Identification and characterization of Achromobacter spanius P-9 and elucidation of its deoxynivalenol-degrading potential.

Deoxynivalenol (DON) poses significant challenges due to its frequent contamination of grains and associated products. Microbial strategies for mitigating DON toxicity showed application potential. Eight bacterial isolates with DON degradation activity over 5% were obtained from various samples of organic fertilizer in this study. One of the isolates emerged as a standout, demonstrating a substantial degradation capability, achieving a 99.21% reduction in DON levels. This isolate, underwent thorough morphological, biochemical, and molecular characterization to confirm its identity, and was identified as a new strain of Achromobacter spanius P-9. Subsequent evaluations revealed that the strain P-9 retains its degradation activity after a 24-h incubation, reaching optimal performance at 35 °C with a pH of 8.0. Further studies indicated that Ca2+ ions enhance the degradation process, whereas Zn2+ ions exert an inhibitory effect. This is the pioneering report of DON degradation by Achromobacter spanius, illuminating its prospective utility in addressing DON contamination challenges.

Determination of ß2-Agonist Residues in Fermented Ham Using UHPLC-MS/MS after Enzymatic Digestion and Sulfonic Resin Solid Phase Purification.

ß2-agonists are a class of synthetic sympathomimetic drugs with acute poisoning effects if consumed as residues in foods. To improve the efficiency of sample preparation and to overcome matrix-dependent signal suppression in the quantitative analysis of four ß2-agonists (clenbuterol, ractopamine, salbutamol, and terbutaline) residues in fermented ham, an enzyme digestion coupled cation exchange purification method for sample preparation was established using ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS). Enzymatic digests were subject to cleanup treatment on three different solid phase extraction (SPE) columns and a polymer-based strong cation resin (SCR) cartridge containing sulfonic resin was found to be optimal compared with silica-based sulfonic acid and polymer sulfonic acid resins based SPEs. The analytes were investigated over the linear range of 0.5 to 10.0 µg/kg with recovery rates of 76.0-102.0%, and a relative standard deviation of 1.8-13.3% (n = 6). The limit of detection (LOD) and the limit of quantification (LOQ) were 0.1 µg/kg and 0.3 µg/kg, respectively. This newly developed method was applied to the detection of ß2-agonist residues in 50 commercial ham products and only one sample was found to contain ß2-agonist residues (clenbuterol at 15.2 µg/kg).

Extraction and Study of Hypoglycemic Constituents from Myrica rubra Pomace.

Myrica rubra pomace accounts for 20% of the fruit's weight that is not utilized when it is juiced. The pomace contains bioactive phenolic substances such as anthocyanins and flavonoids. To improve the utilization value of Myrica rubra pomace, an optimized extraction method for the residual polyphenols was developed using response surface methodology (RSM). The resulting extract was analyzed by high performance liquid chromatography (HPLC), and the in vitro hypoglycemic activity and antioxidant activity of the polyphenolic compounds obtained were also investigated. The optimum extraction conditions (yielding 24.37 mg·g-1 total polyphenols content) were: extraction temperature 60 °C, ultrasonic power 270 W, ethanol concentration 53%, extraction time 57 min, and solid to liquid ratio 1:34. Four polyphenolic compounds were identified in the pomace extract by HPLC: myricitrin, cyanidin-O-glucoside, hyperoside, and quercitrin. DPPH and hydroxyl radical scavenging tests showed that the Myrica rubra polyphenols extract had strong antioxidant abilities. It is evident that the residual polyphenols present in Myrica rubra pomace have strong hypoglycemic activity and the juiced fruits can be further exploited for medicinal purposes.

Determination of Polycyclic Aromatic Hydrocarbons in Traditional Chinese Medicine Raw Material, Extracts, and Health Food Products.

