A procedure to measure subcutaneous adipose (SAT) using brightness-mode ultrasound has recently been standardized and applied to various groups of adults including underweight, overweight and obese adults. High reliability of this procedure was found in each of the examined groups. The purpose of this study was to determine inter-observer reliability of the standardized brightness-mode ultrasound measurement of uncompressed SAT in three to six-year-old children.− 2. Sound speed was set to 1450 ms− 1 for SAT.Three experienced observers independently captured the ultrasound images at the eight standardized measurement sites in each of the 20 children and evaluated their images using an interactive software that detects the SAT contour and automatically measures multiple thicknesses in each image; the mean of these represents SAT thickness at a given site. The children were aged 4.9 ± 1.0 years; their body mass index ranged from 13.6–17.7 kgmDI) ranged from 25.7–86.4 mm, mean DI was 48.1 ± 15.5 mm. For DI, resulting from 160 measurements by each observer, the intra-class correlation coefficient was 0.998 , standard error of the estimate was 1.1 mm, and 95% limits of agreement were within ±2.1 mm. The median difference in DI was 0.8 mm, i.e. about 1.9% of mean DI.SAT thickness sums with fibrous structures included  ID:  Body composition and growth are important determinants of childhood health . AlthougThese estimates were derived from measurements of body mass and height and compared to normative growth standards –3.h) and body mass (m), there are several other anthropometric measures in use for determining relative body weight and body composition in adults and in children [m/h2) [m/(h·s) has been proposed, which considers the individual’s sitting height (s) and thus, implicitly, the leg length [In addition to the analysis of body height  –9, whichn [m/h2) , 4. A sin [m/h2) . As an ag length –12. Noneg length .A widely used approach for assessing body fat, specifically subcutaneous adipose tissue (SAT), is the measurement of skinfolds. Skinfold thickness is composed of a double fold of compressed adipose tissue and skin . SkinfolDI) ranging from DI = 10 to 50 mm, 95% of the values among experienced observers were found to be within ±1.0 mm from the mean [DI ranging from 12 to 245 mm, 95% of repeated observer measurements were within ±2.2 mm from the mean [DI ranging from 12 to 77 mm, 95% of values were within ±1.4 mm from the mean, and in a second subgroup with DI ranging from 53 to 245 mm, 95% of values were within ±2.9 mm from the mean [A new approach has recently been introduced which results in highly accurateand reliable measurements of uncompressed SAT in adults , 18. Thithe mean . In a grthe mean . In a suthe mean .DI was compared, the intra-class correlation coefficient (ICC = 0.994) and its 95% confidence interval (95% CI: 0.983–0.998) indicated excellent intra-observer reliability [DI ranged from 34.8 to 112.3 mm, 95% of measurement differences in DI were within 0.4 to 2.0 mm [In a sample of 274 preschool children, mean SAT thickness significantly differed between boys and girls, while anthropometric characteristics such as body mass, body height, BMI, and waist circumference did not show any significant differences . Additioiability . Thickneo 2.0 mm .intra-observer reliability in children [The standardized ultrasound technique for measuring SAT has repeatedly shown high intra-and inter-observer reliability in various groups of adults –20, and children .inter-observer reliability studies in children are missing. The aim of this study was to bridge this gap and to compare the results found in preschool children aged three to six years to the published results in adult groups. The analysis of the inter-observer reliability will allow a large-scale implementation of this technique.However, Health Survey, an evaluation study of the preschool-based health promotion program Join the Healthy Boat in Southwest Germany, ultrasound measurements of SAT were performed as part of body composition analysis [Health Survey was registered at the German Clinical Trials Register (DRKS) operated by the German Institute of Medical Documentation and Information, Cologne, Germany (ID: DRKS00010089) and approved by the ethics committee of Ulm University (application number 188/15) and is in accordance with the Declaration of Helsinki. Written consent to participate in the reliability analysis was given by the parents of 20 children (40% boys) aged 4.9 ± 1.0 years. Three observers  certified by the International Association of Sciences in Medicine and Sports (www.iasms.org) performed the ultrasound measurements of SAT , sitting height (s) were measured to the nearest 0.1 cm, and body mass (m) to the nearest 0.05 kg. The BMI (m/h2) and the MI (0.53 m/(h·s)) [s for assessing relative body weight. In individuals with long legs, the MI is higher than the BMI and vice versa. For a person with a Cormic index C = s/h = 0.53, representing mean leg length, the BMI and the MI are equal [Anthropometric measurements were performed in accordance with the International Standards for Anthropometric Measurements . Body hem/(h·s)) , 11, 12 re equal , 11, 12.h) [The observers marked the eight standard sites on the right side of the participants’ body. These sites are defined with respect to the individual’s body height (h) . In thish) .Three observers captured the brightness-mode ultrasound images at the eight marked measurement sites in each child independently and evaluated their 160 images. The standardized brightness-mode ultrasound imaging was performed with the participants lying in a supine, prone or rotated position . The opeA thick layer of ultrasound gel, typically 5 mm, was used between the probe and the skin to avoid compression. The gel layer appeared as a black band on top of the ultrasound image, and underneath, the epidermis, dermis, SAT, muscle fascia, and muscle were clearly visible Fig. a and c. Each of the three observers captured the eight images at the standardized sites in 20 children, resulting in three measurement series and a number of 3·8·20 = 480 ultrasound images, which formed the basis for this inter-observer reliability study.www.rotosport.at) to evaluate SAT thicknesses at the eight standard sites. The observers evaluated their sets of images independently. The SAT contour was detected interactively, and multiple thicknesses of SAT, typically 100 per image, were measured automatically. The robust mean of these thicknesses determines the SAT thickness at the given site. Speed of sound was set to c = 1450 ms− 1 for distance determination in SAT [d) including and excluding embedded fibrous structures , and also calculated the sums of the eight individual sites DI and DE, respectively. The sum of embedded fibrous structures was also calculated as DI - DE.The images were imported into the SAT image evaluation software  from their mean (DMEAN), and 95% limits of agreement were calculated as mean difference ± 1.96·SD of the differences [SPSS 21  was used. Values were reported as mean ± standard deviation (SD). Normal distribution was tested using the Shapiro-Wilk test. The ICC and its’ 95% CI were calculated based on a two-way random effects model with average measures . A lineaferences . Similarferences , and in ferences , 18–20. DI,MEAN), calculated from three measurement series per child, ranged from 25.7 to 86.4 mm , the standard error of the estimate was 1.1 mm, and Pearson’s r was 0.997. The inter-observer results for DE were: ICC = 0.998 (95% CI: 0.995–0.999), standard error of the estimate = 1.0 mm, and Pearson’s r = 0.998 . Accordingly for DE: SD = 1.0 mm, 95% limits of agreement were ± 2.0 mm  p > 0.895.The individual observer differences Δ mm Fig. b. VarianI) from DI, MEAN ranged from 0.0 to 2.5 mm, the median was 0.8 mm. ABS (ΔE) also ranged from 0.0 to 2.5 mm, and the median was 0.7 mm. The relative measurement differences from the mean of the three observations were calculated as: Δrel = 100·ABS(Δ)/DMEAN. For DI, the median of the relative differences ΔI,rel was 1.9%, the maximum 4.7%; for DE median ΔE,rel was 2.1%, and the maximum 5.5%.Absolute values of observer differences ABS (ΔI) at the eight individual sites (n = 3·20 = 60), Fig. E). Median values of ABS (δI) ranged from 0.1 to 0.3 mm, maximum deviation was 1.6 mm. Median values of ABS (δE) ranged from 0.1 to 0.4 mm, maximum deviation was 1.7 mm [see Additional file Figure es n = 3·0 = 60, F− 2 . Although the BMI range covered only about four units, the highest DI-value, DI = 86.4 mm, was 3.4 times larger than the lowest DI-value, DI = 25.7 mm. In several cases, almost the same BMI values were associated with large differences in DI, e.g. individuals with a BMI of 15.3 kgm− 2, 15.4 kgm− 2, and 15. 7kgm− 2 showed sums of SAT thicknesses of 32.4 mm, 52.6 mm, and 61.2 mm, respectively.The BMI values ranged from 13.6 to 17.7 kgmTable DI was 0.8 mm, i.e. about 1.9% of mean DI. This is comparable to the high reliability that was previously found in adults [This inter-observer study conducted by three observers (of two research centers) in 20 children aged three to six years resulted in a SEE of 1.1 mm, and the 95% limits of agreement were within ±2.1 mm. The ICC was 0.998 (95% CI 0.980–0.999), and the median difference in n adults , 19, 20.The brightness-mode based ultrasound method for measuring SAT has been standardized  and applPreviously, this method was used in a pediatric sample for the first time to examine sexual dimorphism of adipose tissue in 274 children aged three to five years. The study found that mean SAT thicknesses significantly differed between boys and girls, even though neither the BMI nor the waist circumference differed .DI along with the inter-observer results of the present study. This overview shows that differences in DI were about three times as large when measurements were conducted by novices compared to experienced observers [The application of this technique has revealed high intra- and inter-observer reliability in several adult populations , 18–20, bservers . In thisI) was 0.3 mm at each of the sites lower abdomen, lateral thigh, distal triceps, and brachioradialis. However, the medians of the relative values of the differences varied depending on the SAT thickness at the given site: 4.3, 2.9, 3.4, and 6%, respectively. The relative differences were smaller with increasing SAT thickness , this method is the only one that enables a quantification of fibrous structures (fasciae) embedded in the SAT, which amount to a substantial percentage of the subcutaneous adipose tissue mass. The reliability of SAT thickness measurements when embedded fibrous structures (fasciae) are excluded is comparable to the measurements when these structures are included. This standardized method enables body composition and fat patterning analyses in children on a much finer scale than obtainable with any other method. The reliability results found here indicate that there is high potential for ultrasound to replace or compliment other methods for determining body fat in children. Training is necessary to obtain the high reproducibility and accuracy level possible with this standardized method.Additional file 1. SAT thickness sums excluding fibrous structured of each participant measured by the three observersAdditional file 2. Absolute differences in SAT thickness values to the mean of the three measurements at each individual site


