LC-MS and Transcriptome Analysis of Lipopeptide Biosynthesis by Bacillus velezensis CMT-6 Responding to Dissolved Oxygen.

Dissolved oxygen (DO) is an key factor for lipopeptide fermentation. To better understand the link between oxygen supply and lipopeptide productivity in Bacillus velezensis CMT-6, the mechanism of DO on the synthesis of antimicrobial lipopeptides by Bacillus velezensis CMT-6 was examined. The production of surfactin and iturin of CMT-6 was detected by liquid chromatography-mass spectrometer (LC-MS) under different DO conditions and transcriptome analysis was performed. At 100 and 200 rpm, the lipopeptides productions were 2753.62 mg/L and 3452.90 mg/L, respectively. There was no significant change in the yield of iturin but that of surfactin increased by 64.14%. Transcriptome analysis revealed that the enriched differential genes were concentrated in the GO term of oxidation-reduction process. The marked enrichment of the lipopeptides synthesis pathway, including microbial metabolism in diverse environments and carbon metabolism in the two-component system, were observed. More importantly, the expression levels of the four surfactin synthetase genes increased at higher DO, however, the iturin synthetase gene expression did not. Furthermore, modular surfactin synthetase was overexpressed (between 9- and 49-fold) at 200 rpm but not at 100 rpm, which is suggestive of efficient surfactin assembly resulting in surfactin overproduction. This study provides a theoretical basis for constructing engineering strains with high lipopeptide production to adapt to different DO.

Protective role of l-threonine against cadmium toxicity in Saccharomyces cerevisiae.

Environment and food contamination with cadmium (Cd) can cause serious toxicity, posing a severe threat to agricultural production and human health. However, how amino acids contribute to defenses against oxidative stress caused by Cd in cells is not fully understood. As a model eukaryote with a relatively clear genetic background, Saccharomyces cerevisiae has been commonly used in Cd toxicity research. To gain insight into Cd toxicity and cell defenses against it, 20 amino acids were screened for protective roles against Cd stress in S. cerevisiae. The results showed that threonine (Thr, T) had the strongest protective effect against Cd-induced mortality and membrane damage in the cells. Compared to the antioxidant vitamin C (VC), Thr exhibited a higher efficacy in restoring the superoxide dismutase (SOD) activity that was inhibited by Cd but not by H2 O2 in vivo. Thr exhibited evident DPPH (2,2-diphenyl-1-picrylhydrazyl) activity but weak ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-9 sulfonic acid)) scavenging activity, giving it a weaker effect against Cd-induced lipid peroxidation and superoxide radical O2- , compared to VC. More importantly, compared to the chelating agent EDTA, Thr showed stronger chelation of Cd, giving it a stronger protective effect on SOD against Cd than VC in vitro. The results of the in vivo and in vitro experiments revealed that the role Thr plays in cell defenses against Cd may be attributed to its protection of the SOD enzyme, predominantly through the preferential chelation of Cd. Our results provide insights into the protective mechanisms of amino acid Thr that ameliorate Cd toxicity and suggest that a supplement of Thr might help to reduce Cd-induced oxidative damage.

Effect of T-2 toxin-injected shrimp muscle extracts on mouse macrophage cells (RAW264.7).

