aureus CGMCC 1.1861 proteome. Twenty-two proteins, including some two-component regulatory systems (TCRS), were associated with signal transduction. Differential expression analysis revealed that only 43 proteins were up-regulated and 41 proteins were down-regulated by cFP. The most significantly different pathways were amino acid metabolism, fatty acid degradation, and metabolism of cofactors and vitamins. Results showed that cFP down-regulated virulence factors, up-regulated lipid and amino acid metabolism, promoted acetylation and phosphorylation, and decreased alcohol dehydrogenase expression. A total of 12 significantly differentially expressed proteins (DEPs) were related to signal transduction. Among them, Rot (Q9RFJ6) and SarR (Q9F0R1), which can inhibit transcription of the Agr system, were up-regulated, whereas virulence factors such as ESAT-6 protein A (Q2G189), phenol soluble modulin (Psm, Q2FZA4), and a peptide ABC transporter permease (Q2G168) were down-regulated. AgrA (Q2FWM4) was down-regulated by cFP in S. aureus. Conclusion cFP reduced AgrA and the expression of some exotoxins but increased Rot and SarR expression.

Polyphasic analysis in the description of Sulfitobacter salinus sp. nov., a marine alphaproteobacterium isolated from seawater Abstract Purpose A polyphasic analysis was performed on a novel bacterium, designated strain KMU-143T, which was isolated from seawater collected in the Republic of Korea. Methods A novel marine bacterium KMU-143T was analyzed and described using a polyphasic taxonomic method including 16S rRNA gene sequence analysis, DNA–DNA hybridization, and physiological, biochemical, and chemotaxonomic analyses. Results Strain KMU-143T was Gram-stain-negative, strictly aerobic, oval-shaped, non-motile, and chemoorganoheterotrophic. Phylogenetic analysis based on the 16S rRNA gene sequence demonstrated that the novel marine bacterium belongs to the family Rhodobacteraceae, of the class Alphaproteobacteria, and that it possessed the highest (97.1%) sequence similarity with Sulfitobacter pontiacus ChLG 10T and Sulfitobacter undariae W-BA2T. DNA–DNA relatedness values between strains KMU-143T, S. pontiacus JCM 21789T, and S. undariae KCTC 42200T were less than 70%. The major isoprenoid quinone of the novel isolate was ubiquinone-10 (Q-10) and the major (> 10%) cellular fatty acids were C16:0 and C18:1 ω7c. The genomic DNA G+C content of strain KMU-143T was 56.1 mol%. The polar lipid profile of the strain KMU-143T was found to consist of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, an unidentified aminolipid, and two unidentified lipids. Conclusion Based on the discriminative phylogenetic position and combination of genotypic and phenotypic properties, the strain is considered to represent a new species of the genus Sulfitobacter for which the name Sulfitobacter salinus sp. nov. is proposed. The type strain of S. salinus sp. nov. is KMU-143T (= KCCM 90322T = NBRC 113459T).

Microbiological, biochemical, physicochemical surface properties and biofilm forming ability of Brettanomyces bruxellensis Abstract Purpose Brettanomyces bruxellensis is a serious source of concern for winemakers. The production of volatile phenols by the yeast species confers to wine unpleasant sensory characteristics which are unacceptable by the consumers and inevitably provoke economic loss for the wine industry. This ubiquitous yeast is able to adapt to all winemaking steps and to withstand various environmental conditions. Moreover, the ability of B. bruxellensis to adhere and colonize inert materials can be the cause of the yeast persistence in the cellars and thus recurrent wine spoilage. We therefore investigated the surface properties, biofilm formation capacity, and the factors which may affect the attachment of the yeast cells to surfaces with eight strains representative of the genetic diversity of the species. Methods The eight strains of B. bruxellensis were isolated from different geographical and industrial fermentation origins. The cells were grown in synthetic YPD medium containing 1% (w/v) yeast extract (Difco Laboratories, Detroit), 2% (w/v) bacto peptone (Difco), and 1% (w/v) glucose. Surface physicochemical properties as electrophoretic mobility and adhesion to hydrocarbon of the cells were studied. The ability of the strains to form biofilm was quantified using a colorimetric microtiter 96-well polystyrene plate. Biochemical characteristics were examined by colorimetric methods as well as by chemical analysis. Result Our results show that the biofilm formation ability is strain-dependent and suggest a possible link between the physicochemical properties of the studied strains and their corresponding genetic group. Conclusion The capacity to detect and identify the strains of the spoilage yeast based on their biofilm formation abilities may help to develop more efficient cleaning procedures and preventing methods.

