<Emphasis Type="Italic">In silico</Emphasis> phylogenetic analysis of lactic acid bacteria and new primer set for identification of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> in food samples

Lactic acid bacteria (LAB) include species very closely related both physiologically and genotypically. Therefore, the identification of this bacteria group using conventional phenotypic methods is ambiguous and cumbersome. In this study, we have analyzed a recA gene fragment from 30 bacteria, including LAB and species common in the human gastrointestinal tract, aiming to evaluate the gene conservation among them and the development of primers and PCR conditions able to discriminate Lactobacillus plantarum strains from LAB closely related. The fragment with 995 bp of recA gene has grouped LAB, enterobacteria and bifidobacteria, in different clusters. A novel primer pair, LPrecAF and LPrecAR with 23 and 18 bp, respectively, has allowed the single amplification of a 108 bp fragment of L. plantarum strains contained in culture broth and fermented dairy samples. The observed detection limit for food samples and for cultures broth were 1 × 10³ and 7 × 10² CFU mL⁻¹, respectively. This approach proved to be a simple and efficient method for the identification and monitoring of L. plantarum in food, feeds, and culture broth. Moreover, the assay could be used in the studies from human or environmental microbiota.     

Genetic typification by pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) of wild <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Oenococcus oeni</Emphasis> wine strains

Genetic typification of 120 bacterial isolates of Lactobacillus plantarum and Oenococcus oeni from different Rioja musts and wines was performed by numerical analysis of pulsed-field gel electrophoresis (PFGE) patterns with endonuclease SfiI, and 46 of them were also studied by randomly amplified polymorphic DNA (RAPD)-PCR. A comparative study of both typification methods applied to L. plantarum and O. oeni oenological strains was performed. Bacterial species was determined both by biochemical identification methods and by specific PCR analysis. A wide variety of restriction digest patterns were detected by PFGE among L. plantarum strains (36 unrelated patterns and one closely related pattern, out of 48 isolates), as well as among O. oeni strains (18 unrelated patterns out of 72 isolates). PFGE was shown to be a suitable method for strain differentiation and to determine which strains are present in wine fermentations, with a discriminatory power to type L. plantarum and O. oeni strains higher than that of RAPD-PCR.     

A New Multiplexed Real-Time PCR Assay to Detect <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>, <Emphasis Type="Italic">C. coli</Emphasis>, <Emphasis Type="Italic">C. lari</Emphasis>, and <Emphasis Type="Italic">C. upsaliensis</Emphasis>

A new rapid method based on real-time PCR was developed to detect four thermophilic Campylobacter species (Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis) in food samples. The assay targeted the bipA gene for C. upsaliensis and C. lari, whereas the gene encoding the ATP-binding protein CJE0832 was used to detect C. coli and C. jejuni. These genes were chosen for this assay due to their low variability and mutation rate at a species level. The multiplex PCR showed 100% inclusivity for all 25 thermophilic Campylobacter strains tested and 100% exclusivity for 38 non-targeted strains belonging to closely related species. The newly developed real-time PCR could detect down to 10² genomes/reaction and displayed efficiency above 97% for all species except for C. upsaliensis (90.1%). The method proved to be a reliable tool for food analysis, showing 100% sensitivity, 96% efficiency, and 92.45% specificity when validated against the gold standard method UNE-EN ISO 10272:2006 using 200 diverse food samples (meat, fish, fruits and vegetables, and raw milk). In artificially spiked samples, the detection limit of the method was 10 cfu/g in salad, 5 cfu/g in turkey meat, and 1 cfu/g in the rest of meat samples tested. Consequently, the newly designed molecular tool represents a quick and safe alternative to obtain reliable results concerning the presence/absence of the main thermophilic Campylobacter in any food sample.     

Volatile flavor composition of <Emphasis Type="Italic">gamguk</Emphasis> (<Emphasis Type="Italic">Chrysanthemum indicum</Emphasis>) flower essential oils

The volatile composition of the essential oil from fresh gamguk (Chrysanthemum indicum) flowers was investigated. The volatile constituents were extracted by the hydro distillation method. Volatile compositional changes of gamguk prepared via different drying methods (shade- and freeze-drying methods) were also determined. Total 36, 63, and 55 volatiles constituents were confirmed in the essential oil from fresh and shade-, and freeze-dried flowers. Ketones were predominant in the volatiles of gamguk flowers (%): fresh, 43.8; shade dried, 30.3; and freeze dried, 36.1. Camphor was the most abundant volatile component in all samples, and the content of borneol was also remarkable. The content of camphor was higher in fresh sample than those of dried samples while borneol concentration was significantly increased in the dried samples.     

