Reverse transcription - loop-mediated isothermal amplification assay for the rapid detection of pathogenic Listeria monocytogenes in meat products.

This study reports the use of reverse transcription - loop-mediated isothermal amplification (RT-LAMP) to detect Listeria monocytogenes in meat. The assay was designed to target the iap gene of L. monocytogenes, to which four primers, recognizing six distinct iap sites, were designed. We optimized the RT-LAMP conditions and established the following optimal systems: 60 min, 63 °C, 2.0 mmol/L MgSO4, 1.0 mol/L betaine, 2.0 mmol/L dNTPs, 320 U/mL Bst DNA polymerase, 0.4 µmol/L outer primers, and 0.8 µmol/L inner primers. The RT-LAMP amplification products were identified by a visible white Mg2P2O7 precipitate or electrophoresis on a 2% agarose gel. RT-LAMP has a sensitivity of 7.3 × 101 CFU/mL, which is 2-fold higher than that of LAMP. When commercially available raw meat samples (including beef, pork, mutton, and rabbit) were analyzed simultaneously with RT-LAMP and the Chinese National Standard GB 4789.30-2016, their abilities to detect L. monocytogenes were the same. Samples containing L. monocytogenes killed by 15 psi at 121 °C for 15 min were used to confirm the specificity of RT-LAMP for live microorganisms. Thus, we used RT-LAMP to efficiently detect L. monocytogenes in meat products.

A natural biopreservative: Antibacterial action and mechanisms of Chinese Litsea mollis Hemsl. extract against Escherichia coli DH5α and Salmonella spp.

Chemical preservatives have potential safety hazards, which may pose threats to human health. Safer biopreservatives are therefore urgently required. This study investigated the bacteriostatic activity and mechanism of Litsea mollis Hemsl. essential oil against Escherichia coli DH5α and Salmonella spp. Antibacterial activity of Litsea mollis Hemsl. essential oil 9 (LMEO9) against E. coli DH5α was observed (zone of inhibition was 5.0 ± 0.2 mm; minimum inhibitory concentration was 0.05%). Increases in electrolyte, nucleic acid, and alkaline phosphatase leakage in LMEO9-treated bacteria suggested that the cell envelope had been damaged. Scanning and transmission electron microscopy also demonstrated morphological alterations and content leakage during LMEO9 treatment. According to the kill-time analysis and propidium iodide uptake assay, LMEO9 led to cell death. These results demonstrated that LMEO9, which could affect bacterial cell envelope structural integrity, is a low-cost biopreservative that could be useful for the dairy industry and in fresh storage.

Heterologous expression and purification of dermaseptin S4 fusion in Escherichia coli and recovery of biological activity.

Heterologous expression of dermaseptin S4 (DS4), which has cytolytic activity in vitro against a broad spectrum of pathogenic microorganisms, was examined in Escherichia coli. The plasmid pGEX-4T-1, encoding DS4 fused with glutathione S-transferase (GST), was constructed and cloned into the E. coli strain BL21 (DE3). The fusion protein was overexpressed in this strain after induction with isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified to homogeneity using GST affinity chromatography. To recover biologically active DS4, the purified fusion protein was cleaved using thrombin protease; the liberated DS4 was shown to be bactericidally active against an indicator strain. Since it is less expensive to obtain such a peptide biologically, in this study, we report for the first time a method to express purify DS4 in E. coli using a GST fusion system.

Antifungal activity and mechanism of Litsea cubeba (Lour.) Persoon essential oil against the waxberry spoilage fungi Penicillium oxalicum and its potential application.