Biodegradation kinetics of microcystins-LR crude extract by Lysinibacillus boronitolerans strain CQ5 Abstract Purpose As the most common variant of microcystins (MCs), microcystin-LR (MCLR) is a kind of toxins produced by some species of harmful cyanobacteria and more and more attention has been paid to it. Biodegradation has been extensively investigated and recognized to be a cost-efficient and environmentally benign method for MC clean-up. In order to further research the growth characteristics of strain and the biodegradation characteristics of MCLR, it is necessary to use the dynamic mathematical models as powerful and useful tools. Methods In this study, strain CQ5 was screened and identified by morphological observation, physiological and biochemical tests, and 16S rDNA sequence analysis. The kinetic models of cell growth and MCLR degradation were established with the Gompertz model and revised Monod kinetic model. Results The results showed that strain CQ5 had the closest phylogenetic similarity to Lysinibacillus boronitolerans (T-10a, AB199591) in the phylogenetic tree, with 99% bootstrap support. Strain CQ5 could utilize MCLR as the carbon and nitrogen source for growth. When the initial pH value was 7 and the inoculation amount was 3%, strain CQ5 grew well in MSM, in which the MCLR crude extract was used as the carbon and nitrogen source of strain CQ5. Within 244 h, the MCLR concentration changed from 14.12 to 1.57 μg/L and its degradation rate could reach 88.88%. The growth curve fitted with the Gompertz growth model (Nt = 1.3119 * exp(−0.1237 * exp(−6.6341t)), R2 > 0.99). The process of MCLR degradation agreed with the first-order reaction kinetic equation (lnS = 2.64764 − 0.01537t, R2 > 0.99). The linkage relationship between MCLR concentration, cell density, and MCLR degradation rate was consistent with the revised Monod equation (V = 0.342S, R2 > 0.97) at low substrate concentration, where Vmax/ Ks was 0.342. The dynamic relationship in which strain CQ5 degraded MCLR and used it as the carbon and nitrogen source to promote its own growth could be explained by the equation S = 14.12 e− 0.342 Nt (N = 1.08). Conclusion The growth of strain CQ5 and MCLR concentration in degradation system could be simulated and predicted by the dynamic mathematical models in this study. And the predicted results were very consistent. These results could provide theoretical reference for studying the mechanism of MCLR biodegradation and promote the engineering application of strain CQ5.

Fermentation conditions of serine/alkaline milk-clotting enzyme production by newly isolated Bacillus licheniformis BL312 Abstract Purpose This study was conducted to find a microbial milk-clotting enzyme (MCE) with a high and stable milk-clotting activity (MCA) to proteolytic activity (PA) ratio suitable for the cheese industry. Methods Microbial strains were isolated from soil suspensions cultured in solid casein medium. 16S rDNA of representative isolates were sequenced to identify the microbial species. Nutrition and fermentation conditions were systematically examined to optimize MCA of the selected MCE. Protease inhibitors were used to identify the type of MCE. The casein hydrolysis was analyzed through reversed-phase HPLC (RP-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results The Bacillus licheniformis BL312 was identified from 50 bacterial strains. BL312 MCE achieved a maximal MCA (460 ± 15 SU/mL) at 48 h that was 2.7-fold higher than the control, and the MCA/PA ratio (9.0) and pH (6.6) remained stable throughout the fermentation process. Medium containing 30 g/L wheat bran shorts, 5 g/L glucose, and 3 g/L corn steep liquor was sufficient for optimal BL312 MCE production. Fermentation conditions of an inoculum size of 7.0% (v/v), fermentation temperature of 37 °C, agitation speed of 210 rpm, and initial pH 6.6 were required to achieve maximal MCA. BL312 MCE was inhibited by phenylmethanesulfonyl fluoride (PMSF) and high concentrations of ethylenediaminetetraacetic acid (EDTA) (5–25 mM). The αs-casein (αs-CN) and β-casein (β-CN) hydrolysates generated by BL312 MCE and calf rennet were different. Conclusions BL312 MCE is a serine/alkaline protease that exhibits high MCA and various hydrolysis for caseins in comparison with calf rennet.