The contents of PF, BBR, and SCH were (6.112 ± 0.166) mg/g, (335.1 ± 14.95) μg/g, and (5.867 ± 0.136) μg/g in BSPs. On this basis, PF and BBR were selected as targeted analytes for the pharmacokinetic study of BSPs in rats. Memantine hydrochloride was used as an internal standard (IS), and the plasma samples were processed by liquid-liquid extraction with ethyl acetate. All the analytes were separated on a C18 reversed phase column, eluted with a mobile phase consisting of acetonitrile-formic acid (0.01%) , and detected by ESI in the selected ion mode with multiple reaction monitoring (MRM). The target fragment ions were m/z 525.3 ⟶ 449.5 for PF, 336.2 ⟶ 320.2 for BBR, and 180.1 ⟶ 163.1 for IS. The linear ranges of PF and BBR were 5–500 ng/mL and 0.1–20 ng/mL with good linearity (r2 > 0.99). No obvious matrix effect was observed, and acceptable accuracy, precision, recovery, and stability were obtained. The proposed method has been successfully applied to the pharmacokinetic study of BSPs in rats after a single dose.Bushen Pills (BSPs), as a traditional Chinese medicine (TCM), is widely used in clinic to enrich Yang, nourish Yin, stem essence, and strengthen kidneys. Two chromatographic methods, liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), were applied to analyze the multiple active components of BSPs in dosage form for quality evaluation and in rat plasma for pharmacokinetics study, respectively. Three active constituents of BSPs, including paeoniflorin (PF), berberine hydrochloride (BBR), and schizandrin (SCH), were simultaneously determined by the established LC-MS method with electrospray ionization (ESI) in positive selected ion monitoring (SIM) mode at  Danxi xinfa 500 years ago, is one of the well-known traditional Chinese medicines (TCM) employed to enrich Yang, nourish Yin, stem essence, and strengthen kidneys. It is prescribed in combination with 10 medicinal crude drugs at a certain mass ratio based on the theory of traditional Chinese herbal medical science. The types and ratio of the drugs were as follows: Herba Cynomorii (9.3%), Plastrum Testudinis (9.3%), Radix Rehmanniae Praeparata (23.3%), Fructus Lycii (14.0%), Radix Asparagi (9.3%), Rhizoma Anemarrhenae (9.3%), Radix Paeoniae Alba (9.3%), Rhizoma Zingiberis (2.3%), Cortex Phellodendri Chinensis (9.3%), and Fructus Schisandrae Chinensis (4.6%) + with m/z at 503.1 and 455.2, and the protonated molecules for BBR and IS (sparfloxacin) were [M+H]+ with m/z at 336.0 and 393.1 . Chromatographic separation was carried on a Waters Symmety®Cnd 393.1 . The ana18 column  at 30°C. The mobile phase was composed of 0.01% formic acid and acetonitrile  with a flow rate of 0.5 mL/min. The injection volume was 20 μL, and the run time was 5.0 min. The nebulizer pressure was 50 psi, and the capillary voltage was 4000 V, with a 400 eV delta EMV. The spray gas (nitrogen) temperature was set at 350°C with a gas flow of 11 L/min. Mass spectrometric detection was operated in MRM with positive ion mode from 0 to 3.6 min for BBR and IS (memantine hydrochloride), and then with negative ion mode from 3.6 to 5.0 min for PF. The target fragment ions were m/z 525.3 ⟶ 449.5 for PF with the [M+HCOO]− as the precursor ion, 336.2 ⟶ 320.2 for BBR, and 180.1 ⟶ 163.1 for IS . Chromatographic separation was carried on a Waters Symmety® C1 for IS  with theμg/mL. The IS solution of sparfloxacin was diluted with methanol to yield a concentration of 1.0 μg/mL. For LC-MS/MS assay, mixed working solutions containing 0.05–5.0 μg/mL PF and 1–200 ng/mL BBR were also obtained with methanol. The IS working solution of memantine hydrochloride was diluted with the mobile phase to a concentration of 100 ng/mL. 20 μL of each mixed working solution dried with nitrogen gas and 200 μL blank plasma was added to get final concentration of calibration samples containing PF 5, 10, 20, 50, 100, 200, 500 ng/mL and BBR 0.1, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0 ng/mL. The low, middle, and high quality control (QC) samples containing PF 6, 40, and 400 ng/mL and BBR 0.2, 4.0, and 16.0 ng/mL were prepared independently in the same fashion. All stock and working solutions were stored at 4°C in the refrigerator.A stock solution for each standard  was prepared at a concentration of 1.0 mg/mL in methanol. For LC-MS assay, mixed working solutions of PF, BBR, and SCH were prepared with methanol to six concentrations for construction of calibration plots in the ranges of 5.0–15.0, 0.15–0.45, and 0.06–0.18 v/v) in an ultrasonic bath for 30 min, diluted with methanol-water  to volume and mixed. The extracted solution was centrifuged at 5000 rpm for 10 min. 40 μL of the supernatant was mixed with 100 μL of IS  and diluted to 1.0 mL with methanol-water  to get the final sample solution. Then 5 μL of the solution was injected into the LC-MS system for analysis. Three batches of BSPs samples were selected randomly, and the contents of PF, BBR, and SCH were determined by the newly developed analytical method.The fine powdered BSPs (0.75 g) was accurately weighed and transferred to a volumetric flask of 25 mL, then extracted with 20 mL of methanol-water  were mixed with 2.0 mL of ethyl acetate, vortexed for 3 minutes, and then centrifuged at 4000 rpm for 5 min. The supernatant (1.6 mL) was separated and blown to dryness in a water bath at 40°C with nitrogen. The residue was reconstituted with 100 μL of the mobile phase, and finally, 20 μL was injected into the LC-MS/MS system for analysis.Liquid-liquid extraction was used for extraction of PF, BBR, and IS from plasma. 200 in vivo pharmacokinetic study was carried out with five male Wistar rats. They were allowed to acclimate to the housing conditions for at least 1 week and fasted for 12 h prior to the experiments with free access to water. The BSPs were ground into a powder and suspended in 0.4% carboxymethylcellulose sodium (CMC-Na) solution, then given to the rats at a dose of 5 g/kg  via intragastric gavage. This dose was equivalent to 30.55 mg/kg, 1775.55, and 29.35 μg/kg PF, BBR, and SCH, respectively. 0.5 mL of blood was collected from the subclavian venous sinus at specified time intervals  after the administration of BSPs. Samples were placed into heparinized tubes and separated immediately by centrifugation (3000 rpm for 15 min). The obtained plasma samples were transferred into clean tubes and stored at −20°C for further analysis.The The LC-MS method for quantification of PF, BBR, and SCH in BSPs was validated with specificity, linearity, precision, accuracy, repeatability, and stability. The LC-MS/MS method for plasma samples was validated in compliance with the NMPA guidelines for bioanalytical method validation, including selectivity, matrix effect, linearity, recovery, precision, accuracy, and stability.Considering the content and stability of ingredients, combined with the convenient and accessible analytical methods, the active components, including PF, BBR, and SCH, from the minister and adjuvant herbs of the formula, were selected as the target analytes for quality control and pharmacokinetics study of BSPs.m/z ions identified for PF, BBR, and SCH were 503.1, 336.0, and 455.2 in positive mode.A gradient based LC-MS method was developed and validated for the determination of PF, BBR, and SCH in BSPs samples. Compared with methanol, acetonitrile could provide lower background noise and higher response for the cited analytes. Adding 0.2% formic acid to the aqueous phase could obtain the most satisfactory chromatographic peak shape and the sensitivity by enhancing the ionization of analytes. The gradient elution procedure of acetonitrile-0.2% formic acid in water was finally adopted and could successfully achieve good separation of the index components of BSPs with reasonable analysis time. Additionally, the MS conditions of the cited analytes were investigated in both positive and negative ion modes. The major v/v) for 30 min had better peak shape, less impurity interference, and higher recovery.For the extraction of active components in BSPs, ultrasonication and cold immersion were compared. It was found that ultrasonication with methanol-water , solid-phase extraction (SPE), and liquid-liquid extraction (LLE) were tried to get better plasma preparation. The samples pretreated for PPT with methanol and/or acetonitrile showed obvious endogenous interferences. The SPE and LLE methods were acceptable for the biosample pretreatment. However, the SPE method is extremely expensive for a large number of samples. Although the extraction recoveries for analytes were only around 40%, the LLE method with ethyl acetate was finally adopted for the sample preparation, due to its lower cost, stable extraction rate, better producibility, and lower matrix effect.Representative MRM chromatograms of blank rat plasma, blank rat plasma spiked with PF, BBR, and IS, and extracted plasma sample from a rat dosed with BSPs are shown in Y = 0.0074X + 0.0032 for PF, Y = 0.0796X + 9.2074 × 10−4 for BBR. The resulting correlation coefficients (r2) were both greater than 0.99. The LLOQ for PF and BBR was 5.0 ng/mL and 0.1 ng/mL, respectively. The matrix effect and extraction recovery were evaluated and are summarized in The calibration curves were established, ranging from 5–500 ng/mL for PF to 0.1–20 ng/mL for BBR. Typical equations for the calibration curves were μg/g, and (5.867 ± 0.136) μg/g, respectively.The established LC-MS method was successfully applied to quantitative analysis of the three active components. The content of PF, BBR, and SCH in three batches of commercial BSPs samples was (6.112 ± 0.166) mg/g, (335.1 ± 14.95) The validated LC-MS/MS method was successfully applied to the pharmacokinetic study for the determination of PF and BBR concentration in rat plasma after a single oral dose of BSPs. The mean concentration-time profiles for PF and BBR are shown in Tmax detected in rat plasma were 2.60 h for PF and 0.90 h for BBR, indicating that BBR was absorbed more rapidly in vivo than PF. Besides, the values of t1/2 were 3.64 h for PF, but 9.59 h for BBR, which suggested that PF was quickly eliminated while BBR was more slowly eliminated. In addition, it showed that PF was the constituent with the greater plasma concentration and AUC than that of BBR, possibly associated with the content of PF much higher than BBR in BSPs prescription. Furthermore, a reliable LC-MS/MS method for the determination of SCH in rat plasma was also successfully established. However, the drug concentration was too low to quantify after a single dose of BSPs, resulting in the complete drug concentration-time curve not obtained. Meanwhile, the multipeak phenomenon was observed for PF. The result is similar to the PK study of PF in the previously published articles [As can be seen from articles , 21. PosIn the present study, a selective and reliable LC-MS method was developed to simultaneously detect PF, BBR, and SCH in BSPs, which is suitable for routine analysis in quality control of the pharmaceutical preparation. Another simple, rapid, and specific LC-MS/MS assay for quantification of PF and BBR in biological samples was established and fully validated, and then successfully applied to a pharmacokinetic study of BSPs after a single dose in rats. This research offers experimental evidence and valuable references for quality control and clinical use of BSPs.