Detection of <Emphasis Type="Italic">Campylobacter</Emphasis> colonies using hyperspectral imaging

The presence of Campylobacter in foods of animal origin is the leading cause of bacterially induced human gastroenteritis. Isolation and detection of Campylobacter in foods via direct plating involves lengthy laboratory procedures including enrichments and microaerobic incubations, which take several days to a week. The incubation time for growing Campylobacter colonies in agar media usually takes 24–48 h. Oftentimes the problem is the difficulty of visually differentiating Campylobacter colonies from non-Campylobacter contaminants that frequently grow together with Campylobacter on many existing agars. In this study, a new screening technique using non-destructive and non-contact hyperspectral imaging was developed to detect Campylobacter colonies in Petri dishes. A reflectance spectral library of Campylobacter and non-Campylobacter contaminants was constructed for characterization of absorption features in wavelengths from 400 to 900 nm and for developing classification methods. Blood agar and Campy-Cefex agar were used as culture media. The study found that blood agar was the better culture medium than Campy-Cefex agar in terms of Campylobacter detection accuracy. Classification algorithms including single-band thresholding, band-ratio thresholding and spectral feature fitting were developed for detection of Campylobacter colonies as early as 24 h of incubation time. A band ratio algorithm using two bands at 426 and 458 nm chosen from continuum-removed spectra of the blood agar bacterial cultures achieved 97–99% of detection accuracy. This research has profound implications for early detection of Campylobacter colonies with high accuracy. Also, the developed hyperspectral reflectance imaging protocol is applicable to other pathogen detection studies.     

Development of a multilocus variable-number of tandem repeat typing method for Listeria monocytogenes serotype 4b strains

Listeria monocytogenes serotype 4b strains have been identified as the causative agent in many human listeriosis epidemics as well as in a considerable number of sporadic cases. Due to the genetic homogeneity of serotype 4b isolates, development of rapid subtyping methods with high discriminatory power for serotype 4b isolates is required to allow for improved outbreak detection and source tracking. In this study, multilocus variable-number tandem repeat analysis (MLVA) was developed and used to characterize 60 serotype 4b isolates from various sources. All isolates were also characterized by automated EcoRI ribotyping, single enzyme pulsed-field gel electrophoresis (PFGE) with ApaI, and a multilocus sequence typing (MLST) scheme targeting six virulence and virulence-associated genes. Discriminatory power of MLVA (as determined by Simpson Index of Discrimination) was higher than the discriminatory power of any of the other three methods. MLVA markers targeted were found to be stable and did not change when three isolates were passaged daily for 70 days. Cluster analyses of MLVA, PFGE and MLST consistently grouped the same isolates into three major clusters, each of which includes one of the three major L. monocytogenes epidemic clones (i.e., ECI, ECIa and ECII). We conclude that the MLVA method described here (i) provides for more discriminatory subtyping of L. monocytogenes serotype 4b strains than the other three methods, (ii) identifies three major groups within the serotype 4b, which are consistent with the groups identified by other subtyping methods, and (iii) is easy to interpret. Use of MLVA may thus be recommended for subtyping of serotype 4b isolates, including as a secondary more discriminatory subtyping method that could be used after initial isolate characterization by PFGE or ribotyping.     

Dark chocolates supplemented with <Emphasis Type="Italic">Lactobacillus</Emphasis> strains

Dark chocolate masses and chocolates were supplemented with viable cells of two bacterial strains Lactobacillus caseii and Lactobacillus paracasei with potential probiotic properties, which were lyophilized in milk. Total number of live bacteria in the lyophilizate was 7.9×10⁹ cfu/g. Sucrose or isomalt and aspartame were used as bulking substances and sweeteners. Sensory attributes of these chocolates were not different from that of traditional chocolates. Calorie value of sucrose-free chocolate was lower by approximately 11.1–14.6% (dependent on their formulation) relative to chocolate sweetened with sucrose. Chocolate, which contains isomalt and aspartame can be consumed by diabetics. Numbers of live L. casei and L. paracasei cells in the examined batches of chocolate were very high and approached 10⁶–10⁷ cfu/g after 12 months of keeping at 4 and 18 °C. Neither the texture nor the total and volatile acidity of chocolate masses were changed by addition of the lyophilized preparation of Lactobacillus cells. Casson yield values of dark sucrose-free chocolate masses supplemented with this lyophilizate were decreased by approximately 3–55% (dependently on fat contents in these masses) as compared to that of analogous chocolate masses sweetened with sucrose.     