Litsea cubeba essential oil (LCEO) is a broad-spectrum bacteriostatic substance produced from the fruit of the Litsea tree that has been used for the treatment of various diseases in China for thousands of years. Here, the antifungal activities of LCEO against 10 different fungi (Naganishia diffluens, Fusarium sacchari, Cladosporium tenuissimum, Fusarium proliferatum, Fusarium verticillioides, Fusarium subglutinans, Mucor racemosus, Penicillium oxalicum, Penicillium chrysogenum, and Aspergillus niger) that cause rot to waxberries were assessed. The chemical components of LCEO and its modes of action against P. oxalicum were investigated. Citral (32.62 %) was characterized as the main component of LCEO by gas chromatography-mass spectrometry. LCEO exhibited excellent antifungal activities against all 10 fungi. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration of LCEO against P. oxalicum were 2.24 and 4.48 g/L, respectively. Furthermore, LCEO (MIC) compromised membrane permeability and integrity, caused leakage of the cell components, and increased production of malondialdehyde and reactive oxygen species. Scanning electron microscopy and transmission electron microscopy indicated that the morphology and ultrastructure of the LCEO-treated hyphal cell membrane and organelles were severely damaged. Meanwhile, LCEO increased the shelf life of waxberries from 1-2 to 5-6 d. LCEO is a potential ecologically friendly alternative to commercial fungicides to inhibit postharvest fungal contamination of waxberries during shipment and storage.

Sensitive and rapid detection of three foodborne pathogens in meat by recombinase polymerase amplification with lateral flow dipstick (RPA-LFD).

Foodborne illnesses, caused by harmful microorganisms in food, are a significant global health issue. Current methods for identifying these pathogens are both labor-intensive and time-consuming. In this research, we devised a swift and precise detection technique using recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) for three foodborne pathogens found in meat. By employing a dedicated detection device, RPA-LFD allows for the rapid analysis of DNA from Escherichia coli O157 (E. coli O157), Salmonella, and Shigella-pathogens that are prohibited in food. The detection thresholds for E. coli O157, Salmonella, and Shigella are 0.168 fg/µl (1.04 CFU/ml), 0.72 fg/µl (27.49 CFU/ml), and 1.25 fg/µl (48.84 CFU/ml), respectively. This method provides a short detection window, operates at low temperatures, follows simple procedures, and exhibits high sensitivity. Our study establishes the RPA-LFD method for simultaneously identifying the nucleic acid of three foodborne pathogens, offering an efficient solution for quickly identifying multiple contaminants.

Hypolipidemic effect of polysaccharide from Sargassum fusiforme and its ultrasonic degraded polysaccharide on zebrafish fed high-fat diet.

Sargassum fusiforme is a brown seaweed that grows abundantly along the rocky coastlines of Asian countries. The polysaccharides derived from Sargassum fusiforme (SFPS) have received much interest due to their various bioactivities, such as hypolipidemic, hypoglycemic, and antioxidant activities. In this study, we extracted and purified SFPS, and obtained the ultrasonic degradation product (SFPSUD). The lipid regulatory effects of SFPS and SFPSUD were investigated in a zebrafish model fed a high-fat diet. The results showed that SFPS significantly decreased the levels of total cholesterol (TC) and triglycerides (TG), and increased the activities of lipoprotein lipase (LPL) and hepatic lipase (HL). SFPSUD was more effective than the SFPS in reducing the TC and TG levels in zebrafish, as well as increasing the LPL and HL activities. Histopathological observations of zebrafish livers showed that SFPSUD significantly improved lipid metabolism disorder in the hepatocytes. The possible lipid-lowering mechanism in zebrafish associated with SFPS and SFPSUD may involve acceleration of the lipid metabolism rate by increasing the activities of LPL and HL. Thus, SFPSUD could be tested as a highly effective hypolipidemic drug. Our results suggest that SFPS and SFPSUD have potential uses as functional foods for the prevention and treatment of hyperlipidemia. Ultrasound can be effectively applied to degrade SFPS to improve its physicochemical properties and bioactivities.

Flowering time control in ornamental gloxinia (Sinningia speciosa) by manipulation of miR159 expression.