The four polycyclic aromatic hydrocarbon markers (PAH4) of benzo[a]anthracene (BaA), chrysene (Chr), benzo[b]fluoranthene (BbF), and benzo[a]pyrene (BaP) are indicators showing polycyclic aromatic hydrocarbon (PAH) contamination levels in Chinese medicine raw materials (CMRMs), extracts and health food products; Samples of herbal medicine, herbal extracts, and food supplements were extracted with n-hexane, then cleaned up sequentially on Florisil and EUPAH solid-phase extraction (SPE) columns. A gas chromatography-mass spectrometry method for the determination of four polycyclic aromatic hydrocarbon markers in Chinese medicine raw material, extracts, and health food products was established; In spiked-recovery experiments, the average recovery was about 78.6-107.6% with a precision of 2.3-10.5%. The limit of quantification (LOQ) and limit of detection (LOD) of the PAH4 markers in this method were 2.0 µg/kg and 0.7 µg/kg, respectively. When the developed method was utilized to determine PAH4 contents in 12 locally available health food products, 3 samples contained over 10.0 µg/kg BaP, and 5 samples contained over 50.0 µg/kg PAH4. The European Union (EU) limits for BaP and PAH4 are 10 and 50.0 µg/kg, respectively; therefore, more attention must be drawn to the exposure risk of BaP and PAH4 in CMRMs, their extracts, and health food products. According to the risk assessment based on the Margin of Exposure (MOE) method, it is recognized that the products mentioned in this study pose a low risk.

Quantification of phytic acid in baby foods by derivatization with (trimethylsilyl)diazomethane and liquid chromatography-mass spectrometry analysis.

RATIONALE: Phytic acid (PA) is both a naturally occurring nutrient and a widely used food additive for conferring antioxidant properties to food. PA can be found in baby foods and it is essential to monitor PA content due to its anti-nutritional properties when present in excess. Current methods for determining PA content are unsatisfactory because interference from inositol phosphates and inorganic phosphates complicates PA quantification. METHODS: Baby foods were extracted using aqueous HCl, and the extractant was subjected to derivatization with (trimethylsilyl)diazomethane after de-metalation using a cation exchange resin. The PA derivative was quantified using liquid chromatography-mass spectrometry (LC/MS/MS) with a multi-response monitoring mode (m/z 829 to 451). RESULTS: The linearity of the developed analytical method ranged from 10 to 1000 ng/mL for PA with R2 > 0.999. Reasonable reproducibility was obtained with an intraday relative standard deviation (RSD; N = 5) of 4.5% and an interday RSD (N = 5) of 5.7% at a concentration of 10 ng/mL. The developed method was successfully applied to determine PA content in various baby foods, with PA recovery between 90.6% and 119.8%. CONCLUSIONS: A robust and sensitive method for the determination of PA in baby foods has been developed by methyl esterification with (trimethylsilyl)diazomethane and using LC/MS/MS analysis. The established method showed good anti-interference and precision, and it has been applied for the determination of PA in various baby foods.

Microbial diversity and chemical analysis of the starters used in traditional Chinese sweet rice wine.

Chinese sweet rice wine (CSRW) is a popular alcoholic drink in China. To investigate the effect of the microbial composition in CSRW starters on the final quality of the alcoholic drink, high-throughput sequencing on the fungal internal transcribed spacer II and bacterial 16S rRNA gene of the microflora in 8 starter samples was performed. The sequencing data analysis showed that 10 genera of yeasts and mold, and 11 genera of bacteria were identified. Fungal diversity analyses showed the significant variances in the fungal compositions among the starter samples. Starter microbiota were dominated by the Rhizopus genus in SZ5, LS6, NN8, QD9, DZ10 and DZ11, indicating its important role in starch hydrolysis during CSRW brewing. According to principal coordinate analyses, the bacterial composition had even less similarity among the 8 starter samples. The chemical determination of CSRW fermented with the 8 starters demonstrated that the CSRW quality and flavor were drastically influenced by the taxonomic composition and metabolism of the microbes in the starters. This study suggests it is necessary to standardize rice wine manufacturing and flavor classification by specifying starter and fermentation techniques.

Determination of three kinds of banned drugs in milk powder by hybrid solid-phase extraction purification combined with gas chromatography and mass spectrometry.