Following intramuscular injections of 0.1 mL, 3 mg kg-1 BW-1(1/10 LD50) T-2 toxin (T-2), the tissue concentration of T-2 in shrimp was quantitatively detected using LC-MS/MS. The biological half-time (t1/2) of T-2 in blood was 40.47 ± 0.24 min. The highest number of intramuscular T-2 shrimp could tolerate when given at blood t1/2 intervals was 4. The shrimps which were injected 5 T-2 died. The T-2 toxin highest accumulation was 0.471 ± 0.012 ng g-1 BW-1. The effect of toxic shrimp muscle subjected to different processing conditions (high pressure, trifluoroacetic acid, acid and alkali digestions, artificial digestive juice [to simulate exposure to gastric and intestinal juices]) on mouse macrophage cells (RAW267.4) were evaluated by the MTT assay. The inhibition ratio of 2% muscle extract on RAW267.4 was 85.70 ± 2.63%. The immunocytotoxicity of muscle extracts to RAW264.7 was highest in muscle extracts subjected to physical and chemical digestion (high pressure > NaOH > trifluoroacetic acid > 0.02 M HCl > 0.2 M HCl > controls), and also artificial digestion (artificial intestinal juice > artificial gastric juice > N type intestinal juice > N type gastric liquid > controls). Results showed that high-pressure and artificial intestinal juice were most effective in the release of modified T-2 to free T-2 thus enhancing toxicity. These results can be interpreted as measurement of T-2 in food being of little value because of enhanced toxicity of T-2-contaminated food as they pass through the gastrointestinal tract.

A sensitive method for simultaneous quantitative determination of surfactin and iturin by LC-MS/MS.

Surfactin and iturin are antimicrobial lipopeptides produced from Bacillus spp. and have significant prospective applications in many fields. Therefore, accurate analysis of these lipopeptides in the fermented product of some Bacillus strains is important. A sensitive method for simultaneous quantitative determination of surfactin and iturin fermented by Bacillus natto NT-6 was developed and validated using liquid chromatography-tandem mass spectrometry. Crude extracts of antimicrobial lipopeptide samples were dissolved in a mixture of acetonitrile/water (7:3, v/v) in 0.1 % (v/v) formic acid and eluted with acetonitrile/water (7:3, v/v) containing 5 mmol L-1 ammonium acetate and 0.1 % (v/v) formic acid. The target compounds were detected by mass spectrometry (ESI+) using selective ion monitoring. A good linear regression in the range of 0.20-10.0 mg L-1 for both surfactin and iturin (R 2 ≥ 0.9995) was observed with spiked recoveries of 93.3-108.2 %, RSD values less than 15 %, precision 4.14-13.30 %, and a detection limit of 0.374 mg L-1. This method has a simple preprocessing operation, good repeatability, and provides an accurate quantitative analysis of surfactin and iturin. Graphical Abstract Surfactin and iturin from Bacillus natto NT-6 extraction and detection procedure.

Preparation of PCL/lecithin/bacteriocin CAMT6 antimicrobial and antioxidant nanofiber films using emulsion electrospinning: Characteristics and application in chilled salmon preservation.

Multi-functional packaging materials are an important development for food preservation. Emulsion electrospinning is a novel and simple method that can be used to prepare multi-functional packaging materials, which can effectively protect the loaded active substances during the preparation process. In this study, PCL/lecithin/bacteriocin CAMT6 nanofiber films with antimicrobial and antioxidant activity were prepared by emulsion electrostatic spinning. The morphology and homogeneity of the prepared nanofibrous membranes could be improved by optimising the formulation of the emulsion for electrospinning. Analytical testing of the prepared nanofiber films revealed that the nanofibers had a core-shell structure, with bacteriocin CAMT6 effectively encapsulated in the core layer and the PCL and phospholipids homogeneously mixed to form the shell layer. Additionally, the nanofiber films had acceptable tensile properties and water absorption capacity. In chilled salmon meat, the nanofiber film effectively inhibited the growth of bacteria, slowed the oxidation of oil and slowed water loss, which was a good protective effect. This study provides a reference for the encapsulation application of food-active packaging materials and bacteriocins.

An "off-on" fluorescent nanosensor for the detection of cadmium ions based on APDC-etched CdTe/CdS/SiO2 quantum dots.