Dear Editor,I have read with interest the published article entitled “Mortality rate of acute kidney injury in SARS, MERS, and COVID-19 infection: a systematic review and meta-analysis” by Chen et al. [Firstly, the mortality rate for COVID-19 patients with AKI is different in the text  from one reported in the authors’ Figure 1 . The authors might need to clarify this discrepancy.I2 = 97.1%, P < 0.0001, instead of 53.99% (CI 52.34–55.65), I2 = 98.4%, P < 0.0001, had the authors sorted the duplicate-entry problem.Secondly, the authors mistakenly made a duplicate entry of the study by Chen et al. (2020) in the COVID-19 forest plot. This mistake resulted in a pooled AKI mortality rate of 78.0% (CI 63.0–90.0), I2 = 97.1%, P < 0.0001. This strongly impacts the reliability of the conclusion drawn [Thirdly, the authors concluded the mortality rate for COVID-19 patients with AKI from an otherwise a high heterogeneity of I2 = 7.4%, P < 0.375, see Fig. I, on the other hand, reanalyzed authors’ data and performed sensitivity analysis according to the Cochrane Library recommendation . I exclu I2 = 0.0%, P = 0.589) and MERS , but there was for COVID-19 infection .There was no evidence of statistical heterogeneity among studies reporting AKI mortality in SARS  patients with acute kidney injury (AKI) is different from the text and Figure 1c in the original publication of our article [In addition, our research letter aimed to overview the AKI mortality in patients with different coronaviruses, but the clinical heterogeneity between studies should be also noted. One of the important factors may be the racial difference between studies as Dr. Joel Swai noted. Even if the recent study from Fisher et al. indicated the races are not associated with mortality in COVID-19 patients developing AKI , future


High-resolution melting analysis (HRMA) is a novel molecular technique based on the real-time PCR that can be used to detect vancomycin resistance Enterococcus (VRE). The purpose of this study was to identify VRE species with HRMA in clinical isolates.E. faecium isolates and 19 (38.77%) E. faecalis isolates were detected. Average melting temperatures for divIVA in E.faecalis, alanine racemase in E.faecium, and vanA in VRE strains were obtained as 79.9 ± 0.5 °C, 85.4 ± 0.5 °C, and 82.99 ± 0.5 °C, respectively. Furthermore, the data showed that the HRMA method was sensitive to detect 100 CFU/ml for the divIVA, alanine racemase, and vanA genes. Also, out of 49 Enterococcus spp., which were isolated by HRMA assay, 8 isolates (16.32%) of E. faecium and 18 isolates (36.73%) of E. faecalis were detected. The vanA gene was reported in 2 isolates (25%) of E. faecium and 9 isolates (50%) of E. faecalis.Out of 49 Enterococcus isolates, 11 (22.44%) E. faecium, E. faecalis, and the vanA gene with high sensitivity and specificity.This study demonstrated that using the HRMA method, we can detect  High-resolution melting analysis (HRMA) is an innovative technology that has the potential to distinguish bacterial types  based on variations in DNA methylation patterns. The HRMA is a simpler and more cost-effective way to characterize multiple bacterial samples and antibiotic resistance, comparing to other conventional methods such as culture and biochemical identification . HRMA reEnterococcus faecalis and Enterococcus faecium were identified as the two most prevalent Enterococcus spp. causing healthcare-associated infections (HAI) [E. faecium has been reported as the most common Enterococcus spp. [vanA, vanB, vanC, vanD, vanE, and vanG, with a high prevalence reported for vanA and vanB. The vanA is the most common phenotype of VRE and demonstrates higher level of resistance to vancomycin and teicoplanin. The spread of the vanA gene from Enterococcus to Staphylococcus aureus in the same patient demonstrates the mobility of these resistance genes [divIVA gene encodes a multifunctional protein, which plays an important role in cell division, biofilm formation, resistance, and viability in E. faecalis. The divIVA gene has a conserved N-terminal domain in E. faecalis which can be used as a detection target [Vancomycin resistance Enterococcus (VRE) is an important cause of nosocomial infections . Enterocns (HAI) , 6. E. fce genes , 7. Moren target , 9.Given the importance of different Enterococcus spp. in human, accurate and rapid identification of these species is important. Phenotypic tests, in addition to their time-consuming procedures and high costs, may be associated with some errors , 11. HenE. faecium and E. faecalis. Further, by optimizing the HRMA method, the vanA gene was identified in these species.The purpose of this study was to evaluate HRMA as a rapid and accurate method for the identification of . isolated in this experimental-analytical study and collected from 374 clinical specimen including blood, sputum, urine, ulcers, and secretions from therapeutic centers of Hamadan University of Medical Sciences  during April 2015 to August 2016. The samples were cultured on sheep blood agar  and incubated at 37 °C overnight. Then, the small, smooth, cream colonies were subcultured to bile esculin azide agar , and also, biochemical tests including leucyl aminopeptidase (LAP), pyrrolidonyl arylamidase (PYR), and growth in 6.5% NaCl broth were performed. All clinical Enterococci were further identified to the species using conventional biochemical tests devised by Mahon, Textbook of Diagnostic Microbiology [Forty-nine (49) human clinical Enterococcus sppobiology .To extract the DNA, a boiling method was used according to Tongeren et al. study . FirstlydivIVA , alanine racemase (for E. faecium), and vanA (for VRE strains) primers listed in Arabestani et al. [2. DNA amplification was performed  with thermal cycling conditions consisting of an initial denaturation step at 94 °C for 5 min, followed by 30 amplification cycles that included denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min, and extension at 72 °C for 10 min.E. faecalis NCTC13779, E. faecium NCTC7174, and E. faecium ATCC51559 (for vanA gene) were used as positive controls. Forward and reverse primers for divIVA, alanine racemase, and vanA were used as sequencing primers using the Eppendorf Thermocycler (Germany). All PCR products were sent to Pishgam Company  for sequencing.PCR was carried out using i et al.   were serially diluted, starting from 107 to 100 CFU/ml (0.5 McFarland 1.5 × 108 CFU/ml). Two microliters of each serially diluted DNAs were applied in triplicate with each primer in singleplex/multiplex for the real-time PCR. PCR efficiency was calculated from the slope of the standard curve using the formula E = 10(−1/slope)−1. The primer efficiency range between 90 and 110% was considered as reliable. In this study, the efficiency of each gene was calculated by the ABI® 96 Software 3.2.0 .A two-step amplification PCR assay was performed on a Step One-Plus® 96 instrument  using the EvaGreen dye . The amplification starts with an initial denaturation at 95 °C for 15 min, followed by 40 cycles of 95 °C for 30 s and 57 °C for 30 s. One cycle of melt curve step was conducted by ramping the temperature from 60 °C to 90 °C. The melt curve plot was prepared by plotting the negative derivative of fluorescence (− Rn) versus temperature. To determine the sensitivity of each primer in the real-time PCR assay, the DNAs isolated from Normalized and difference graphs were generated to assess the ability of the HRMA method to differentiate among bacterial strains. HRMA assay amplification was performed, using a real-time PCR instrument . Reactions were prepared in a total volume of 20 μl that included 4 μl of 5X HRMA Master Mix (HOT FIREPol® EvaGreen HRMA Mix), 1 μl of each primer (20 pmol), 1 μl of bacterial DNA, and 13μl of DEPC-treated water . The amplification protocol involved a 10 min hold at 95 °C followed by 40 amplification cycles comprising of 95 °C denaturation for 15 s, annealing at 58 °C for 30 s, and extention at 72 °C for 10 s. The HRMA reactions were carried out in triplicate for each isolate tested. After amplification, high-resolution melting was carried out on the PCR amplicons which were generated. The first part of the melting process involved a brief denaturation of samples at 95 °C for 1 min and rapid annealing. Following this, the temperature was again slowly increased and HRMA curve data obtained from 60 °C to 95 °C.R2) and slope values were obtained from the standard curve, and the efficiency (E) of PCR was calculated according to the equation E = 10–1/slope−1.The correlation coefficients  generated the amplification and melt curves, cycle threshold (CT), products’ melt temperature (Tm), standard curve, and unknown sample quantification data. HRMA data analyzed by the ABI Step One-Plus High-Resolution Melt (HRMA) software V3.01 .E. faecium and 19 (38.8%) E. faecalis were isolated. Nineteen isolates (38.8%) were not detectable by phenotypic tests. Out of 11 isolates of E. faecium, 8 isolates (72.72%) were obtained from female patients, and 3 isolates (27.27%) were collected from male patients. Also, 2 isolates (27.27%) were collected from blood, 1 isolate (9.09%) was isolated from ulcer, 1 isolate (9.09%) was obtained from sputum, and 7 isolates (63.63%) were collected from urine. Among 19 E. faecalis isolates, 13 isolates were isolated from female patients and 6 isolates were obtained from male patients. Moreover, 7 isolates (36.84%) were obtained from blood, 2 isolates (10.05%) were isolated from ulcer, 2 isolates (10.52%) were collected from sputum, and 9 isolates (57.89%) were isolated from urine were collected.Out of 49 Enterococcus spp., 11 (22.4%) E. faecalis NCTC13779 with NZ_UGJA01000003.1 accession number, E. faecium NCTC7174 with LR134337.1 accession number, and E. faecium ATCC51559 with accession number of JSVT00000000.The results of sequencing were blasted and determined as divIVA efficiency = 99.89% for E. faecalis NCTC13779, alanine racemase efficiency = 100.01% for E. faecium NCTC7174, and vanA efficiency = 101.09% for E. faecium ATCC51559. The sensitivity of the real-time PCR assay was found to be 107 to 100 CFU/ml for all standard strains (0.5 McFarland 1.5 × 108 CFU/ml)    of E. faecium  obtained from blood and 6 isolates (75%) collected from urine were detected by the HRMA method. The HRMA-detected isolates of E. faecium were obtained from blood , ulcer , sputum , and urine .The results showed that out of 11 clinical isolates 22.5%) of % of E. fsolates 1.32%  have bedivIVA primer was 79.9 ± 0.5 °C and for alanine racemase primer was 85.4 ± 0.5 °C, and HRMA assay detected 100 CFU/ml of E. faecalis and E. faecium. Ozbak [In Figs. m. Ozbak , Sweden m. Ozbak , and Finm. Ozbak  determin2 for the real-time PCR was determined as 2.5 mM. A proper MgCl2 concentration in a PCR reaction is one of the most important factors in building a distinguishable plot for determining Enterococcus species [The optimal concentration of MgCl species , 19. On  species  and EiscStaphylococcus aureus, which has acquired the vanA gene, emphasizes the importance of VRE infection control. The detection of VRE strains by HRMA is the most specific test and is useful for the detection of VRE with low tolerance. Therefore, the present paper showed that the vanA gene was positive in 2 isolates (25%) of E. faecium and 9 isolates (50%) of E. faecalis by HRMA technique, also, primer specificity for vanA gene was 100.01% and melting temperature for this gene observed at 79.9 ± 0.5 °C. Lata et al. [3 genomic equivalents. Cha et al. [2 CFU/ml. Young et al. [4 CFU/ml and 102 CFU/ml, respectively. In the present study, a detection limit of 101 and 100 CFU/ml equivalents was achieved in singleplex and multiplex assay format, respectively. The detection limit of the real-time PCR assay developed in this study for detection of VRE comparable to results obtained by Ozbak [Rapid and accurate detection of VRE is critical to block the transmission of the infection. Moreover, the emergence of vancomycin-resistant a et al.  reporteda et al.  reportedg et al.  and Bababy Ozbak .E test), poor diffusion of vancomycin has been observed and caused false negative (indicating susceptibility rather than resistance) results. In one study, Váradi et al. [Identification of different Enterococcus species and antibiotic resistance by phenotypic method has several disadvantages. To explain, the conventional susceptibility methods require at least 24 h following an overnight inoculation (16–20 h) and in the case of agar-based methods  limited dilutions were used to determine the sensitivity and (b) using a two-step reaction instead of a one-step reaction can affect the results. Hence, using more dilutions and low concentration of bacterial suspensions could give better results. Although the use of HRMA technique in some cases is at high cost, use of the real-time multiplex PCR method can play an important role to reduce the costs. Therefore, it is suggested that in future studies, the effectiveness of HRMA method in comparison with phenotypic methods for the diagnosis of other bacteria involved in hospital infections should be considered.Real-time PCR technique as an alternative test has been widely employed in detections of Enterococcus spp. during recent decades, as it offers accurate, rapid, and low-cost detection. The developed HRMA assay is easy operating and requires no use of probe, which makes it a useful tool for epidemiology analysis and laboratory use. Overall, HRMA-based PCR methods worked effectively in detecting Enterococcus spp. in various clinical samples. Hence, this study provided rapid detection methods for the patient to detect VRE strains in clinical isolates. Owing to the simplicity and ease of use, HRMA application in the areas of clinical diagnostics has also increased. This assay can be further tested with a large amount of pure or mixed strains and contaminated clinical samples.