BACKGROUND AND AIMS: Gloxinia (Sinningia speciosa) is a popular commercial plant for its attractive and colourful flowers. However, the genetic mechanism of flowering time regulation in gloxinia is largely unknown. Recent studies on model plants have elucidated that miR159 acts as a negative regulator of floral transition in short-day photoperiods. The aim of this study was to investigate whether genetic modification of miR159 expression can offer an effective approach for regulation of flowering characteristics in gloxinia. METHODS: Transgenic gloxinia plants were generated that over-express or suppress miR159 by means an efficient Agrobacterium-mediated transformation system in order to study the effect of miR159 on flowering time. In addition, the full-length cDNA of gloxinia GAMYB (SsGAMYB) was also cloned, and was verified to be a target of miR159 by modified RNA ligase-mediated 5' rapid amplification of cDNA ends. KEY RESULTS: Transgenic gloxinia plants that over-express or suppress miR159 exhibited significantly late or early flowering, respectively. During flower development, the expression level of miR159 was negatively correlated with SsGAMYB in gloxinia. MiR159-mediated SsGAMYB expression affected the expression levels of SsLEAFY (SsLFY) and three MADS-box genes (SsAP1, SsAP3 and SsAG), which regulated floral transition downstream of GAMYB. In addition, suppression of miR159 caused a conversion of petals and sepals in a few transgenic plants. CONCLUSIONS: miR159-regulated GAMYB expression is an effective pathway of flowering time control in gloxinia. Transgenic manipulation of miR159 can be used as an applicable strategy to regulate flowering time in commercial ornamental plants.

Effects of lactobacillus plantarum ZJ316 on pig growth and pork quality.

BACKGROUND: Lactobacillus plantarum is a plant-associated bacterial species but it has also been found in human, mouse and porcine gastrointestinal tracts. It can ferment a broad spectrum of plant carbohydrates; it is tolerant of bile salts and low pH, and it has antagonistic potential against intestinal pathogens. However, experiments reporting the use of L. plantarum as a probiotic are limited. In this study, the effects of L. plantarum ZJ316 isolated from infant fecal samples on pig growth and pork quality were investigated. RESULTS: One hundred and fifty newly weaned pigs were selected randomly and divided into five groups. Group 1 was fed a diet supplemented with the antibiotic mequindox; Groups 2, 3 and 4 were fed a diet supplemented with L. plantarum and no antibiotic; and Group 5 was fed a mixture of mequindox and L. plantarum. After a 60 days initial treatment, samples were collected for evaluation. The results showed that, the L. plantarum ZJ316 has probiotic effects on pig growth and that these effects are dose dependent. The effects of a dose of 1 × 109 CFU/d were more pronounced than those of a dose of 5 × 109 CFU/d or 1 × 1010 CFU/d. In Group 2 (1 × 109 CFU/d), the diarrhea (p = 0.000) and mortality rates (p = 0.448) were lower than in antibiotic-treated pigs (Group 1), and the daily weight gain (p = 0.001) and food conversion ratios were better (p = 0.005). Improved pork quality was associated with Lactobacillus treatment. pH (45 min, p = 0.020), hardness (p = 0.000), stickiness (p = 0.044), chewiness (p = 0.000), gumminess (p = 0.000) and restoring force (p = 0.004) were all significantly improved in Lactobacillus-treated pigs (Group 2). Although we found that L. plantarum exerted probiotic effects on pig growth and pork quality, the mechanisms underlying its action require further study. Polymerase chain reaction-denaturing gradient gel electrophoresis results showed that the gut bacterial communities in Lactobacillus- and antibiotic-treated pigs were very similar and the quantity of L. plantarum ZJ316 was below the detection limits of DGGE-band sequencing. The concentration of short-chain fatty acids in Lactobacillus- and antibiotic-treated fecal samples were not significantly different (p = 0.086). However, the villus height of ilea (p = 0.003), jejuna (p = 0.000) and duodena (p = 0.036) were found to be significantly improved by Lactobacillus treatment. CONCLUSION: L. plantarum ZJ316 was found to have probiotic effects, improving pig growth and pork quality. The probiotic mechanism might not involve L. plantarum colonization and alteration of the gut bacterial community. Rather, it might be related to the inhibition of the growth of opportunistic pathogens and promotion of increased villus height.

Surface expression of Helicobacter pylori urease subunit B gene E fragment on Lactococcus lactis by means of the cell wall anchor of Staphylococcus aureus protein A.