Bacillus halotolerans strain LYSX1-induced systemic resistance against the root-knot nematode Meloidogyne javanica in tomato Abstract Purpose Determination of the nematicidal potential and mode of action of bacteria isolated from tobacco rhizosphere soil against the root-knot nematode Meloidogyne javanica in tomato plants. Methods Antagonistic bacteria were isolated from rhizosphere soil of tobacco infested with root-knot nematodes. Culture filtrate was used to examine nematicidal activity and ovicidal action of bacterial strains. Biocontrol of M. javanica and growth of treated tomato plants were assessed in pot experiments. To clarify whether secondary metabolites of bacteria in tomato roots induced systemic resistance to M. javanica, bacterial culture supernatants and second-stage juvenile nematodes were applied to spatially separated tomato roots using a split-root system. Bacterial strains were identified by 16S rDNA and gyrB gene sequencing and phylogenetic analysis. Results Of the 15 bacterial strains isolated, four (LYSX1, LYSX2, LYSX3, and LYSX4) demonstrated nematicidal activity against second-stage juveniles of M. javanica, and strain LYSX1 showed the greatest antagonistic activity; there was dose-dependent variability in nematicidal activity and inhibition of egg mass hatching by strain LYSX1. In vivo application of LYSX1 to tomato seedlings decreased the number of egg masses and galls and increased the root and shoot fresh weight. Treatment of half of the split-root system with LYSX1 reduced nematode penetration to the other half by 41.64%. Strain LYSX1 was identified as Bacillus halotolerans. Conclusion Bacillus halotolerans LYSX1 is a potential microbe for the sustainable biocontrol of root-knot nematodes through induced systemic resistance in tomato.

Thioredoxin1 regulates conidia formation, hyphal growth, and trap formation in the nematode-trapping fungus Arthrobotrys oligospora Abstract Purpose Arthrobotrys oligospora, a model nematophagous fungus that produces specific adhesive networks to capture nematodes, has been proposed as a potentially effective biological agent to control harmful plant-parasitic nematodes. Although thioredoxin has been characterized as playing important roles in many cellular processes in other species, its function in nematophagous fungi has not been studied. Here, the function of a thioredoxin homolog, Aotrx1, was investigated in A. oligospora. Methods The encoding gene of Aotrx1 in the nematophagous fungus A. oligospora was knocked out by homologous recombination; strain growth was assessed. Results The ΔAotrx1 strain of A. oligospora showed a significant decrease in growth rate on different media (PDA, CMY, and TG), a 70% decrease of conidia production, and a lower germination rate compared with the wild type. The mutant strain was unable to form traps to capture nematodes and was more sensitive to SDS and H2O2. Conclusion Thioredoxin is involved in conidia development, trap formation, normal mycelial growth, and resistance to environmental stresses in the nematode-trapping fungus A. oligospora.

Characterization of edible swiftlet’s nest as a prebiotic ingredient using a simulated colon model Abstract Purpose Edible bird’s nest (EBN) has been considered as one of the nutritious foods and was also claimed to aid in digestion problems. Potential prebiotic of the EBN for gut health by the presence of glycan within the complex structure of the EBN glycoprotein to date has not been reported. The gut health can contribute to the overall consumers’ health in the improvement of the gut beneficial bacterial growth. In this study, the potential prebiotic of the EBN was conducted using a simulation of in vitro human colon model system. Methods The EBN-extracted glycan and EBN glycoprotein (crude sample) were digested using in vitro oral, gastric and duodenal model system. Prebiotic activities of the undigested EBN glycan and EBN glycopeptide compounds were studied with the fructooligosaccharide as a positive control, using inoculum of 10% (w/v) faecal bacteria in the in vitro fermentation system. Result The fermentation of EBN glycan and EBN glycopeptide had shown significant increases of the gut beneficial bacteria and was comparable with fructooligosaccharide fermentation, with each sample presented different profiles of bacterial growth. The fermentation of EBN glycan and EBN glycopeptide demonstrated an increase in the total short-chain fatty acid production, particularly acetate, propionate and butyrate. Conclusion These findings suggested that the EBN can be functioned as a natural prebiotic upon consumption, thus providing a potential as prebiotic ingredients.

Correction to: Identification and characterization of ectoine-producing bacteria isolated from Can Gio mangrove soil in Vietnam After publication of this paper, the authors determined an error in the funding information. It was written as Grant B2007-SHP-32, but it should be Grant B2017-SPH-32.