Kyphosus vaigiensis (herbivore), Stegastes rectifraenum (omnivore), and Balistes polylepis (carnivore) at two mining sites. Metal concentrations were analyzed by atomic absorption spectrophotometry and stable nitrogen and carbon isotopes were estimated using mass spectrophotometry. Also, we assessed the risk to human health from the consumption of these three species based on permissible limits; although only two of them  are consumed as food. Metal concentrations differed among fish species; except for iron. The highest concentrations of metals were not always recorded in the species at the highest trophic level; i.e., Balistes. The highest concentrations (dry weight) recorded were cadmium (0.21 ± 0.03 µg g−1) and lead (1.67 ± 0.26 µg g−1), in S. rectifraenum; copper (1.60 ± 0.49 µg g−1) and zinc (67.30 ± 8.79 µg g−1), in B. polylepis; and iron (27.06 ± 2.58 µg g−1), in K. vaigiensis. Our findings show that each element accumulates differently in particular marine organisms; depending on the physiology of the species and the biogeochemistry of its habitat; which in turn is affected by the anthropogenic activities in adjacent areas. No risk of heavy metals toxicity is expected from the human consumption of the species and sites studiedIn the Gulf of California; mineral deposits have contributed to high metal contents in coastal environments. This study examined cadmium; lead; copper; zinc; and iron contents in three fish species;  Trace elements can cause harmful health effects when they accumulate in organisms at concentrations above those required for the metabolic functions . Zinc, c−1) in Chloroscombrus chrysurus, lead (8.49µg g−1) in Galeoides decadactylus, and zinc and iron  in Ilisha africana [Ctenopharyngodon idellus thought to be affected by copper mining in the district of Purple Mountain, China, for over 30 years, recorded concentrations (dry weight) of up to 8.5 ± 0.75 µg g−1 of copper, 27.7 ± 2.7 µg g−1 of zinc, and 0.705 ± 0.155 µg g−1 of lead, and no detectable concentrations of cadmium [−1, respectively) were recorded in Eugerres brasilianus, and the highest concentration of zinc (477 µg g−1) was recorded in Genidens genidens. Cadmium levels were below the limit of detection in fish [Local mineral deposits in the environment may contribute to increase the content of one or more chemicals in sediments relative to the levels considered as typical of the Earth’s crust . The cheus, lead .49µg g−1−1) is higher than levels recorded in industrialized areas of Russia [−1) have been found in sediments [−1) [Deposits of various minerals are located in the Gulf of California, some of which have been exploited through mining operations at sites adjacent to the coast . Two minf Russia . Iron anf Russia , and higediments . The secediments . Besidesediments . Reportsnts [−1) .Sargassum. These algae cover large areas, providing habitat, shelter, and food to numerous species of invertebrates and fish [Kyphosus vaigiensis , the Cortez damselfish Stegastes rectifraenum , and the fine scale triggerfish Balistes polylepis . S. rectifraenum is highly appreciated as an ornamental fish for aquarists, while the other two species are captured by artisanal fishers for direct human consumption [The coastal areas off STR and LAP are home to macroalgae beds with the capacity to remove trace elements suspended in the water column , which mand fish , includisumption .K. vaigiensis confirm that this species is a functional herbivore that feeds primarily on macroalgae, including phaeophytes, chlorophytes, and rhodophytes, and almost no animal material [Kyphosus, such as K. bigibbus, K. cinerascens, K. sydneyanus, K. azureus, and K. elegans  [S. rectifraenum (family Pomacentridae) was first described as an herbivore [B. polylepis  has been classified as predominantly carnivorous, although some algae are consumed occasionally as incidental food [Sargassum along the Gulf of California [Almost all members of the Kyphosidae family are herbivorous . Studiesmaterial ,18 as otifornia) ,19,20,21erbivore , but laterbivore ,24. B. ptal food ,26. The lifornia , suggestK. vaigiensis, K. cinerascens and K. sp. sampled in China, Australia, or New Caledonia  and Bahía de La Paz (LAP), Baja California Sur, on the west coast of the Gulf of California . This coKyphosus vaigiensis, Stegastes rectifraenum, and Balistes polylepis, were caught in STR and LAP by free diving using a Hawaiian-type harpoon. In STR, three sites were sampled during the dry (May–June 2015) and rainy (September–October 2015) seasons: Punta Gorda , Las Cuevitas , and Los Frailes   . Las Cue.101″ W) .Kyphosus vaigiensis , 68 of Stegastes rectifraenum , and 61 of Balistes polylepis  were captured and transported on ice to the laboratory. The total length (±1 mm) of each fish was recorded. Afterward, fish were dissected, and a sample of white muscle from the anterior dorsal region was excised from each specimen. Muscle samples were rinsed with distilled water and dried in Petri dishes in a dry-heat oven for 24–48 h at 75 °C to constant weight. Then, tissues were ground using a Wig-L-Bug (model 30) electro-mechanical stirrer until a homogeneous powder was obtained. Each sample was transferred to a separate vial and stored in a drying chamber until tested for trace elements. A subsample of muscle from each specimen was saved for isotopic analysis.A total of 70 specimens of −1) respectively were as follows: Cd: 0.01 and 0.02; Pb: 0.07 and 0.10; Cu: 0.017 and 0.020; Zn: 0.021 and 0.060; Fe: 0.65 and 1.35.Samples of 0.5 g of muscle tissue were digested with nitric acid and hydrogen peroxide in a microwave oven . After acid digestion, 1 mL of hydrochloric acid (HCl) was added to each sample and the volume was brought to 50 mL with deionized water. Cadmium (Cd), lead (Pb), copper (Cu), zinc (Zn), and iron (Fe) concentrations were determined by atomic absorption spectrophotometry  using an air/acetylene flame . Each sa15N of an organism typically shows a 3.4 ‰ (±1‰) increase at each higher trophic level; thus, it can be used to determine the trophic position of an organism [13C changes little as carbon moves through the food web and can be used to evaluate the ultimate sources of energy for an organism [2 and CO2 in tin capsules using a PDZ Europe ANCA-GSL elemental analyzer interfaced to a PDZ Europe 20–20 isotope ratio mass spectrometer  at the Stable Isotope Laboratory, University of California Davis. It was not necessary to account for lipids in fish samples, as the average C:N ratio of all samples (C:N = 3.3 ± 0.1) was below 3.5 [15N) are reported in parts per thousand (‰) relative to N2 in air, while carbon isotope ratios (δ13C) are reported relative to Pee Dee Belemnite (PDB) using the following equation:15N or 13C and R = 15N/14N or 13C/12C. Samples were analyzed interspersed with several replicates of at least four different laboratory reference materials. These reference materials were previously calibrated using international reference materials, including IAEA-600, USGS-40, USGS-41, USGS-42, USGS-43, USGS-61, USGS-64, and USGS-65. The standard deviation was ±0.3 ‰ for 15N and ±0.2 ‰ for 13C.Stable isotope ratios of nitrogen and carbon are powerful tools for evaluating the trophic structure and energy flow across aquatic ecosystems. The δorganism . In contorganism . Sampleselow 3.5 . NitrogeK. vaigiensis, S. rectifraenum, B. polylepis). ANOVA were followed by the post hoc Tukey tests [Before the statistical tests, to each sample that presented an undetectable concentration of some metal it was assigned a value that corresponds to half the detection limit of such metal . Metal cey tests .a is the intercept, b takes positive values for STA and negative for LAP, c takes positive values for the rainy season and negative for dry, d is the coefficient of the dummy variable Sp1 and takes positive values for K. vaigiensis, and negative for others, e is positive for S. rectifraenum and negative for B. polylepis, f is the partial coefficient of normalized standard fish length, and g and h are the coefficients for the stable isotopes. Before the analyses, fish length was normalized by species, and the data were arranged according to the categorical variable’s location, season, and species, and also according to the continuous variables fish standard length and isotopic readings . The multiple categorial regression was applied to evaluate and compare the relative weight of each of the factors in determining the concentration of the different elements in fish muscle. The models were fitted in the R statistical package using a significance level of p < 0.05.Multiple categorial regression models of the general form:−1 BW day−1) was calculated as follows [−1); CR = Mean per-capita daily consumption rate of fish muscle (30 mg day−1); BW = mean body weight of an adult person (65 kg) [−1), zinc (11 mg day−1), and copper (0.9 mg day−1) to assess the contribution of the estimated EDI for these elements. No RDI has been set for cadmium and lead since these metals are considered toxic to humans. To assess the potential toxicological risk of the intake of the elements studied through the consumption of muscle from each of the three species, each EDI was compared with its respective reference oral dose (RfD), namely: cadmium: 1 μg kg−1 bodyweight day−1; copper: 40.0 μg kg−1 bodyweight day−1; iron: 700 μg kg−1 bodyweight day−1, and zinc: 300 μg kg−1 bodyweight day−1 [−1 bodyweight day−1 [One of our objectives was to compare the results obtained in this study with standards and results of previous works and assess the potential health risk of human consumption of muscle of the three species studied. To this end, element concentrations were converted from dry weight (dw) to fresh weight (fw) as follows:8–79.4%) . The Est follows :EDI = (C (65 kg) . From a  (65 kg) ) for iroht day−1 . An RfD ure) 100)using thht day−1 ,54,57.K. vaigiensis, 38  showed undetectable cadmium levels (<0.01 µg g−1), 48  had undetectable lead levels (<0.07 µg g−1), and 16  showed undetectable copper levels (<0.017 µg g−1). Of the 68 samples of S. rectifraenum, 20  showed undetectable cadmium levels, 34  had undetectable lead levels, and 26  showed undetectable copper levels. Of the 61 B. polylepis organisms, 24  showed undetectable cadmium levels, 48  had undetectable lead levels, and 18  showed undetectable levels of copper.Of the 70 specimens of S. rectifraenum (STR = 11.09 ± 0.16 cm and LAP = 22.97 ± 0.89 cm) and B. polylepis (STR = 12.47 ± 0.13 cm and LAP = 26.62 ± 1.05 cm); the opposite occurred for K. vaigiensis (STR = 31.82 ± 1.32 cm and LAP = 23.70 ± 1.42 cm). Concerning the metal concentrations between locations, the annual average concentrations of cadmium (0.19 ± 0.03 µg g−1)  = 13.64, p = 0.000), copper (1.57 ± 0.20 µg g−1)  = 6.74, p = 0.012), and iron (32.60 ± 3.65 µg g−1)  = 8.90, p = 0.004) in K. vaigiensis fish caught in STR were higher  than concentrations in fish from LAP, while the concentrations of lead (0.59 ± 0.16 µg g−1) and zinc (42.09 ± 2.96 