Metabolism of Fructooligosaccharides in Lactobacillus plantarum ST-III via Differential Gene Transcription and Alteration of Cell Membrane Fluidity.

Although fructooligosaccharides (FOS) can selectively stimulate the growth and activity of probiotics and beneficially modulate the balance of intestinal microbiota, knowledge of the molecular mechanism for FOS metabolism by probiotics is still limited. Here a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth of Lactobacillus plantarum ST-III using FOS or glucose as the sole carbon source. A total of 363 genes were differentially transcribed; in particular, two gene clusters were induced by FOS. Gene inactivation revealed that both of the clusters participated in the metabolism of FOS, which were transported across the membrane by two phosphotransferase systems (PTSs) and were subsequently hydrolyzed by a ß-fructofuranosidase (SacA) in the cytoplasm. Combining the measurements of the transcriptome- and membrane-related features, we discovered that the genes involved in the biosynthesis of fatty acids (FAs) were repressed in cells grown on FOS; as a result, the FA profiles were altered by shortening of the carbon chains, after which membrane fluidity increased in response to FOS transport and utilization. Furthermore, incremental production of acetate was observed in both the transcriptomic and the metabolic experiments. Our results provided new insights into gene transcription, the production of metabolites, and membrane alterations that could explain FOS metabolism in L. plantarum.

Development of a Method for the Analysis of Multiclass Antibiotic Residues in Milk Using QuEChERS and Liquid Chromatography-Tandem Mass Spectrometry.

A precise and simplified method of sample preparation for the simultaneous quantification of the antibiotics ß-lactam, macrolide, tetracycline, sulfonamide, and quinolone in bovine milk was developed. The central composite design of response surface methodology was used to design and optimize the method for the determination of six different antibiotic residues in milk. The recovery of each antibiotic was studied using a quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. Octadecylsilane (C18), primary secondary amine (PSA), and sodium acetate (Na acetate) were the main factors affecting the recovery of each antibiotic. After optimization, the maximum predicted recovery rate was 84.18% for erythromycin under the optimized conditions of 101.20 mg C18, 52.00 mg PSA, and 1.01 g Na acetate. The recovery rates of the five other antibiotic residues ranged from 86.09% to 115.99%. The results suggested that modified QuEChERS could effectively be implemented in the analysis of antibiotic residues in milk.

Cracking Streptococcus thermophilus to stimulate the growth of the probiotic Lactobacillus casei in co-culture.

Lactobacillus casei, a probiotic, and Streptococcus thermophilus, a fast acidifying lactic acid bacterial strain, are both used in the food industry. The aim of this study was to investigate the interaction between L. casei and S. thermophilus in the presence or absence of S. thermophilus-specific bacteriophage during milk fermentation. The acidification capability of L. casei co-cultured with S. thermophilus was significantly higher than that observed for L. casei or S. thermophilus cultured alone. However, the probiotic content (i.e., L. casei cell viability) was low. The fastest acidification and the highest viable L. casei cell count were observed in co-cultures of L. casei and S. thermophilus with S. thermophilus phage. In these co-cultures, S. thermophilus compensated for the slow acid production of L. casei in the early exponential growth phase. Thereafter, phage-induced lysis of the S. thermophilus cells eliminated the competition for nutrients, allowing L. casei to grow well. Additionally, the ruptured S. thermophilus cells released intracellular factors, which further promoted the growth and function of the probiotic bacteria. Crude cellular extract isolated from S. thermophilus also significantly accelerated the growth and propagation of L. casei, supporting the stimulatory role of the phage on this micro-ecosystem.

Improvement of exopolysaccharide production in Lactobacillus casei LC2W by overexpression of NADH oxidase gene.

Lactobacillus casei LC2W is an exopolysaccharide (EPS)-producing strain with probiotic effects. To investigate the regulation mechanism of EPS biosynthesis and to improve EPS production through cofactor engineering, a H2O-forming NADH oxidase gene was cloned from Streptococcus mutans and overexpressed in L. casei LC2W under the control of constitutive promoter P23. The recombinant strain LC-nox exhibited 0.854 U/mL of NADH oxidase activity, which was elevated by almost 20-fold in comparison with that of wild-type strain. As a result, overexpression of NADH oxidase resulted in a reduction in growth rate. In addition, lactate production was decreased by 22% in recombinant strain. It was proposed that more carbon source was saved and used for the biosynthesis of EPS, the production of which was reached at 219.4 mg/L, increased by 46% compared to that of wild-type strain. This work provided a novel and convenient genetic approach to manipulate metabolic flux and to increase EPS production. To the best of our knowledge, this is the first report which correlates cofactor engineering with EPS production.