Fragment E of ureB (ureBE) was cloned from a clinical isolate of Helicobacter pylori. A prokaryotic expression vector, pAMJ399, with the ureB fragment E and the Staphylococcus aureus protein A anchor fragment (spaX), was constructed. The fusion protein was expressed under the control of the P170 promoter in Lactococcus lactis. Western blot assay of lactococcal cell wall extracts with a polyclonal chicken antiserum confirmed the immunity of the expressed recombinant protein which was located on the cell surface. These results provide the first report of a surface display system in lactic acid bacteria for the delivery of oral vaccines against Helicobacter pylori.

[Optimization of plantaricin production by Lactobacillus plantarum ZJ316].

OBJECTIVE: To enhance the production of plantaricin by Lactobacillus plantarum ZJ316 isolated from infant feces. METHODS: We analyzed fermentation parameters influencing cell growth and plantaricin production with different medium composition and under different cultivation conditions. RESULTS: MRS (DE MAN, ROGOSA, SHARPE) medium was more suitable for producing bacteriocin than other media. The maximum plantaricin production was obtained in modified MRS medium containing 10 g/L maltose and 10 g/L glucose, 10 g/L yeast extract, 10 g/L tryptone and 2g/L tri-ammonium citrate, 1 mL/L Tween80, 6 g/L K2HPO4.3H2O, 5 g/L sodium acetate, 0.2 g/L magnesium sulfate and 0.05 g/L manganese sulfate. The optimal initial pH and temperature for plantaricin production were 6.5 and 30 degrees C for 24 h. CONCLUSION: After optimizing, the production of plantaricin was increased 2.3-fold using the optimized medium, compared with in the basic MRS medium.

Purification and Characterization of Plantaricin ZJ316, a Novel Bacteriocin against Listeria monocytogenes from Lactobacillus plantarum ZJ316.

Bacteriocins are known to be natural preservatives, which are becoming increasingly necessary in many types of food to control the proliferation of pathogenic bacteria. In this study, a novel bacteriocin produced by Lactobacillus plantarum ZJ316, called plantaricin ZJ316, was purified by ammonium sulfate precipitation, gel chromatography, and high-performance liquid chromatography. By mass spectrometry, the molecular mass of plantaricin ZJ316 was determined to be 2,366.06 Da. No homologous sequences were found in databases based on comparisons with the N-terminal amino acid sequencing. The bacteriocin was heat resistant and stable after incubation at pH 2.0 to 10.0. It was sensitive to α-chymotrypsin, trypsin, and proteinase K. Plantaricin ZJ316 had a broad inhibitory activity against gram-negative and gram-positive bacteria, especially Listeria monocytogenes. Our results suggested that this bacteriocin has the potential to inhibit pathogenic bacteria in food products.

Comparative genomic analysis of Lactobacillus plantarum ZJ316 reveals its genetic adaptation and potential probiotic profiles.

OBJECTIVE: In previous studies, Lactobacillus plantarum ZJ316 showed probiotic properties, such as antimicrobial activity against various pathogens and the capacity to significantly improve pig growth and pork quality. The purpose of this study was to reveal the genes potentially related to its genetic adaptation and probiotic profiles based on comparative genomic analysis. METHODS: The genome sequence of L. plantarum ZJ316 was compared with those of eight L. plantarum strains deposited in GenBank. BLASTN, Mauve, and MUMmer programs were used for genome alignment and comparison. CRISPRFinder was applied for searching the clustered regularly interspaced short palindromic repeats (CRISPRs). RESULTS: We identified genes that encode proteins related to genetic adaptation and probiotic profiles, including carbohydrate transport and metabolism, proteolytic enzyme systems and amino acid biosynthesis, CRISPR adaptive immunity, stress responses, bile salt resistance, ability to adhere to the host intestinal wall, exopolysaccharide (EPS) biosynthesis, and bacteriocin biosynthesis. CONCLUSIONS: Comparative characterization of the L. plantarum ZJ316 genome provided the genetic basis for further elucidating the functional mechanisms of its probiotic properties. ZJ316 could be considered a potential probiotic candidate.