A gradient clean-up method for the quantification of five kinds of banned drugs (two hormones, two sedatives, and one chloramphenicol) in milk powder was developed. We used the combination of solid-phase extraction purification with gas chromatography and mass spectrometry. Milk powder was initially hydrolyzed by ß-glucuronidase/arylsulfatase, and then the hydrolyzed solution was concentrated and purified using a C8 and cation resin solid-phase extraction column. To isolate hormones and chloramphenicol drugs, products from the previous step were diluted with methanol and further purified using a silica and diatomite solid-phase extraction column. After derivatization, the drugs were analyzed by gas chromatography with mass spectrometry, and the hydrolyzed solution was diluted with 5% ammoniated methanol to purify sedatives before gas chromatography with mass spectrometry analysis. Results showed that after adding the banned drugs at concentrations of 0.3-10.0 µg/kg, the average recovery range was 78.2-97.3% with relative standard deviations of 5.3-12.5%. The limit of quantification of the banned drugs (S/N ≥ 10) was 0.3-5.0 µg/kg, whereas the limit of detection (S/N ≥ 3) was 0.1-2.0 µg/kg. The solid-phase extraction gradient purification system was simple, rapid, and accurate, and could satisfy the detection requirements of hormone, sedatives, and chloramphenicol drugs when used together with gas chromatography and mass spectrometry.

Identification of 19 phthalic acid esters in dairy products by gas chromatography with mass spectrometry.

A detection method for 19 kinds of phthalic acid ester compounds analyzed by n-hexane/ether/acetonitrile 1:7:8 v/v/v mixed solvent extraction, quick, easy, cheap, effective, rugged, and safe purification and internal standard method of quantitative gas chromatography with mass spectrometry was established. This method can effectively remove interfering materials, such as lipids, fatty acids, and pigments, from dairy products. The 19 kinds of phthalic acid ester compounds were within a 0.025-0.2 mg/kg range, the recovery rate was 65.2-125.7%, relative standard deviation was 7.9-15.4% (n = 6), and the limit of detection was 0.005-0.02 mg/kg. Concentrations of the 19 kinds of phthalic acid ester compounds ranged between 0.01 and 0.12 mg/kg in ten dairy materials and 20 dairy products. The established method is simple, rapid, accurate, and highly sensitive.

Acute toxicity of deoxynivalenol and bioremediation of a highly effective deoxynivalenol degrading Achromobacter spanius P-9 on zebrafish embryos and adults.

Deoxynivalenol (DON) is one of the mostly concerned mycotoxins and several microbes showed bioremediation effects on DON toxic effects. In this study, the acute toxicity of a new DON degrading strain Achromobacter spanius P-9 with DON on zebrafish embryos and adults were firstly performed. For zebrafish embryos, bacterial concentrations of 2.5 × 107 CFU/mL and 5.0 × 107 CFU/mL had no significant effects on growth and development. However, at 7.5 × 107 CFU/mL, some effects were observed, and at 10.0 × 107 CFU/mL, the embryo survival rate decreased to 70%, with 3.3% teratogenicity. Higher bacterial concentrations correlated with faster heart rates. DON (100 µg/mL) significantly reduced embryo survival to 36.7% in 96 h. Bacterial solutions at 7.5 × 107 CFU/mL and 10.0 × 107 CFU/mL expanded the zebrafish intestinal tissue wall, while DON at 100 µg/mL negatively impacted intestinal morphology. Liver tissue in zebrafish exposed to Achromobacter spanius P-9 showed no significant differences from the control group. However, exposure to DON solution increased liver fluorescence intensity and caused liver cell changes, including edema, vacuolization, and blurred boundaries. For adult zebrafish, the ROS and 8-OHdG contents in the exposure group increased with the increase of bacterial solution concentration, the SOD enzyme activity, CAT enzyme activity, GST enzyme activity and MDA was not significantly different with the control group. Compared with the control group, the content of ROS, GST enzyme activity, MDA and 8-OHdG after DON treatment showed an upward trend, SOD and CAT enzyme activities showed a decreasing trend. Achromobacter spanius P-9 has no obvious inhibitory effect on the growth and development of zebrafish embryos and has no obvious death and toxicity during the growth of adult fish, providing data support for the future application of this strain in the biodegradation of DON.