Molecular characteristics of an exopolysaccharide from Lactobacillus rhamnosus KF5 in solution.

The molecular weight and chain conformation of an exopolysaccharide (EPS), S2, from Lactobacillus rhamnosus KF5 were determined by size-exclusion chromatography combined with multi-angle laser light scattering (SEC-MALLS), dynamic light scattering (DLS), viscometry and atomic force microscopy (AFM). The weight-average molecular weight (Mw), intrinsic viscosity [ƞ], radii of gyration (R(g)) and hydrodynamic radii (R(h)) were 7.5 × 10(5) Da, 2.331 dL/g, 44.3 nm and 29.4 nm, respectively. The conformational parameters were calculated from the above data according to the theory of dilute polymer solutions. The Mark-Houwink-Sakurada exponent α was found to be 0.687, and the value of the structure-sensitive parameter ρ (R(g)/R(h)) was 1.51. The results revealed that the EPS S2 existed as a random coil conformation in 0.1M NaNO3 aqueous solution. AFM further confirmed the random coil morphology of this molecule in aqueous solution. EPS S2 present extended chains (fibrous morphology), with circular side chains, dispersed in SDS solution.

A new comprehensive index for discriminating adulteration in bovine raw milk.

This paper proposes a new comprehensive index, called Q, which can effectively discriminate artificial adulterated milk from unadulterated milk. Both normal and adulterated samples of bovine raw milk were analysed by Fourier transform infrared spectroscopic instrument to measure the traditional indices of quality, including fat (FAT), protein (PRO), lactose (LAC), total solids (TS), non-fat solid (NFS), freezing point (FP) and somatic cell counts (SCC). From these traditional indices, this paper elaborates a method to build the index Q. First, correlated analysis and principle component analysis were used to select parameter pairs TS-FAT and FP-LAC as predominant variables. Second, linear-regression analysis and residual analysis are applied to determine the index Q and its discriminating ranges. The verification and two-blind trial results suggested that index Q could accurately detect milk adulteration with maltodextrin and water (as low as 1.0% of adulteration proportions), and with other nine kinds of synthetic adulterants (as low as 0.5% of adulteration proportions).

Dextran synthesized by Leuconostoc mesenteroides BD1710 in tomato juice supplemented with sucrose.

The characteristics of the growth of Leuconostoc mesenteroides BD1710 and the synthesis of dextran in tomato juice supplemented with 15% sucrose were assayed. L. mesenteroides BD1710 could synthesize approximately 32 g L(-1) dextran in the tomato-juice-sucrose medium when cultured at 28 °C for 48 h, which was on the same level as the dextran yield in a chemically defined medium. The viscosity of the cultured tomato-juice-sucrose medium with various dextran contents was also measured. The results of the monosaccharide composition, molecular-weight distribution, Fourier transform infrared spectra (FTIR) and nuclear magnetic resonance spectra (NMR) showed that the polysaccharide synthesized by L. mesenteroides BD1710 in the tomato-juice-sucrose medium was dextran with a peak molecular weight of 6.35 × 10(5)Da, a linear backbone composed of consecutive α-(1 → 6)-linked d-glucopyranosyl units and approximately 6% α-(1 → 3) branches.

Partial characterization and immunostimulatory activity of exopolysaccharides from Lactobacillus rhamnosus KF5.

Lactobacillus rhamnosus KF5, a strain newly isolated from the faeces of a healthy human volunteer, has been shown to produce the exopolysaccharides (EPS) in skim milk. Two EPS fractions were separated from the fermented skim milk by removing proteins, ethanol precipitation, anion-exchange and gel permeation chromatography. Fraction S1, with a low average molecular weight of 1.36 × 10(4)Da, was composed of glucose, arabinose, glucosamine, galactosamine and galactose in an approximate molar ratio of 2.03:1.29:1.25:0.72:0.61. Such monosaccharide composition of the exopolysaccharide by lactic acid bacteria has not been reported so far, and S1 is likely to be an unusual type of microbial EPS. Whilst Fraction S2, with a high average molecular weight of 1.23 × 10(6)Da, contained rhamnose, glucose and galactose in a molar ratio of approximately 1.73:1.47:1.00. Both of EPS fractions could significantly stimulate splenocyte proliferation in vitro, indicating two EPS fractions had the potential immunomodulatory activity.

Development of a PCR assay for rapid detection of Cronobacter spp. from food.