µg g−1) were 0.6-fold or similar to those recorded in fish from LAP. In the case of S. rectifraenum, Cd, Cu, and Fe concentrations were not significantly different in fish between STR and LAP, while the levels of Pb (2.76 ± 0.49 µg g−1)  = 14.88, p = 0.000) and Zn (23.88 ± 0.77 µg g−1)  = 10.87, p = 0.002) were higher  in fish caught in LAP versus STR. Regarding B. polylepis, Zn concentrations (93.72 ± 14.77 µg g−1)  = 12.74, p = 0.001) were significantly higher (2.6-fold) in samples from STR versus LAP; no significant differences were observed for any of the remaining metals  was higher than in the rainy season (16.78 ± 1.93 µg g−1)  = 28.25; p = 0.000). In fish from LAP, the concentrations of Cd (0.06 ± 0.03 µg g−1) = 5.17, p = 0.03), Pb (2.08 ± 0.71 µg g−1)  = 8.89, p = 0.006), Cu (1.71 ± 0.21 µg g−1)  = 69.63, p = 0.000), and Zn (55.01 ± 4.72 µg g−1)  = 19.79, p = 0.000) were higher in samples obtained in the dry season than in the rainy season; no significant differences were observed for Fe concentrations. Regarding S. rectifraenum caught in STR, significant differences were detected only in Cu concentration, being higher in fish from the dry season (1.05 ± 0.12 µg g−1) versus the rainy season  = 76.46, p = 0.000). In LAP, Pb (4.16 ± 0.43 µg g−1)  = 11.42, p = 0.002) and Cu (0.88 ± 0.14 µg g−1)  = 10.58, p = 0.003) levels were higher in fish caught in the dry season versus the rainy season; no significant differences were observed in the other metals. As for B. polylepis, significant differences between seasons were detected in Zn concentrations in fish caught in STR, with higher concentrations in the rainy season (123.54 ± 20.58 µg g−1) than in the dry season  = 8.89, p = 0.005) and in Pb and Fe concentrations, being higher in fish from dry season  = 12.55, p = 0.001 and F = 12.38, p = 0.001 respectively); no significant differences in metal concentrations were observed in fish caught in LAP (t in LAP .p < 0.05) in S. rectifraenum (11.66 ± 0.14 cm), and no differences were observed between B. polylepis (24.65 ± 0.71 cm) and K. vaigiensis (28.69 ± 1.08 cm). Metal concentrations of Cd  = 4.32, p = 0.015), Pb  = 4.52, p = 0.012), Cu  = 3.68, p = 0.027) and Zn  = 20.81, p = 0.000) and both stable isotopes δ13C  = 32.20, p = 0.000) and δ15N  = 20.95, p = 0.000) in fish muscle differed between fish species. The highest mean concentrations recorded were cadmium and lead in S. rectifraenum, copper and zinc in B. polylepis, and iron in K. vaigiensis. B. polylepis showed no significant differences in mean cadmium, lead, copper, and iron contents relative to the other two species. As regards stable isotopes, the average value of δ13C showed significant differences between the three species, with the lowest values found in B. polylepis (−16.25 ± 0.21), followed by S. rectifraenum (−14.99 ± 0.19) and K. vaigiensis (−13.66 ± 0.27); for δ15N, the mean value for K. vaigiensis was significantly lower (16.77 ± 0.16) versus S. rectifraenum (18.12 ± 0.13) and B. polylepis (17.73 ± 0.17).13C), and trophic level (δ15N). The only factor that significantly explains the cadmium concentrations observed is the location (Cd in STR). For lead, significantly contributing factors were season (higher Pb in the dry season), fish species (higher Pb in S. rectifraenum), and fish size (higher Pb in larger fish). For copper, the location (higher Cu in STR) and season (higher Cu in the dry season). For zinc, the season (higher Zn in the rainy season), fish species (higher Zn in B. polylepis), fish size , and food source (negative relationship with δ13C); finally, for iron, the factors explaining the concentration are location (higher Fe in LAP), fish size (higher Fe in larger organisms), and trophic level (inverse relationship with δ15N).Multiple linear regression models  were runB. polylepis . In the three fish species, the estimated EDI of copper, zinc, iron, and cadmium are below their respective RfD  of copper, zinc, iron, and cadmium in humans from the consumption of muscle of each of the fish species studied is shown in olylepis  was the tive RfD . In the ed value .Is there a detectable, consistent effect of mining activities in the bioaccumulation of trace metals in fish, or are local oceanographic and geochemical dynamics the key drivers? Does bioaccumulation depend on the type of minerals exploited or the unique characteristics of each fish species? Our observations reveal that there is no clear pattern of differences in trace metal concentrations between STR (copper mining) and LAP (phosphorite mining); instead, the differences appear to be mostly related to the physiology of the sampled fish species and the local biogeochemical dynamics.Tripneustes depressus (30.9 ± 5.5 µg g−1) and Eucidaris thouarsii (38.3 ± 2.3 µg g−1) had cadmium concentrations nearly 3-fold higher than peak levels in macroalgae [−1 in the sea urchin Eucidaris thouarsii attributed to the consumption of crustose coralline (Lithophyllum) or articulated coralline (Amphiroa) algae or other organisms such as mollusks, barnacles, filamentous, or turf-forming algae attached to macroalgae or rocks [K. vaigiensis, which is the species at the lowest trophic level of the three fish species studied. Undetectable or very low concentrations of copper (<0.03 µg g−1) in muscle have been previously reported [K. vaigiensis) suggests that those differences might be associated with differences in the geochemical dynamics between localities [Padina durvillaei increase after upwelling events [The presence of cadmium in some localities of the Gulf of California has been attributed to natural land-based sources, most likely supplied by weathering and transport from phosphorite-containing rocks  that maycroalgae . Also, hor rocks ,66. Therreported ,68,69. Creported  and inflreported . The tracalities , probablg events .K. vaigiensis, but not for B. polylepis or S. rectifraenum. Copper content in the three fish species was several orders of magnitude lower than the mean Cu levels in the algae P. durvillaei (74 ± 29 µg g−1 and 82 ± 31 µg g−1) collected in the central and southern sites of STR, respectively [Scorpaena mystes (0.7 ± 0.4 µg g−1) sampled in the same area [−1) [P. durvillaei (63 ± 43 µg g−1) collected from these same sites; this finding has been attributed to the low geochemical mobility and bioavailability of zinc [B. polylepis, especially in specimens from STR. In a study carried out in Bahía Santa Maria on the Pacific coast of Baja California Sur that included the analysis of trace elements in homogenized samples of head, gut, and abdomen of 40 fish species, B. polylepis showed the highest zinc content (2.65 µg g−1) [−1 have been reported for this species [Lutjanus argentiventris and Haemulopsis leuciscus have recorded lower zinc  and copper  contents relative to herbivorous species [K. vaigiensis, S. rectifraenum, and B. polylepis from LAP than in other organisms inhabiting the same area, and these three species yielded copper levels within the range reported for these same species in other studies   are not  of zinc . Zinc wa µg g−1) . Zinc le species . In cont studies . All the15N in coastal ecosystems [15N was significantly related to zinc content in the three fish species; thus, nitrogen levels in these species are likely influenced by the presence of nitrogen-enriched discharges that may include zinc particulate matter [15N in fish muscle is strongly related to the species [K. vaigiensis and S. rectifraenum, the relationship between δ15N and zinc is probably more closely associated with the species than with the environment [B. polylepis showed significant differences in zinc levels related to the season of the year and locality. In contrast, the relationship between iron and δ13C suggests that iron may have a benthic origin [Anthropogenic discharges of nitrogen compounds have been associated with significant increases of δe matter . This mae matter . During e matter . On the  species , similarironment . Only B.c origin , influenc origin , rather S. rectifraenum and particularly B. polylepis, would have higher metal levels than species described as herbivores or omnivores with a preference for plants, like K. vaigiensis. However, this expectation was not met in the present study, nor has it been confirmed in other similar studies [K. vaigiensis, an herbivore, had similar cadmium, lead, and copper concentrations than B. polylepis, a species classified either as a carnivore or an omnivore [B. polylepis than in K. vaigiensis, which is contrary to the findings reported in other trophic webs where carnivorous fish have shown lower zinc contents [S. rectifraenum [K. vaigiensis, whereas copper and zinc concentrations were significantly higher in K. vaigiensis. The amount of a given metal accumulated in an aquatic organism depends on the particular element and the species involved and may vary across species according to the particular ecological niche, swimming behavior, and metabolic activity of each [The feeding habits of a species can influence the accumulation of trace elements in its tissues . Therefo studies ,76. K. vomnivore ,26. Zinccontents . The omnifraenum  showed s of each . Most of of each . These pBalistes polylepis specimens showed a higher cadmium content (2.57 µg g−1 and 2.94 µg g−1) than their predators: striped marlin Kajikia audax (0.63 ± 0.70 µg g−1) and Makaira nigricans (0.29 ± 0.18 µg g−1), all of them captured at the southern tip of the Gulf of California [However, other non-essential elements such as cadmium compete for the functional groups in these proteins. By displacing zinc from these proteins, cadmium becomes non-bioavailable; thus, metallothioneins also act as detoxifiers by decreasing the bioavailability of non-essential elements, thereby reducing their toxicity . Accordilifornia .13C and iron content in organisms revealed by the MRL. Therefore, iron content is likely associated with benthic feeding habits, so that food may also involve the incidental intake of organic and inorganic matter from the substrate and water, which may be influenced by the composition of sediments and rocks [K. vaigiensis and copper in S. rectifraenum decrease in the rainy season, especially in STR. This pattern is likely associated with the content of these elements in their diet , as the species composition and abundance of macroalgae change throughout the year [Iron is an essential element, and thus no maximum permissible levels have been set in regulations. This element was found within the range of concentrations reported for these same species in other studies, which is consistent with the direct relationship between the carbon isotope nd rocks . The conthe year , as wellthe year .Atherina hepsetus specimens and copper, lead, and iron contents. This inverse relationship between body size and metal content was observed in S. rectifraenum in the present study, as larger specimens of this species lower copper level. However, for K. vaigiensis and S. rectifraenum, a direct relationship was found