Transcriptome Analysis of Deoxynivalenol (DON)-Induced Hepatic and Intestinal Toxicity in Zebrafish: Insights into Gene Expression and Potential Detoxification Pathways.

The effects of deoxynivalenol (DON, 50 µg/mL) on the zebrafish liver and intestine were studied. Differentially expressed genes (DEGs) from mRNA and lncRNA were analyzed by RNA seq. Gene Ontology (GO) and signaling pathways were studied where the top 30 DEGs of each type of RNA were involved. The results showed there were 2325 up-regulated and 934 down-regulated DEGs of lncRNA in the intestinal tract, and 95 up-regulated genes and 211 down-regulated genes in the liver, respectively. GO functional annotation analysis showed that lncRNA was enriched in the biological processes, involving the RNA splicing, CSF1-CSF1R complexes, and MAP kinase activity. DEGs of lncRNA located in the KEGG signal pathways include the C-type lectin receptor signaling and the NOD-like receptor signaling pathways. Metabolism involves the biosynthesis of indole alkaloids, cancer pathways for human disease, MAPK and Rap1signaling pathways for environmental information processing, necroptosis and focal adhesion for cell processes. The mRNA gene expression analysis showed there were 1939 up-regulated, 1172 down-regulated genes and 866 up-regulated, 1211 down-regulated genes in the intestine and liver of zebrafish, respectively. This study provides transcriptome analysis and toxicological investigation of DON in the zebrafish liver and intestine, offering insights into gene expression patterns and potential detoxification pathways.

Lightweight tea bud recognition network integrating GhostNet and YOLOv5.

Aiming at the problems of low detection accuracy and slow speed caused by the complex background of tea sprouts and the small target size, this paper proposes a tea bud detection algorithm integrating GhostNet and YOLOv5. To reduce parameters, the GhostNet module is specially introduced to shorten the detection speed. A coordinated attention mechanism is then added to the backbone layer to enhance the feature extraction ability of the model. A bi-directional feature pyramid network (BiFPN) is used in the neck layer of feature fusion to increase the fusion between shallow and deep networks to improve the detection accuracy of small objects. Efficient intersection over union (EIOU) is used as a localization loss to improve the detection accuracy in the end. The experimental results show that the precision of GhostNet-YOLOv5 is 76.31%, which is 1.31, 4.83, and 3.59% higher than that of Faster RCNN, YOLOv5 and YOLOv5-Lite respectively. By comparing the actual detection effects of GhostNet-YOLOv5 and YOLOv5 algorithm on buds in different quantities, different shooting angles, and different illumination angles, and taking F1 score as the evaluation value, the results show that GhostNet-YOLOv5 is 7.84, 2.88, and 3.81% higher than YOLOv5 algorithm in these three different environments.

Deoxynivalenol Degradation by Various Microbial Communities and Its Impacts on Different Bacterial Flora.

Deoxynivalenol, a mycotoxin that may present in almost all cereal products, can cause huge economic losses in the agriculture industry and seriously endanger food safety and human health. Microbial detoxifications using microbial consortia may provide a safe and effective strategy for DON mitigation. In order to study the interactions involving DON degradation and change in microbial flora, four samples from different natural niches, including a chicken stable (expJ), a sheep stable (expY), a wheat field (expT) and a horse stable (expM) were collected and reacted with purified DON. After being co-incubated at 30 °C with 130 rpm shaking for 96 h, DON was reduced by 74.5%, 43.0%, 46.7%, and 86.0% by expJ, expY, expT, and expM, respectively. After DON (0.8 mL of 100 µg/mL) was co-cultivated with 0.2 mL of the supernatant of each sample (i.e., suspensions of microbial communities) at 30 °C for 96 h, DON was reduced by 98.9%, 99.8%, 79.5%, and 78.9% in expJ, expY, expT, and expM, respectively, and was completely degraded after 8 days by all samples except of expM. DON was confirmed being transformed into de-epoxy DON (DOM-1) by the microbial community of expM. The bacterial flora of the samples was compared through 16S rDNA flux sequencing pre- and post the addition of DON. The results indicated that the diversities of bacterial flora were affected by DON. After DON treatment, the most abundant bacteria belong to Galbibacter (16.1%) and Pedobacter (8.2%) in expJ; Flavobacterium (5.9%) and Pedobacter (5.5%) in expY; f_Microscillaceae (13.5%), B1-7BS (13.4%), and RB41 (10.5%) in expT; and Acinetobacter (24.1%), Massilia (8.8%), and Arthrobacter (7.6%) in expM. This first study on the interactions between DON and natural microbial flora provides useful information and a methodology for further development of microbial consortia for mycotoxin detoxifications.