In this study, we have developed a novel fluorescent "OFF-ON" quantum dots (QDs) sensor based on CdTe/CdS/SiO2 cores. Ammonium pyrrolidine dithiocarbamate (APDC), ethylenediamine tetraacetic acid (EDTA), and 1,10-phenanthroline (Phen) served as potential chemical etchants. Among these three etchants, APDC exhibited the most pronounced quenching effect (94.06%). The APDC-etched CdTe/CdS/SiO2 QDs demonstrated excellent optical properties: the fluorescence of the APDC-etched CdTe/CdS/SiO2 QDs system (excitation wavelength: 365 nm and emission wavelength: 622 nm) was significantly and selectively restored upon the addition of cadmium ions (Cd2+) (89.22%), compared to 15 other metal ions. The linear response of the APDC-etched CdTe/CdS/SiO2 QDs was observed within the cadmium ion (Cd2+) concentration ranges of 0-20 µmol L-1 and 20-160 µmol L-1 under optimized conditions (APDC: 300 µmol L-1, pH: 7.0, reaction time: 10 min). The detection limit (LOD) of the APDC-etched CdTe/CdS/SiO2 QDs for Cd2+ was 0.3451 µmol L-1 in the range of 0-20 µmol L-1. The LOD achieved by the QDs in this study surpasses that of the majority of previously reported nanomaterials. The feasibility of using APDC-etched CdTe/CdS/SiO2 QDs for Cd2+ detection in seawater, freshwater, and milk samples was verified, with average recoveries of 95.27%-110.68%, 92%-106.47%, and 90.73%-111.60%, respectively, demonstrating satisfactory analytical precision (RSD ≤ 8.26).

Iturin A Strongly Inhibits the Growth and T-2 Toxin Synthesis of Fusarium oxysporum: A Morphological, Cellular, and Transcriptomics Study.

Fusarium oxysporum (F. oxysporum) is a common contaminant of dried fish, and the T-2 synthesis by this organism in dried fish products poses a serious public health risk. In this study, we investigated the effects of iturin A, a cyclic lipopeptide produced by Bacillus subtilis, on the growth and synthesis of the T-2 toxin of F. oxysporum, and transcriptomics was conducted. Results showed that the inhibitory effect of iturin A on F. oxysporum was significantly enhanced with an increase in iturin A concentrations. More specifically, compared with the control group, all indexes in the iturin A treatment group with 50 µg/mL were decreased to 24.84 mm, 0.33 × 106 cfu/mL, and 5.86 ng/mL for the colony diameter, number of spores, and concentration of T-2 toxin, respectively. Furthermore, iturin A was proven to destroy the integrity of cell membranes and cause a significant increase in ROS at 25 µg/mL or 50 µg/mL. Transcriptomic analysis revealed that with the treatment of iturin A, the genes of the oxidation-reduction process were up-regulated, while the gene expression of mycelial growth, cell integrity, transmembrane transport, energy metabolism, and others were down-regulated. More importantly, the Tri5 gene cluster was significantly inhibited. This study provided new insights into the mechanism for the inhibitory effect of iturin A on the growth and T-2 toxin synthesis of F. oxysporum and theoretical guidance for the application of iturin A in the preservation of dried aquatic products.

Walnut protein hydrolysates, rich with peptide fragments of WSREEQEREE and ADIYTEEAGR ameliorate UV-induced photoaging through inhibition of the NF-κB/MMP-1 signaling pathway in female rats.

Skin photoaging is a complicated pathological process, and the imbalance of inflammatory regulation is associated highly with photoaging progression. Previously, prepared walnut protein hydrolysates (WPH), rich with peptide fragments of WSREEQEREE and ADIYTEEAGR demonstrated desirable photoprotection. However, it remains unclear if the photoprotection is mediated by the targeted inhibition of the NF-κB signaling pathway. Herein, we examined the regulation of WPH on inflammatory cytokine expression, and elucidated the modulation of the NF-κB/MMP-1 signaling pathway by WPH in a photoaging SD rat model. WPH significantly reduced the expression level of inflammatory cytokines IL-1ß and IL-6, but significantly increased the level of IL-2 (all P < 0.05). Furthermore, WPH dramatically inhibited the activation of the NF-κB signaling pathway by mitigating the phosphorylation of IκB and p-65 proteins in a dose-dependent manner. The histopathological results indicated that WPH predominately attenuated epidermal hyperplasia, reduced the inflammatory filtration, and promoted collagen deposition in the photoaging skin tissue. Furthermore, WPH significantly stimulated the expression of TGF-ß and procollagen type I, and inhibited the MMP-1 activities (all P < 0.05). Overall, the underlying mechanism of WPH ameliorating skin photoaging may be attributed to the synergistic modulation via reversing the inflammatory imbalance, suppressing the activation of the NF-κB signal pathway, stimulating procollagen type I synthesis, and inhibiting MMP-1 activities. According to these results, it can be concluded that WPH has the potential as an anti-photoaging agent in functional foods.