between both length and lead and iron content, contrasting with the inverse relationships reported by [In addition to the biogeochemical characteristics of a site and the feeding habits of a species, the level of metals bioaccumulated in an organism may also depend on its body size. Canli and Atli  observedorted by  for otheSparus auratus showed no relationship between metal content and body size, which coincides with our findings for B. polylepis in the present study. Canli and Atli [Mugil cephalus, which was not recorded in any of the species included in the present study. Although copper content was inversely related to body size in S. rectifraenum, no significant differences were observed between sites for this species. This result indicates that copper content in S. rectifraenum may be more closely related to its metabolism and indicates the ability of this species to regulate this element even in copper-rich marine environments as STR. Other studies conducted in polluted sites where copper levels were expected to be high in sciaenidae fish [Canli and Atli  reportedand Atli  also obsdae fish ,81, showdae fish ,81. The dae fish ,79,82.K. vaigiensis and B. polylepis are species of commercial importance for human consumption, while S. rectifraenum is an aquarium fish [−1 wet weight  and highly bioavailable essential micronutrients—vitamins (B and D) and minerals such as calcium, phosphorus, iodine, zinc, iron, and selenium . Howeverium fish . Cadmiumy weight ). Lead ly weight ) for Pb y weight . The ingy weight . In the lifornia ,71,76 isMugil cephalus from the eastern coast of the Gulf of California [K. vaigiensis and B. polylepis contributes nutritionally an important amount of copper, zinc, and iron that do not represent a risk for human health. Except for few cases, such as lifornia , the cadThe mining activities examined in this study are not directly reflected on the metal contents in fish, instead of geochemical dynamics and fish physiology. Fish species in the same trophic level and inhabiting the same sites showed significant differences in the levels of the same elements; on the other hand, no differences in metal content were observed between species from different trophic levels. Each element accumulates differently in individual species.According to this report, there is currently no risk of heavy metals toxicity derived from the human consumption of fish of the species and sites studied, except for lead in LAP. Further research is needed to assess the bioavailability of trace elements in different marine species to understand the mechanisms underlying bioaccumulation and assess the ecotoxicological risk of a site. Meanwhile, it is important to maintain permanent seafood safety monitoring programs in place. Also, as a rule of thumb, the consumption of a diverse diet  may reduce the risk of food safety problems.


Vitis vinifera L.) using spectrometric and chromatographic techniques. Ten vine varieties  obtained from the years 2015, 2016, and 2017 were selected for the study. The antioxidant activity was determined spectrophotometrically using four fundamentally different methods; the content of total polyphenolic compounds was determined using the Folin–Ciocalteu method. In 2015, the content of 14 antioxidants  were studied. The results of the study show the high content of antioxidant components in grape seeds and the differences in content in individual varieties and in individual years.The paper deals with the study of antioxidant properties of extracts from vine seeds ( Wine is an important source of phenolic substances, but there are many studies focused on the content of polyphenols not only in wine or grapevine, but also in byproducts of winemaking industry, such as grape seeds or grape stems ,2,3.Vine seeds contain a significant source of polyphenolic substances 20–55%), which are important mainly in the production of red wines . Phenoli5%, whichFlavonoids form a substantial part of the phenolic compounds present in grapes and include several classes ,6. They The solid waste generated by the wine industry is about 30% total weight and consists mainly of grape marc . Large amounts of valuable compounds, such as dietary fiber, seed oil, anthocyanins, and phenolic compounds, remain inside the marc . PhenoliThe usage of this plant material as a source of bioactive polyphenolic compounds promises an opportunity to obtain added-value products from winemaking byproducts and merits the attention of the food/pharma industries to provide valuable quality bioactive extracts and innovative added-value co-products . In receThe search of new added-value applications to these byproducts requires the performance of an additional and accurate assessment of its polyphenol profile and the antioxidant capacity of the extracts. This will allow their employment by the food/pharma industry .Antioxidants represent a large and diverse group of substances which work on different principles; therefore, the methodology and possibilities of their determination are often complex. For this reason, the aim of this study was to determine in more detail the polyphenol and antioxidant profile of selected grapevine varieties using a chromatographic method and different spectrometric methods. The novelty of this study is the comparison of interspecific varieties with common grapevine varieties within three years.The graphs below show the results of antioxidant activity and content of polyphenolic compounds during the years 2015–2017 in ten vine varieties. These are the average values from the three measurements.The average value of antioxidant activity determined using the DPPH method was 9432 µg/g GAE in 2015; 10,828 µg/g GAE in 2016, and 11,624 µg/g GAE in 2017. All results are presented in The average value of antioxidant activity determined using the FRAP method was 12,217 µg/g GAE in 2015; 13,724 µg/g GAE in 2016; and 14,807 µg/g GAE in 2017. All results are presented in The average value of antioxidant activity determined using the ABTS method was 3695 µg/g GAE in 2015; 4702 µg/g GAE in 2016; and 6518 µg/g GAE in 2017. All results are presented in 1 GAE in 2017. All results are presented in −1 GAE in 2015; 1673 µg/g GAE in 2016; and 2479 µg/g GAE in 2017). The highest values of antioxidant activity were reached in 2017 and the lowest in 2015. Seeds from blue varieties showed higher values than seeds from white varieties.The average value of antioxidant activity determined using the chlorophyllin free radical method was 2272 µg/g GAE in 2015; 2366 µg/g GAE in 2016; and 3084 µg/g The average value of the content of total polyphenolic compounds was 7831 µg/g GAE in 2015; 8796 µg/g GAE in 2016; and 9782 µg/g GAE in 2017. All results are presented in r = 1 means a perfect positive correlation and the value r = −1 means a perfect negative correlation. Results are shown in The Pearson correlation coefficient was used to measure the strength of a linear association between two methods for each year. The value The values of correlation coefficients from the years 2015–2017 are in the range from 0.79 to 0.97, which shows a positive correlation between the individual methods. The highest values of the Pearson correlation coefficient were 0.97 between the FRAP and DPPH methods in 2015 and between CHFR and ABTS in 2016. In 2017, the highest value of the coefficient 0.92 was between the ABTS and FRAP methods and between TP and CHFR. The lowest value of the coefficient 0.79 was between the FRAP and DPPH methods in 2016 and between the TP and FRAP methods.Using HPLC with UV-VIS detection, the content of selected antioxidant components was determined in the 2015 samples. Attention was focused on the content of gallic acid, caffeic acid, coumaric acid, coutaric acid, ferulic acid, fertaric acid, quercetin, trans-piceid, trans-piceatannol, rutin, quercetin-3-β-D-glucoside, quercitrin, myricetin, catechin, and epicatechin. The graphs below  show thep < 0.01, caffeic acid: p < 0.01, coumaric acid: p < 0.01, coutaric acid: p < 0.01, ferulic acid: p < 0.01, fertaric acid: p < 0.01, trans-piceid: p < 0.01, trans-piceatannol: p < 0.01, rutin: p < 0.01, quercetin-3-β-D-glucoside: p < 0.05, quercitrin: p < 0.01, myricetin: p < 0.05, catechin: p < 0.01, epicatechin: p < 0.01.Significance levels within individual years of gallic acid: The most represented compound of the monitored antioxidants was gallic acid; its average content in all monitored varieties was 225.4 µg/g. It was present at the highest levels in the Cerason variety (298 µg/g) and was least represented in the Palava variety (160 µg/g ). The second most common compound was epicatechin, with an average content of 138.2 µg/g. The Kofranka variety showed the highest average content of epicatechin: 193 µg/g. The lowest epicatechin content, 107 µg/g, was determined in the Palava variety. Average concentrations of all phenolic compound are presented in The least represented compound was rutin, which had an average content of 0.326 µg/g. The highest content was recorded in the Kofranka variety (0.51 µg/g) and the lowest in the Palava variety (0.22 µg/g). The second least represented compound was coumaric acid; its average value across all monitored varieties was 1.76 µg/g. Its highest content was recorded in the Blaufrankisch variety (2.42 µg/g) and the lowest in the Erilon variety (1.25 µg/g). From these results, it is again clear that seeds from blue varieties showed higher values of antioxidant components than seeds from white varieties.For ease of comparison with other studies, the results determined by HPLC with UV-VIS for the Cerason variety were used; the values of antioxidant components from 2015 to 2017 were averaged and are summarized in In a 2016 study by Farhadi et al. , polypheAn experiment by Rockenbach et al.  used HPLVitis vinifera L. grown in Greece. They were tested for their content of characteristic polyphenols. The compounds found were major low molecular weight components, including gallic acid, catechin, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, and procyanidins B1 and B2. The total content of polyphenolic compounds ranged from 55.1 to 964 µg/g of seeds, with an average of 380 µg/g. The most abundant polyphenol was catechin, which accounted for 49.8% of the total content, followed by epicatechin (26.0%), epicatechin gallate (9.3%), procyanidin B1 (5.8%), and procyanidin B2 (5.1%). Epigallocatechin and galactic acid contents were minor [1 (Welschriesling 2015) to 11,272 µg/g Cerason 2017).Research by Guendez et al.  examinedre minor . The aveVitis vinifera L.). The study examined the phenolic antioxidants of extracts prepared from the seeds, husks, and tufts of Pusa Navarang and Merlot varieties. HPLC was used to determine the catechin  and epicatechin (9299 µg/g) content of the Pusa Navarang seeds, more higher than in our study, where the catechin concentration was only 181.3 µg/g and epicatechin concentration was 138.2 µg/g of dry matter.Doshi et al.  analyzedIn a study by Hassanpour et al. , the