Enhancing vitamin B12 content in soy-yogurt by Lactobacillus reuteri.

More attention from the aged and vegetarians has been paid to soy-product due to its taste, easy digestibility, as well as the association with health. However, soy-product has a defect of low vitamin content, mainly the water-soluble vitamin B12. This study was to investigate co-fermentation of glycerol and fructose in soy-yogurt to enhance vitamin B12 production by Lactobacillus reuteri. After a serial combination experiments, the co-fermentation was confirmed to enhance the production of vitamin B12 up to 18 µg/100mL. Both supplementations induced the expression of cobT and cbiA and functioned to balance the redox reaction. Meanwhile, high content of fructose supplementation reduced the production of vitamin B12 and suppressed expression of cobT in bacteria. It was proved that the vitamin B12 content of this soy-yogurt is higher than other fermented soybean based food and thus can be served as an alternative food for the aged and vegetarians.

Purification and characterization of Plantaricin ZJ5, a new bacteriocin produced by Lactobacillus plantarum ZJ5.

The aim of this study is to investigate the antimicrobial potential of Lactobacillus plantarum ZJ5, a strain isolated from fermented mustard with a broad range of inhibitory activity against both Gram-positive and Gram-negative bacteria. Here we present the peptide plantaricin ZJ5 (PZJ5), which is an extreme pH and heat-stable. However, it can be digested by pepsin and proteinase K. This peptide has strong activity against Staphylococcus aureus. PZJ5 has been purified using a multi-step process, including ammonium sulfate precipitation, cation-exchange chromatography, hydrophobic interactions and reverse-phase chromatography. The molecular mass of the peptide was found to be 2572.9 Da using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The primary structure of this peptide was determined using amino acid sequencing and DNA sequencing, and these analyses revealed that the DNA sequence translated as a 44-residue precursor containing a 22-amino-acid N-terminal extension that was of the double-glycine type. The bacteriocin sequence exhibited no homology with known bacteriocins when compared with those available in the database, indicating that it was a new class IId bacteriocin. PZJ5 from a food-borne strain may be useful as a promising probiotic candidate.

Complete genome sequence of the probiotic Lactobacillus plantarum strain ZJ316.

Lactobacillus plantarum strain ZJ316, a probiotic strain with several functions, was isolated from healthy newborn infant fecal samples. Here we report the finished and annotated genome sequence of this organism.

Oral vaccination of mice against Helicobacter pylori with recombinant Lactococcus lactis expressing urease subunit B.

To determine whether a protective immune response could be elicited by oral delivery of a recombinant live bacterial vaccine, Helicobacter pylori urease subunit B (UreB) was expressed for extracellular expression in food-grade bacterium Lactococcus lactis. The UreB-producing strains were then administered orally to mice, and the immune response to UreB was examined. Orally vaccinated mice produced a significant UreB-specific serum immunoglobulin G (IgG) response. Specific anti-UreB IgA responses could be detected in the feces of mice immunized with the secreting lactococcal strain. Mice vaccinated orally were significantly protected against gastric Helicobacter infection following a challenge with H. pylori strain SS1. In conclusion, mucosal vaccination with L. lactis expressing UreB produced serum IgG and UreB-specific fecal IgA, and prevented gastric infection with H. pylori.

[Effects of cu2+ on biosynthesis of camptothecin in cell cultures of Camptotheca acuminata].

Camptothecin is a strong anti-tumor compound isolated from Camptotheca acuminata. One of the most important way for the production of Camptothecin is by cell cultures of Camptotheca acuminata. The effect of Cu2+ on camptothecin accumulation in Camptotheca acuminata cell line was described in this paper. The results showed that the optimum CuCl2 concentration in B5 medium was 0.008 mg/mL, which increased camptothecin production for 30 times compare to the control while has no inhibitive effects on cell growth, at the same time, the peroxidase activity was increased and the anthocyanidin accumulation was inhibited. The promotive effects of Cu2+ on camptothecin accumulation in light was higher than that in dark.