Simultaneous Quantification of Aflatoxin B1, T-2 Toxin, Ochratoxin A and Deoxynivalenol in Dried Seafood Products by LC-MS/MS.

Mycotoxins are secondary metabolites produced by fungi. These contaminate dried seafoods during processing and storage and represent a potential health hazard for consumers. A sensitive, selective and accurate liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was established for simultaneous quantification of four common mycotoxins (aflatoxin B1 (AFB1), T-2 toxin (T-2), ochratoxin A (OTA) and deoxynivalenol (DON)) in dried shrimp, dried fish and dried mussel products. Mycotoxins were extracted from dried seafood samples by acetonitrile/water (85/15, v/v), subjected to ultrasound for 60 min at 20 °C and cleaned up by defatting with n-hexane. The sample matrix affected the linearity of detection (R2 ≥ 0.9974). The limit of detection (LOD) and limit of quantification (LOQ) in dried seafood products varied from 0.1 to 2.0 µg·kg-1 and 0.3 to 5.0 µg·kg-1, respectively. The method was validated by spiking samples with specific mycotoxin levels, and the recoveries, intra-relative standard deviation (RSDs) and inter-RSDs ranged between 72.2-98.4%, 2.8-10.6%, and 5.5-15.4%, respectively. This method was used to analyze 40 dried seafood products purchased from the Zhanjiang seafood market. Results of this product sampling showed that while no DON was detected, AFB1, T-2 and OTA were detected in 30.8%, 17.5% and 33.3% of the samples, respectively. AFB1, T-2 and OTA concentrations varied at 0.58-0.89, 0.55-1.34 and 0.36-1.51 µg·kg-1, respectively. Relatively high frequency of contamination and the presence of AFB1, OTA and T-2 residues indicate the need to monitor mycotoxins in dried seafood products.

Effect of media and fermentation conditions on surfactin and iturin homologues produced by Bacillus natto NT-6: LC-MS analysis.

Lipopeptides possess excellent broad spectrum antimicrobial activity. Different lipopeptides have their own unique chemical structures, properties and biological activities. Quantitative analysis of the lipopeptides iturin and surfactin and their homologues produced by Bacillus natto NT-6 subjected to different culture media, shaking speed of rotary shaker, and liquid and solid fermentation methods was conducted using LC-MS. For iturins, liquid-state fermentation in Landy medium at a shaking speed of 160 r min-1 was the most suitable for maximal homologue production. Addition of 0.4% attapulgite powder increased production by 1.92-fold; activated carbon significantly reduced production. For surfactin homologues, solid-state fermentation in potato dextrose broth medium at shaking speed > 160 r min-1 was the best. Addition of 0.4% attapulgite powder increased production by 1.96-fold; activated carbon had no effect. Thus it is clear that fermentation conditions can be manipulated to maximize iturin and surfactin production.

Analysis of T-2 Toxin Removal Factors in a Lactococcus Fermentation System.