The occurrence of outbreaks of necrotizing meningitis caused by Cronobacter spp. in neonates highlights the need for rapid detection and accurate identification of this pathogenic species. The gold standard for isolation and identification of Cronobacter spp. from powdered infant formula is time consuming and labor intensive. The gyrB gene that encodes the B subunit of DNA gyrase (topoisomerase type II) was found to be suitable for the identification of Cronobacter spp. A region of the gyrB gene of 38 Cronobacter spp. strains and 5 Enterobacter spp. strains was amplified and sequenced, and a pair of primers was designed and synthesized based on the sequence of the gyrB gene. A polymerase chain reaction (PCR) system was developed and optimized to detect Cronobacter spp. The PCR assay amplified a 438 bp DNA product from all 38 Cronobacter spp. strains tested but not from 34 other bacteria. The detection limit was 1.41 pg/PCR (equivalent 282 genomic copies) when the genomic DNA of Cronobacter sakazakii ATCC 29544 was 10-fold diluted. Infant formula powders from 3 different commercial brands were inoculated with strains ATCC 29544 at a level of 56 colony-forming units, and the target fragment were produced after samples were enriched for 6 h at 37 °C. Twenty-five food samples were evaluated by the PCR assay and the conventional method. A PCR product of the expected size was obtained from 3 samples; however, Cronobacter spp. strains were isolated from only 2 samples by the conventional method. This method is a useful tool for rapid identification of Cronobacter spp. in food and potentially environmental samples.

Aggregation and adhesion properties of 22 Lactobacillus strains.

In this paper, the autoaggregating, coaggregating, hydrophobicity, and adhering abilities of 22 Lactobacillus strains belonging to different species were assessed. No correlation existed between autoaggregation and adhesion of the strains belonging to different species, whereas a positive correlation existed between autoaggregation and adhesion of the strains belonging to the same species. After treating with guanidine HCl, the autoaggregating and adhering abilities of some Lactobacillus strains decreased, indicating that surface-bound proteins and other macromolecules played a role in the adhering and autoaggregating abilities. The strains Lactobacillus plantarum 20 and 66 had higher adhesion and coaggregation abilities and should be further studied for their probable probiotic properties. Aggregating, coaggregating, and adhering abilities of Lactobacillus strains could be used as the preliminary criteria for selecting strains having probiotic potential.

Complete nucleotide sequence of plasmid pST-III from Lactobacillus plantarum ST-III.

The complete nucleotide sequence of the 53,560-bp plasmid pST-III from Lactobacillus plantarum ST-III has been determined. The plasmid contains 42 predicted protein-coding sequences, and the functions of 34 coding sequences could be assigned. Homology analysis for the replication protein and the typical features of the origin of replication suggested that pST-III replicates via the theta-type mechanism. Among the predicted genes, we identified a kdp gene cluster (a high-affinity K(+)-transport system) for the first time in the Lactobacillus genus and a system for osmolyte transport. Analysis of the plasmid-encoded functions and the plasmid-cured experiment showed that the genes of pST-III could serve for the niche adaptations of L. plantarum ST-III and make significant contributions to its viability under hyperosmotic conditions. Furthermore, the relative copy number of pST-III was determined to be 6.79±1.55 copies per cell.

Complete genome sequence of the probiotic strain Lactobacillus casei BD-II.

Lactobacillus casei BD-II, a patented probiotic strain (U.S. patent 7,270,994 B2), was isolated from homemade koumiss in China and has been implemented in the industrial production as starter cultures. Here we report the complete genome sequence of BD-II, which shows high similarity with the well-studied probiotic BL23.

Complete genome sequence of the probiotic bacterium Lactobacillus casei LC2W.

Lactobacillus casei LC2W, a patented probiotic strain (Z. Wu, European patent EP 1642963 B1, February 2009), has been isolated from Chinese traditional dairy products and implemented in industrial production as starter culture. Here we present the complete genome sequence of LC2W and the identification of a gene cluster implicated in the biosynthesis of exopolysaccharides.

All 4 bile salt hydrolase proteins are responsible for the hydrolysis activity in Lactobacillus plantarum ST-III.

UNLABELLED: In vertebrates, bile salt hydrolysis plays an essential role in fat metabolism. Bile salts are synthesized in the liver. And in the small intestine glycine and taurine are de-conjugated from bile salts by the enzyme bile salt hydrolase (BSH) from intestinal microbes. However, the mechanism of bile salt hydrolysis in Lactobacillus plantarum is still ambiguous. Four predicted bile salt hydrolase (bsh) genes from L. plantarum ST-III were cloned into Escherichia coli. The function of these genes was explored by overexpression. All 4 proteins that were studied showed activity against glycine- or taurine-conjugated bile salts. Substrate preference was also observed in BSH proteins, especially for the enzyme BSH1, which had high hydrolysis activity for glycodeoxycholic acid. These results suggest that all 4 bile salt hydrolases may be responsible for the bile salt hydrolysis activity in L. plantarum ST-III. PRACTICAL APPLICATION: Hypercholesterolemia is considered one of the major risk factors for coronary heart disease. More interest has focused on intestinal microbes because of their role in the decrease of serum cholesterol. BSH proteins play an important role in the reduction of cholesterol. This paper adds to a better understanding of BSH proteins of intestinal microbes. It gives a great hint that probiotics can be used to solve hypercholesterolemia one day.