conYilmaz et al.  focused Garcia-Jares et al.  used LC/1 to 11,272 µg/g GAE; this variety also reached the highest AOX values, measured by different methods. A study by Guendez et al. [r2 = 0.6499, p < 0.01). However, correlations between the individual compounds showed that procyanidin B1 may be one of the most important scavengers of radicals in grape extracts  despite its small share in the total polyphenol content [The results of our study show that the highest concentration of polyphenols in the Cerason variety ranged from 8799 µg/g z et al.  assessed content . A study by Rockenbach et al.  determinIn a study by Doshi et al. , the antIn a 2017 study, Hassanpour et al.  examinedIn a study by Özcan et al. , phenoliVitis vinifera L. and Vitis labrusca from Brazilian viticulture were studied in Pinot Noir, Isabella, and Cabernet Sauvignon. The seeds had a higher concentration of phenolic compounds ) than the concentration in the husks (660 to 1839 µg.g−1 (CE)). The highest values of antioxidant activity, determined using the DPPH radical and the FRAP method, were found in the seeds of the Pinot Noir variety  and in the peel extracts of the Isabella variety (DPPH: 3640 μmol TE/100 g and FRAP: 4362 μmol Fe2/100 g). In an extensive study by Rockenbach et al. , polypheJayaprakasha et al.  evaluateAntioxidant activity (determined using the DPPH method) and the content of total polyphenols were analyzed in the extracts from the seeds of selected white varieties . The values of antioxidant activity ranged from 31.0 to 41.3 mmol Trolox/g dry matter. The content of total polyphenolic compounds values corresponded to the study by Garcia-Jares et al.  and rangUsing three different methods , the antioxidant activity and the content of polyphenolic compounds in Çalkarası grape seeds obtained from a Turkish winery were determined. Antioxidant activity was expressed in µmol Trolox/g dry matter. The value of antioxidant activity was 19.96 µmol TE/g for the FRAP method, 19.30 µmol TE/g for the DPPH method, and 16.45 µmol TE/g for the ABTS method. The content of total polyphenolic compounds was 27.92 mg GAE/g dry matter .In our experiments, the average value of antioxidant activity of grapevine seeds determined by the DPPH method reached 10,628 µg/g GAE. It was 13,583 µg/g GAE when measured by the FRAP method; 4972 µg/g GAE with the ABTS method and 2574 µg/g GAE with the FR method. The average value of the content of total polyphenolic compounds was 8803 µg/g GAE.The values found in the studies, which are focused on the determination of antioxidant components, differ considerably from each other. The reason for this is that each of the studies used a different methodology of extraction procedures. Thus, the varieties were macerated at different times, with different solvents , with a different seed-to-supernatant dilution ratio, and with different sample drying procedures, etc. Of course, the variations are also due to different collection years, varieties, seed ripening times, and collection areas, etc. In terms of conversions, there are differences related to expression in other units (liters versus kilograms) as well as differences between dried and fresh samples. Last but not least, there were variances due to the use of different analytical methods. Due to such different testing methods, any comparison between studies is not relevant. However, what can be established from the studies is that, to a large extent, catechin and epicatechin are abundant compounds. This information corresponds to our study, in which epicatechin was the most common component.In addition, the methods for determining antioxidant activity and standards were different; specifically, the antioxidant activity expressed in (1) equivalents of different standards , (2) relative percentage, and (3) time loss of absorbance. In addition, the expression of antioxidant activity values changes over time as the radicals for determining antioxidant activity are highly reactive .The differences between the individual studies are surprising. At the end of the discussion, we can say that our results were in the range of values given by other studies. None of our results showed extreme deviations from the results of other studies.Vitis vinifera L.)—was obtained from varieties grown around the Institute of Viticulture and Enology (Mendel University), Czech Republic. These varieties were Cerason, Laurot, Kofranka, Gewürztraminer, Hibernal, Blaufrankisch, Zweigeltrebe, Erilon, Palava, and Welschriesling. This biological material was obtained from the years 2015, 2016, and 2017.Experimental material—vine seeds .A separation machine was used to separate the w/w). After this time, the extract was transferred into 2 mL microtubes  and centrifuged . The supernatant of centrifuged sample was separated into the another microtube, and thus prepared was taken for spectrometric and chromatographic analysis.The ground seeds were extracted in 75% ethanol for 120 h, in a ratio of 1 part ground seeds to 10 parts ethanol (3), acetate buffer, sodium acetate, Folin–Ciocalteu reagent, and sodium carbonate (Na2CO3) decahydrate. Chemicals from Sigma-Aldrich (Germany) were used for the experiments.The following chemicals were used: ethanol 96%, deionized water, stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), radical cation 2,2´-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), methanol 96%, acetic acid 0.2%, liquid nitrogen, 2,4,6-tripyridyl-s-triazine (TPTZ), hydrochloric acid (HCl), ferric chloride . All samples were analyzed in triplicate, the resulting value corresponding to the average of these measurements.A solution for the determination of antioxidant activity by ABTS assay was prepared by mixing two solutions—solution 1: 7 mM solution of ABTS prepared by weighing m = 9.60 mg per 5 mL of distilled water; and solution 2: 4.95 mM solution of potassium persulfate prepared by weighing m = 1.67 mg per 5 mL of distilled water. The resulting solution was diluted 1:10 with distilled water and left in the dark and cold for 12 h.−1), and the results were expressed as gallic acid equivalents.150 µL of reagent R1 (7 mM ABTS and 4.95 mM potassium persulfate) was pipetted into plastic cuvettes, followed by the addition of 3 µL of sample and measured in spectrophotometer (λ = 660 nm) for 12 min. According to the calibration curve, the absorbance was converted to the equivalent content of gallic acid; the antioxidant activity was calculated from the calibration curve using gallic acid as a standard  were pipetted into plastic cuvettes, followed by the addition of 15 µL of sample to be measured. The DPPH test is based on the ability of the stable free radical 2,2-diphenyl-1-picrylhydrazyl to react with hydrogen donors. DPPH shows strong absorption in the ultraviolet-visible spectroscopy (UV-VIS) spectrum. Absorbance was measured for 12 minutes at λ = 505 nm. According to the calibration curve, the absorbance was converted to an equivalent gallic acid content.3 solution: 20 mM FeCl3 (m = 135.13 mg) dissolved in 25 mL of distilled water; and (3) acetate buffer solution: 0.02 M acetate buffer pH 3.6 . The three solutions were mixed in the ratio 1:1:10 (TPTZ:FeCl3:acetate buffer).Three solutions were used to determine the antioxidant activity using the ferric reducing antioxidant power (FRAP) method—(1) TPTZ solution: 10 mM TPTZ (m = 78.02 mg) dissolved in 25 mL of 40 mM HCl; (2) FeCl-1). Results were expressed as gallic acid equivalent.150 µL of reagent were pipetted into plastic cuvettes and then 3 µL of sample were added. Absorbance was measured for 12 minutes at λ = 605 nm. Antioxidant activity was calculated from the calibration curve using gallic acid as a standard  were pipetted into plastic cuvettes and then 8 µL of sample were added. This method uses the ability of chlorophyllin  to accept and donate electrons while stably changing the absorption maximum. This process is conditioned by the alkaline environment and the addition of a catalyst. Absorbance was measured for 12 min at λ = 450 nm. The last measurement values were used for the calculation. Results were expressed as gallic acid equivalent.The Folin–Ciocalteu method was used to determine total polyphenolic compounds. All samples were analyzed in triplicate, and the resulting value was obtained as the average of these measurements.2CO3 decahydrate solution were added. The reaction mixture was shaken and incubated at 22°C for 120 minutes. After this time, the absorbance  was measured at λ = 750 nm against a blank. Results were expressed as gallic acid equivalent.A 40 µL sample was pipetted into a cuvette (3 mL) and diluted with 1960 µL of distilled water. Subsequently, 50 µL of Folin–Ciocalteu reagent was added to the cuvette. The mixture was shaken thoroughly and, after 3 minutes, 300 µL of 20% Na4) and then used for HPLC analysis.Determination of selected antioxidant components using high-performance liquid chromatography (HPLC) with UV/VIS was performed by direct sample injection method. The prepared samples were diluted 10x with 100 mM perchloric acid  and Statistica 10 statistical software (Copyright © StatSoft). Analysis of variance (ANOVA) was used to verify the differences between the variants of experiment, specifically using the nonparametric Kruskal–Wallis test. Pearson’s correlation coefficient was used to determine the correlations between the variants.Grape seeds are rich in antioxidants. Evidence for this claim includes many scientific studies pointing to their antioxidant potential.The average value of antioxidant activity of grapevine seeds determined by the DPPH method was 10,628 µg/g GAE. It was 13,583 µg/g GAE when measured by the FRAP method; 4972 µg/g GAE by the ABTS method; and 2574 µg/g GAE by the FR method. The average value of the content of total polyphenolic compounds was 8803 µg/g GAE. The highest values of antioxidant activity were achieved by the Cerason variety; the lowest values were measured in the Riesling variety. The highest values of antioxidant activity and content of total polyphenolic compounds were reached in 2017; the lowest values were reached in 2015. Seeds from blue varieties showed higher values than seeds from white varieties.The content of 14 phenolic compounds  was determined by HPLC/UV-VIS. The most represented compound was gallic acid, with an average content of 225.4 µg/g in all monitored varieties. It was most present in the Cerason variety (298 µg/g) and least present in the Palava variety (160 µg/g). In contrast, the least represented compound was trans-piceatannol, which had an average content of 0.326 µg/g. It was most present in the Kofranka variety (0.51 µg/g); the lowest content was recorded in the Palava variety (0.22 µg/g).The data obtained in this study are an important indicator of the potential nutraceutical and economic utility of this waste material in the future. Among other things, these results provide useful information for real potential use in the different branches of the food industry.