<Emphasis Type="Italic">In vitro</Emphasis> characterization study of <Emphasis Type="Italic">Bacillus mojavensis</Emphasis> KJS-3 for a potential probiotic

The identification and characterization of Bacillus mojavensis KJS-3 was performed by in vitro tests. A 16S rDNA sequence and phylogenetic tree demonstrated that this isolate belongs to the B. mojavensis group. B. mojavensis KJS-3 supplies nutrients by synthesizing several vitamins. B. mojavensis KJS-3 produces α-amylase and protease. B. mojavensis KJS-3 is cultured well under aerobic conditions without gas production. B. mojavensis KJS-3 allows for assimilation of cholesterol and bile salt hydrolase activity. Finally, adhesion experiments using Caco-2 cells revealed that the adherence of B. mojavensis KJS-3 to Caco-2 cells was approximately 51.2±8.14%.     

Tyramine production of technological important strains of <Emphasis Type="Italic">Lactobacillus</Emphasis>, <Emphasis Type="Italic">Lactococcus</Emphasis> and <Emphasis Type="Italic">Streptococcus</Emphasis>

The aim of this paper was to study the biogenic amines (histamine, tyramine, putrescine, cadaverine, agmatine, spermine and spermidine) production of selected technological important lactic acid bacteria (strains of the genera Lactococcus, Lactobacillus and Streptococcus). Three methods (ion-exchange chromatography (IEC), PCR and cultivation method with pH indicator) were used. Within the 39 strains of lactic acid bacteria tested, the production of tyramine (formed by tyrosine decarboxylase) was detected in eight strains (3 strains of Lactococcus lactis subsp. lactis, three strains of Lactococcus lactis subsp. cremoris, 1 strain of Streptococcus thermophilus and 1 strain of Lactobacillus delbrueckii subsp. bulgaricus). The other tested biogenic amines were not detected. Cultivation in decarboxylation broth seems to be the least accurate method for the detection of biogenic amines due to enhanced risk of false-positive reactions. Therefore, in order to detect bacteria producing biogenic amines, the combination of PCR and chromatographic methods (e.g. IEC) can be recommended.     

Characterization of moisture content and specific gravity of branchwood and stemwood of <Emphasis Type="Italic">Aningeria robusta</Emphasis> and <Emphasis Type="Italic">Terminalia ivorensis</Emphasis>

Aningeria robusta and Terminalia ivorensis of diameters ranging from 10 cm to 25 cm were examined. The following results were obtained: The overall (sapwood and heartwood combined) moisture contents of the branchwood of both Aningeria robusta and Terminalia ivorensis were significantly greater than those of their corresponding stemwood, with the branchwood of Terminalia ivorensis being the highest. It was also observed that the overall specific gravity of the branchwoods of both species was higher than that of the corresponding stemwood, with the branchwood of Aningeria robusta exhibiting the highest specific gravity. Detailed analyses of moisture content distribution and specific gravity of the heartwood and sapwood of Aningeria robusta and Terminalia ivorensis are also presented.     

Prevalence,genetic diversity,and antibiotic susceptibility of <Emphasis Type="Italic">Cronobacter</Emphasis> spp. (<Emphasis Type="Italic">Enterobacter sakazakii</Emphasis>) isolated from <Emphasis Type="Italic">Sunshik</Emphasis>, its ingredients and soils

The prevalence, genetic diversity, and antibiotic susceptibility of Cronobacter species (Enterobacter sakazakii) isolated from sunshik products, its ingredients, and root vegetable farm’s soils were investigated to analyze main reservoirs and contaminated sources of Cronobacter spp. Cronobacter spp. was isolated from 9 of 15 sunshik products, 26 of 72 its ingredients, and 2 of 39 soils. The root vegetables such as sweet potato and carrot showed higher contamination rate (70%) than the other sunshik ingredients. All isolates showed 929 bp band amplified from 16S rRNA and resistant to ampicillin, amoxicillin/clavulanic acid, and cefazolin. All isolates showed diverse random-amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) band patterns. However, 3 cases of RAPD banding patterns between clinical strains and isolates from sunshik products and root vegetables (yam, carrot) were related with similarities level of 80%. These studies indicated that root vegetables can be an important contamination source of Cronobacter spp. in sunshik products. Thus, the preparation of root vegetables for manufacturing sunshik products used as a weaning diet was handled with more care than the other sunshik ingredients.     