Complete genome sequence of the probiotic Lactobacillus plantarum ST-III.

Lactobacillus plantarum strain ST-III, a probiotic strain with several functions, was isolated from kimchi. Here we report the complete genome sequence of ST-III and compared it with two published L. plantarum genomes.

[Screening of a Lactobacillus casei with monoamine oxidase-inhibitory activity].

OBJECTIVE: To screen for monoamine oxidase (MAO)-inhibiting lactobacillus from healthy human dejecta in an in vitro model, and to provide reference for anti-ageing study in vivo of lactobacillus in the future. METHODS: The monoamine oxidase inhibitory model in vitro was applied to screen fermented supernatant and cell-free extracts originated from lactic acid bacteria, and two indices based on screened samples were determined, including biological dosage-effect and the effect of pre-incubated time between MAO and inhibitor on suppression rate. Meanwhile, with membrane separation technology, MAO inhibition from different molecular weight range of samples was measured. Screened strain JH-23 was investigated as target probiotic lactobacillus, identified through the sequence analysis of 16S rDNA gene and API system. RESULTS: The MAO inhibitory rate of cell-free extracts produced by strain JH-23 reached 33.7%. After samples being vacuum freeze-dried, MAO inhibitory rate was up to 53.2% when the reactive concentration was 16 mg/mL. The inhibition was significantly enhanced with the duration of pre-incubation increased, and then the inhibitory effect became steady after 30 minutes. Crude samples were dialyzed for 48 hours, MAO inhibitory effect of dialysates was significantly increased compared with pre-dialysis. The result of bacteria identification demonstrated that strain JH-23 was ascribed to Lactobacillus casei. CONCLUSION: A new screening model in vitro, regarding monoamine oxidase as a target enzyme, was explored in this study. This model was characterized by convenience, rapidity and high sensitivity, and it could be useful for the following anti-aging research in vivo. The cell-free extracts of Lactobacillus casei JH-23 was inhibitory to MAO, and the intracellular small-molecules played a major role in the inhibition.

[Analysis of bacterial diversity of kefir grains by denaturing gradient gel electrophoresis and 16S rDNA sequencing].

Kefir is an acidic, mildly alcoholic dairy beverage produced by the fermentation of milk with a grain-like starter culture. These grains usually contain a relatively stable and specific balance of microbes that exist in a complex symbiotic relationship. Kefir grains can be considered a probiotic source as it presents anti-bacterial, anti-mycotic, anti-neoplasic and immunomodulatory properties. The microorganisms in Kefir grains are currently identified by traditional methods such as growth on selective media, morphological and biochemical characteristics. However, the microorganisms that isolate by these methods can not revert to Kefir grains which indicate that there are some other bacteria that are not isolate from it. In this study, PCR-based Denaturing gradient gel electrophoresis(DGGE) and sequence analysis of 16S ribosomal RNA gene (16S rDNA) clone libraries was used for the rapid and accurate identification of microorganisms from Kefir grains. The PCR primers were designed from conserved nucleotide sequences on region V3 of 16S rDNA with GC rich clamp at the 5'-end. PCR was performed using the primers and genomic DNAs of Kefir grains bacteria. The generated region V3 of 16S rDNA fragments were separated by denaturing gel, and the dominant 16S rDNA bands were cloned, sequenced and subjected to an online similarity search. Research has shown that regions V3 of 16S rDNAs have eight evident bands on the DGGE gel. The sequence analysis of these eight bands has indicated that they belong to different four genera, among them three sequences are similar to Sphingobacterium sp. whose similarities with database sequences are over 98%, three sequences are similar to Lactobacillus sp. whose similarities with database sequences are over 96%, the other two sequence are similar to Enterobacter sp., and Acinetobacter sp. whose similarities with database sequences are over 99% respectively. Although the DGGE method may have a lower sensitivity than the ordinary PCR methods, because when universal bacterial PCR primers are used, only the dominant microbiota of an ecosystem will be visualized on a DGGE gel, producing complex banding patterns. However, it could visualize the bacterial qualitative compositions and reveal the major species of the Kefir grains. Among them Sphingobacterium can be found in Kefir grains as the predominant flora which is reported for the first time. PCR-based DGGE and sequence analysis of 16S rDNA proved to be a valuable culture-independent approach for the rapid and specific identification of the microbial species present in microecosystem and probiotic products.