Structural characterization, antioxidant and antimicrobial activity of water-soluble polysaccharides from bamboo (Phyllostachys pubescens Mazel) leaves.

One water-soluble neutral polysaccharides (NPs), with an average molecular weight of 5.77 × 103 Da, was isolated from bamboo (Phyllostachys pubescens Mazel) leaves and the yield was 10.2% based on weight of raw material. Successively, two fractions (NPs-A and NPs-B) could be obtained after being eluted by DEAE-Sepharose. Structural analysis indicated that NPs was formed by 1,4-ß-linked xylp backbone. Monosaccharide composition and methylation analysis suggested that glucose, arabinose, galactose and rhamnose constituted side chains with the molar ratio of 1.22:1.79:1.89:4.46 with the glycosidic linkages of →4)-Glc-(1→, →3,5)-Ara-(1→, →4)-Gla-(1→, →4)-Rha-(1 → and → 2,4)-Rha-(1→. Antioxidant assays demonstrated that NPs exhibited relatively high activity in a dose-dependent manner with the highest 85.9% DPPH and 99.78% ABTS free radical scavenging rate when the concentration was 4.0 and 3.0 mg/mL, respectively. Moreover, NPs exhibited obvious growth inhibitory against E. coli, S. aureus and B. subtilis when the NPs concentration was in range of 0.50-50.0 mg/mL. Therefore, we concluded that NPs from bamboo leaves could be used as a potential and natural bacteriostat and antioxidant.

Medium optimization for keratinase production in hair substrate by a new Bacillus subtilis KD-N2 using response surface methodology.

Culture medium for keratinase production from hair substrate by a new Bacillus subtilis strain, KD-N2, was optimized. Effects of culture conditions on keratinase production were tested, and optimal results were obtained with 10% inocula (v/v), 16 g/L hair substrate, an initial pH value of 6.5 and a culture volume of 20 mL. Several carbon sources (sucrose, cornflour) and nitrogen sources (yeast extract, tryptone and peptone) had positive effects on keratinase production, with sucrose giving optimal results. To improve keratinase yield, statistically based experimental designs were applied to optimize the culture medium. Fractional factorial design (FFD) experiments showed that MgSO4 and K2HPO4 were the most significant factors affecting keratinase production. Further central composite design (CCD) experiments indicated that the optimal MgSO4 and K2HPO4 concentrations were 0.91 and 2.38 g/L, respectively. Using an optimized fermentation medium (g/L: NaCl 1.0, CaCl2 0.05, KH2PO4 0.7, sucrose 3, MgSO4 0.91, K2HPO4 2.38), keratinase activity increased to 125 U/mL, an approximate 1.7-fold increase over the previous activity (75 U/mL). Human hair was degraded during the submerged cultivation.

Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis.