Molecular diversity among natural populations of <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>/<Emphasis Type="Italic">paraplantarum</Emphasis> strains isolated from autochthonous dairy products

The diversity of 87 Lactobacillus paracasei and Lactobacillus plantarum/paraplantarum strains, previously identified from different autochthonous dairy products, was investigated by phenotypic and genotypic approaches. The increased resolution obtained using phenotypic and genotypic characterization allowed the level of strain heterogeneity detection to be widened. Phenotypic diversity was evaluated by studying biochemical characteristics of technological interest, including antimicrobial and proteinase activities, resistance to nisin, aggregation ability, production of exopolysaccharides, acetoin and diacetyl, citrate utilization, and antibiotic susceptibility. Genotypic diversity was generally evaluated by PCR amplification of repetitive bacterial DNA element fingerprinting using the (GTG)₅ primer [(GTG)₅-PCR]. Moreover, in cases where strains were not discriminated by (GTG)₅-PCR combined with phenotypic analysis, pulsed-field gel electrophoresis (PFGE) analysis was performed. The results indicate that L. plantarum/paraplantarum and L. paracasei natural isolates from artisanal dairy products are a gold mine in terms of diversity of strains and could be potentially interesting to dairy companies for the formulation of functional starter cultures in the production of innovative foods.     

Comparative typing of <Emphasis Type="Italic">L. delbrueckii</Emphasis> subsp<Emphasis Type="Italic">. bulgaricus</Emphasis> strains using multilocus sequence typing and RAPD–PCR

Comparative typing analysis of 25 Lactobacillus delbrueckii subsp. bulgaricus strains, isolated from traditional yoghurts in Turkey, was performed by RAPD–PCR (randomly amplified polymorphic DNA–PCR) and MLST (multilocus sequence typing). RAPD–PCR analyses were performed using two primers; M13 and 1254. Primer 1254 produced better results than primer M13. The bands produced by primer 1254 were brighter and easier to interpret, and a higher number of bands were produced. In addition, clusters produced by primer 1254 were grouped according to the source of isolation. MLST analysis was performed using three genes, β-gal, pheS and rpoA, and isolates were successfully characterized at strain level. To our knowledge, MLST analyses were used for the first time for strain level discrimination in L. delbrueckii subsp. bulgaricus. It enabled a detailed understanding of L. delbrueckii subsp. bulgaricus strains by using allele and sequence types’ analysis. Both MLST and RAPD allowed for the typing of clusters according to the isolation source, while RAPD provided an increased differentiation. However, by increasing the number of genes analyzed, the discriminatory power of MLST could be increased.     

RAPD typing of <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> isolated from various food products from Korea

In the present study, the fingerprinting technique, random amplified polymorphic DNA-PCR was evaluated to characterize 13 strains of Lactobacillus brevis, isolated from different vegetable products of South Korea. Two primers i.e. 239 and KAY3 were used. The primer 239 produced bands ranged from 500-4,000 bp and KAY3 primer produced bands with sizes from 600-4,000 bp. Both primers produced thirteen different RAPD profiles. Phylogenetic dendrogram showed that all the isolates could be divided into six major clusters both the primers. However, a few strains of L. brevis had similar profiles and were not well differentiated by RAPD.     