The objective of this work was to determine the bacterial strains and factors that most efficiently degrade T-2 toxin in foods or animal feed. To determine the most efficient strain and optimal incubation times for degradation of T-2, the rate of T-2 removal by three lactic acid bacteria strains was quantified by liquid chromatography plus tandem mass spectrometry after incubation in de Man Rogosa Sharpe broth with 50 ng mL-1 T-2 at 37°C for 96 h. Various components of the most efficient degradation strain fermentation systems were extracted, and the ability to remove T-2 was assayed. Lactococcus lactis CAMT22361 was the most efficient degradation strain for removing T-2. Yeast extract powder interfered with L. lactis CAMT22361 in the degradation process. T-2 toxin was removed by various components of the L. lactis CAMT22361 cells in the following order: nonprotein material of the extracellular fraction > protein in the extracellular fraction > whole cell ≈ cell wall > cell intracellular matrix fluid. T-2 removal rates were 54.08% ± 0.79%, 43.65% ± 0.84%, 43.09% ± 0.87%, 41.98% ± 0.8%, and 23.45% ± 0.66%, respectively. The nonprotein fraction in the extracellular fluid was most likely the key component in L. lactis CAMT22361 and hence would be the most desirable cellular component to be used to remove T-2 from food or feed.

Antimicrobial peptide AMPNT-6 from Bacillus subtilis inhibits biofilm formation by Shewanella putrefaciens and disrupts its preformed biofilms on both abiotic and shrimp shell surfaces.

Shewanella putrefaciens biofilm formation is of great concern for the shrimp industry because it adheres easily to food and food-contact surfaces and is a source of persistent and unseen contamination that causes shrimp spoilage and economic losses to the shrimp industry. Different concentrations of an antimicrobial lipopeptide, the fermentation product of Bacillus subtilis, AMPNT-6, were tested for the ability to reduce adhesion and disrupt S. putrefaciens preformed biofilms on two different contact surfaces (shrimp shell, stainless steel sheet). AMPNT-6 displayed a marked dose- and time-dependent anti-adhesive effect>biofilm removal. 3MIC AMPNT-6 was able both to remove biofilm and prevent bacteria from forming biofilm in a 96-well polystyrene microplate used as the model surface. 2MIC AMPNT-6 prevented bacteria from adhering to the microplate surface to form biofilm for 3h and removed already existing biofilm within 24h. Secretion of extracellular polymeric substances incubated in LB broth for 24h by S. putrefaciens was minimal at 3× MIC AMPNT-6. Scanning electron microscopy showed that damage to S. putrefaciens bacteria by AMPNT-6 possibly contributed to the non-adherence to the surfaces. Disruption of the mature biofilm structure by AMPNT-6 contributed to biofilm removal. It is concluded that AMPNT-6 can be used effectively to prevent attachment and also detach S. putrefaciens biofilms from shrimp shells, stainless steel sheets and polystyrene surfaces.

Neutralizing effect of hemolymph from the shore crab, Thalamita crenata, on paralytic shellfish toxins.

Several species of crabs are resistant to paralytic shellfish toxins (PSTs) and/or pufferfish toxin, tetrodotoxin, regardless of toxification by the toxins. The shore crab Thalamita crenata, which inhabits Leizhou Peninsula, China, is tolerant to PST toxicity, and the hemolymph has neutralizing effects against the lethal activity of PST. In the present study, we investigated the PST neutralizing factors in the hemolymph from T. crenata and successfully separated PST-binding proteins by PST-ligand affinity chromatography. The neutralization factors, obtained in the fraction with a molecular weight over 10 kDa by ultrafiltration, were susceptible to proteases such as alcalase, animal complex proteases, pancreatin, and papain. The PST-binding protein had high dose-dependent neutralization effects on PST toxicity. The PST-binding activity of the protein was stable at 25 °C and then decreased with an increase in temperature; heating at 65 °C for 60 min eliminated the initial activity by two-thirds. The PST-binding activity was strongly inhibited in the presence of Mg(2+) and Ca(2+), but not Na(+) and K(+). The PST-binding capability of the protein differed among PST components in descending order of neosaxitoxin, gonyautoxins 1 and 4, saxitoxin, and gonyautoxins 2 and 3, suggesting a structure-activity relationship in PST binding.