A new feather-degrading bacterium was isolated from a local feather waste site and identified as Bacillus subtilis based on morphological, physiochemical, and phylogenetic characteristics. Screening for mutants with elevated keratinolytic activity using N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis resulted in a mutant strain KD-N2 producing keratinolytic activity about 2.5 times that of the wild-type strain. The mutant strain produced inducible keratinase in different substrates of feathers, hair, wool and silk under submerged cultivation. Scanning electron microscopy studies showed the degradation of feathers, hair and silk by the keratinase. The optimal conditions for keratinase production include initial pH of 7.5, inoculum size of 2% (v/v), age of inoculum of 16 h, and cultivation at 23 degrees C. The maximum keratinolytic activity of KD-N2 was achieved after 30 h. Essential amino acids like threonine, valine, methionine as well as ammonia were produced when feathers were used as substrates. Strain KD-N2, therefore, shows great promise of finding potential applications in keratin hydrolysis and keratinase production.

Purification and characterization of keratinase from a new Bacillus subtilis strain.

The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50 degrees C was 8.5 and the optimum temperature at pH 8.5 was 55 degrees C. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT), mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO) stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.

Pentapeptide Prosequence Enhances Expression and Structure Folding of Recombinant Thermomyces lanuginosus Lipase in Pichia pastoris.

BACKGROUND: Propeptides of lipases have been demonstrated to influence many properties of their mature proteins as intramolecular chaperones. However, the working mechanism of propeptides may be different. OBJECTIVES: The main objective of this study was to determine the role of pentapeptide prosequence of Thermomyces lanuginosus lipase (TLL) through its effect on the recombinant expression of TLL in P. pastoris, and explore the possible function mechanism with its hydrophobicity. METHODS: We have synthesized a codon-optimized TLL gene coTLL with a Kex2 cleavage site "- KREAEA-" directly after the propeptide "SPIRR-", and obtained expression vector pP-kTL through cloning it into pPIC9K. TL gene without the propeptide and pTL gene with a propeptide directed linked to mature TLL lipase, were also amplified and cloned into pPIC9K to obtain the expression vector pP-TL and pP-pTL. pTL-P and pTL-VP gene variants with mutation in pentapeptide prosequence of TLL were obtained used the site-directed mutation with the pP-pTL plasmid as the template. The recombinant proteins were expressed in P. pastoris GS115. Lipase activity was determined by a spectrophotometric method previously reported using para-nitrophenyl palmitate (pNPP) as the substrate and the thermostability of lipases of TLL was analyzed by incubating at different temperatures (70°C and 80°C) for 5h and molecular dynamics simulation. RESULTS: The average lipase activity of recombinant strains GS-pTL reached 434.32 U/mL, higher than that of GS-TL 377.71 U/mL. The fermentation result of the recombinant strains with modified propeptide showed that the extracellular lipase activity of GS-pTL-VP variant reached 483.29 U/mL, increasing by 11.27% compared with that of GS-pTL (434.32 U/mL). Further analysis performed on the lipase stabilities with propeptide variants by molecular dynamics simulation showed that the RMSD of variant pTL-VP was similar to that of pTL. CONCLUSION: This study revealed that the propeptide "SPIRR-" sequence is beneficial for enhancing TLL expression. In addition, the function of TLL propeptide was identified to be related to its hydrophobicity, implying that propeptide might play a role in assisting the formation of the hydrophobic protein core and accelerate the protein folding process. This work inspired us to attach more emphasis on the propeptide of other lipases for improving their recombinant expression, structure folding and enzymatic properties.

Formation of ethyl carbamate and changes during fermentation and storage of yellow rice wine.

Ethyl carbamate (EC) was analyzed during yellow rice wine production and storage. EC increased slowly during fermentation and rapidly after frying and sterilization. Less amount of EC was formed when cooled rapidly to 30 °C than when cooled naturally. High temperature and long storage time increased EC formation. After 400 days storage, EC increased from 74.0 to 84.2, 131.8 and 509.4 µg/kg at 4 °C, room temperature and 37 °C, respectively, and there was significantly difference between the fried wine and the wine on sale from 2011 (p<0.01). Urea increased during yellow rice wine fermentation and was above 20 mg/kg after the wine was fried; urea contributed to EC formation when the fried wine was cooled slowly. These results indicate that it is necessary for industry to optimize the wine frying conditions, such as temperature, time and cooling process in order to decrease EC formation.