Fourier transform infrared spectroscopy (FTIR) as a tool for discriminating <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> contaminated beef

Detection of beef contamination from harmful pathogens will be helpful in protecting the consumer safety and controlling the outbreaks. In this paper, the potential of Fourier transform infrared spectroscopy (FTIR) was investigated to discriminate the Salmonella contaminated packed beef. A suitable headspace sampling system was designed and used to collect the headspace volatiles from the packed meat to the FTIR gas cell. Spectral signatures of headspace volatiles of meat packages were used to classify the packed meat samples as contaminated or not. FTIR spectrum was divided into several regions in order to reduce the dimensionality as well as to select the regions based on the absorbance properties of various volatiles present in headspace of meat package. Principal component analysis was performed on the entire spectrum (4000–500 cm⁻¹) as well as on the selected sub-regions of entire spectrum. Two statistical classification techniques (linear and quadratic discriminate analysis) were used to develop classification models. The statistical models were validated using bootstrap cross validation technique. The total average classification accuracies were evaluated in terms of coefficient of variance (% CV). Based on the mean of total average classification accuracies and its % CV calculated from five similarly conducted experiments, it was found that the statistical models developed on a part of the spectra (500–850 cm⁻¹) and full spectra (4000–500 cm⁻¹) can be used as potential classification models for non-destructive discrimination of Salmonella contaminated packed beef samples from uncontaminated ones. These results need to be further validated on dataset with larger sample size.     

Sensitive and rapid detection of freshwater crustacean <Emphasis Type="Italic">Spiroplasmas</Emphasis> by ISRs-sequence-targeted species-specific primers

For the rapid and sensitive identification of the Spiroplasmas in the Chinese mitten crab Eriocheir sinensis, a species-specific PCR primer-pair was designed according to Spiroplasma sp. strain CRAB-specific segments of the previously sequenced 16S–23S rRNA intergenic spacer region (ISR). The PCR-mediated assay allowed identification of Spiroplasmas from different aquatic crustacean hosts, E. sinensis and red swamp crayfish Procambarus clarkia, in different geographical areas in China. No cross-reaction could be observed with other three available terrestrial plants/insects Spiroplasma strains (Spiroplasma sp. CH-1, Spiroplasma sp. CR-1 and Spiroplasma sp. M10). The sensitivity of detection of the Spiroplasma sp. strain CRAB could get as little as 100 fg of DNA template. The presented PCR method is useful for sensitive and specific detection of Spiroplasma from freshwater cultured crustaceans in the trade of aquatic products.     

Molecular typing of Vibrio parahaemolyticus isolates from the middle-east coastline of China

The occurrence of outbreaks of Vibrio parahaemolyticus gastroenteritis in China highlights the need for strain characterization and subtyping of this pathogenic species. A total of 56 epidemiologically-unrelated strains of V. parahaemolyticus were isolated from clinical samples, seafood and various environmental sites in the middle-east coastline of China from 2006 to 2008. The isolates were characterized using four molecular typing methods, including ribotyping, enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), pulsed-field gel electrophoresis (PFGE), and sequence analysis of the gyrB gene. Genetic profiles of cluster analysis from these molecular typing tests clearly showed that there were differences in potential pathogenicity among isolates from seafood and its environments. Genetic characterization of two isolates (F13 and QS2) that originated from seafood demonstrated that they were potentially pathogenic. Discriminatory indices of four typing methods for the 56 V. parahaemolyticus isolates were differentiated by Simpson's Index of Diversity. The discriminatory index of ERIC-PCR typing was maximal (D = 0.942), while that of sequence analysis of the gyrB gene was minimal (D = 0.702). The discriminatory ability was greatly enhanced (D = 0.966) when ERIC-PCR was coupled with sequence analysis of the gyrB gene. These results suggest that ERIC-PCR combined with sequence analysis of gyrB gene may be a reliable, rapid typing strategy for V. parahaemolyticus strains.     

Disinfection effect and its mechanism of electrolyzed oxidizing water on spores of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> var. <Emphasis Type="Italic">niger</Emphasis>

A study was carried out on the disinfection efficiency of electrolyzed oxidizing water (EOW) on spores of Bacillus subtilis var. niger. The results showed a remarkable fungicidal rate of 100% after 20 min duration of 191 mg/L active available chlorine (ACC). The disinfection effect was improved with increased ACC or prolonged disinfection time, while organic interferents exerted a strong concentration-dependent inhibition against the disinfection. The disinfection mechanism was also investigated at bio-molecular level. EOW decreased dehydrogenase activity, intensified membrane permeability, elevated suspension conductivity, and caused leakage of intracellular K⁺, proteins, and DNA, indicating a damage of cell walls and membranes. Effects of EOW on microbiological ultra-structures were also verified by transmission electronic microscopy (TEM) images, showing that EOW destroyed protective barriers of the microbe and imposed some damages upon the nucleus area.