Botrytis cinerea were investigated for the first time. AJ was rich in lactic acid (4.46 g/L), acetic acid (1.52 g/L), Lactobacillus (72.45%) and Acetobacter (15.23%), which was a microbial ecosystem consisting of acid-based substances (AS) and beneficial microorganisms (BM). The results of the antifungal assays suggested that B. cinerea was effectively inhibited by AJ, with the half-maximal inhibitory concentration (IC50) of 9.24%. AJ showed the strongest and most-lasting inhibitory effect compared to cell-free supernatant and microbial solution of AJ, indicating that AS and BM and their synergistic effect contributed to the antifungal activity of AJ. Two-step inhibition’ is an antifungal mode of AJ. Firstly, AS not only inhibited the pathogen directly but also provided a dominant niche for BM of AJ. Then, BM in AJ, especially Acetobacter, proliferated and metabolized acetic acid continuously. Thus, AJ achieved high-efficiency and long-acting inhibition. AJ is a promising biological agent considering its features of an eco-friendly, low-cost and easy-to-operate biological agent in rural areas.Synthetic fungicides are eco-unfriendly to agriculture and the environment. Agricultural Jiaosu (AJ), which originates from organic wastes, has the potential to be a substitute for synthetic fungicides. In this study, the characteristics of AJ and its antifungal activity against  B. cinerea was studied for the first time.The antifungal activity of AJ against AJ was an ecosystem and achieved an effective and lasting antifungal activity.AS, MB and their synergistic effect contributed to higher inhibitory rate.Botrytis cinerea is a common and extremely harmful soil-borne pathogen in agricultural production, which can attack the leaves, flowers and fruits of crops  attracts widespread attention and large-scale application as a biological agent in agricultural practice in China. AJ has many advantages, such as sustainability, stabilisation, handleability, low-cost and use of simple equipment. This novel biotechnology can break the bottleneck of fungicides. In general, the AJ is diluted 1:200-500 times and applied to farmland once every 3–7 days. The dosage is about 2–3 L/mB. cinerea were investigated for the first time. The research was divided into five aspects: (1) physicochemical properties and microbial characteristics of AJ, (2) changes in the antifungal activity of AJ with dosage, (3) dynamic changes in the inhibitory effect of AJ with cultivation time, (4) main inhibitory factors of AJ and (5) antifungal mode by the synergistic effect between AS and BM of AJ.There is no relevant literature on the antifungal potential of AJ on phytopathogens. In this study, the antifungal activity and mechanism of AJ against Brown sugar (400 g) and jujube wastes (1200 g) were mixed with 4 L distilled water in air-tight containers for anaerobic fermentation at 30 °C for 3 months and stirred once a week. A large number of jujube wastes were produced when farmers harvest at an organic orchards in Jishan County, Shanxi Province, China. Then these wastes were collected to fermentation. The composition of jujube wastes was described by Guo . AJ was Botrytis cinerea (ACCC 36028) was purchased from the Agricultural Culture Collection of China . The pathogen was cultured on potato dextrose agar (PDA) medium at 25 °C before the experiments.The pH values of AJ were determined by a micro-pH metre . LA and AA were measured using high-performance liquid chromatography with a diode array detector . The samples were mixed with acetonitrile at a 1:1 ratio for 10 min and then filtered through a 0.22 μm filter. The details of the chromatographic procedure and conditions were described by Cai et al. , b.3, 0.015 g bromocresol purple, 15 g agar, pH 6.8 ± 0.2). High-throughput sequencing was performed on an MiSeq platform . The V3–V4 hypervariable region of the bacteria was amplified using the following universal primer set: 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The community structure was analysed by Silva database at the phylum and gene levels , which is indicative of antifungal activity, and Dck and Di represent the colony diameter of the pathogen of CK and treatments (mm), respectively.Blank control (CK) was prepared without the addition of AJ, whereas 5, 15, 25 and 50 mL AJ were added into 95, 85, 75 and 50 mL autoclaved PDA medium to create the dosage of 5%, 15%, 25% and 50%, respectively, and then shaken gently and poured into the dish. After agar gelling, the mycelial plugs (circle disks) of 50) refers to the concentration required for a drug to inhibit cell growth and virus replication by 50%, which is one of the important parameters commonly used in toxicology. Taking the 72 h colony diameter as the y-value and the dosage of AJ as the x-value, quadratic regression analysis was performed to obtain the fitting curve and polynomial equation. Half of the colony diameter of CK was taken as the y-value and entered it into the formula to get the corresponding x-value, which is the IC50 of AJ against B. cinerea  and T3, the precipitate of T2 was washed with sterile water and centrifuged at 12,000 rpm for 10 min, and the supernatant was removed. This operation was repeated three times. Finally, the pellet was mixed with 3 mL sterile water. The same amount of sterile water was used as control (CK). Under the premise of 5% dosage, 19 mL sterile PDA medium was poured into each culture dish. After the medium has cooled and solidified, 1 mL AJ with different treatments was spread onto the surface of fresh PDA medium until drying and then the 5 mm plug was placed at the centre . PCR consisted of 10 µL using SYBR® Green PCR SuperMix-UDG Kit , DNA template (1.0 µL) and double-distilled water to a total volume of 20 µL. The reported units were copies/mL. The PCR primers and the amplification protocol are given in Additional file The gene concentrations of P < 0.05 using Statistix 8.1.1 software . Excel 2016  was used to compute the mean ± standard deviation (SD) and graphical presentation.Statistical analyses were evaluated based on completely randomised variance at To evaluate the antifungal activity of AJ, its characteristics were determined. The main parameters are shown in Table Firmicutes and Proteobacteria dominated the entire community, with relative abundances of 72.80% and 15.10%, respectively. At the genus level, Lactobacillus (72.45%) and Acetobacter (15.23%) occupied the dominant niche.High-throughput analyses were used to detect the bacterial diversity of AJ. Figure Lactobacillus and Acetobacter). It was speculated that AJ had antifungal potential based on its characteristics.AJ was an acidic ecosystem consisting of AS  and BM , which indicated that AJ had a negative impact on the growth of B. cinerea. The pathogen was almost completely suppressed when the dosage of AJ was more than 15% of the whole medium. Also, the inhibitory rate was positively correlated with dosage and cultivation time. At 48 h, the value of this parameter at dosages of 5%, 15%, 25% and 50% was 10.95%, 58.79%, 74.89% and 73.17%, respectively, but there was no significant difference between the latter two treatments (P > 0.05). Interestingly, when the time was prolonged to 72 h, the inhibitory rate climbed to 42.36%, 74.54%, 84.27% and 85.98%, respectively. The inhibition rates of the latter two treatments (25% and 50%) were significantly higher than those of the former two treatments , but there was no significant difference between them (P > 0.05). At this moment, the antifungal activity was 2.87, 0.27, 0.13 and 0.18 times higher than those at 48 h.Dosage is a key factor that affects the antifungal activity in toxicological studies. The effect of the dosage of AJ on its inhibitory effect against ted Fig. . CK show50 was calculated according to the above experimental data using Excel 2016. With the colony diameter on the y-axis and the dosage on the x-axis, the fitting formula was obtained as follows: Y = 0.0382x2 − 2.7089x + 48.021, R2 = 0.9519. Half of the colony diameter of CK at 72 h (26.245 mm) was introduced into the formula as the y-value, and then IC50 was taken as the corresponding x-value. The IC50 of AJ against B. cinerea was 9.24%  but significantly larger than that of T1 and T2 (P < 0.05). At 48 h, the colony diameter of T1 was significantly smaller than that of the other three treatments (P < 0.05), but there was no significant difference between T3 and CK (P > 0.05). The colony diameter at 72 h of CK, T1, T2 and T3 were 49.27 ± 0.24, 35.78 ± 0.49, 40.29 ± 0.56 and 47.30 ± 0.41 mm, respectively. The difference in treatments was significant (P < 0.05). Visually, it was obvious that T1 showed the smallest colony diameter as compared with the other.To determine the main inhibitory factors, AJ was divided into two fractions and the antifungal assay was performed Table . T2 and P < 0.05). The inhibitory rate of T2 was between T1 and T3 and the value increased at the beginning and then decreased (P < 0.05). The peak appeared at 48 h, with 22.11% . The concentration of LA showed a decline with cultivation time from 0.52 to 0.31 g/L. As for AA, there was a trend of increase first and then gradually decreased to 0.29 g/L. Finally, the concentration of AA was only 15.18% of T1. The main component of cell-free supernatant was AS, so the pH value was only 5.09 at the beginning. Subsequently, the value dropped temporarily and then increased. The lowest pH value was observed at 48 h (4.21). Microbial solution consisted of BM of AJ, such as Lactobacillus and Acetobacter. Compared to T1, LA and AA in T3 increased slightly. The concentration of AA was only 0.36 g/L at 72 h, which was 18.85% that of T1. The pH value dropped from 6.66 to 5.08, which was equivalent to the initial value of T1.To further reveal the antifungal mechanism of AJ, the concentrations of LA and AA and the pH were dynamically measured during the inhibition process Fig. . OrganicIn summary, the pH value and the concentration of AA displayed an apparent fluctuation during the inhibition process. AJ showed the lowest pH value and the strongest acid production capacity, especially AA.B. cinerea and the main strains of AJ (Lactobacillus and Acetobacter) in different periods were quantitated by qPCR  from 1.68E+08 copies/mL at 0 h. Cell-free supernatant (T2) showed an inhibitory effect at the early stage. The gene concentration of the pathogen decreased to 2.89E+03 copies/mL within 48 h and then recovered to 3.16E + 04 copies/mL. Microbial solution (T3) had a weak influence on the growth of B. cinerea, with the highest gene concentration of 3.27E + 04 copies/mL at 72 h.PCR Fig. . B. cineAcetobacter rapidly proliferated and occupied the dominant niche, thus inhibiting the growth of the pathogen by metabolising a large amount of AA. This conclusion was consistent with the results of Fig. In summary, AJ could inhibit the pathogen effectively and persistently. B. cinerea.In Fig. When the inhibitory rates of T2 and T3 were added, the sum was less than the inhibitory rate of T1. This difference was considered as a synergistic effect of AS and BM. Figure In summary, AS were the main inhibitory factor at the early stage. BM gradually played a vital role at the late stage, which led to an increased proportion of the synergistic.B. cinerea were investigated. The following points were discussed based on the comprehensive analysis of the results.AJ not only has a strong antifungal activity but also provides a high-value-added treatment strategy of organic wastes, which achieves the dual role of resource utilisation and safety production. This product has the advantages of green, low-cost and easy operation and has been widely applied in rural China in recent years  and BM (Lactobacillus and Acetobacter). When AS and BM of AJ were separated, the two fractions still showed an inhibitory effect. These results suggested that AS and BM are the key inhibitory factors. Acetobacter was the antagonistic microorganism that played a major role during the late stage of the inhibition process . When AJ was added to the medium dominated by phytopathogens, AS inhibited the growth of the pathogen directly at the early stage (48 h) and provided a dominant niche for BM in AJ simultaneously. With the consumption or transformation of AS, the inhibitory effect gradually weakened. However, BM in AJ, especially Acetobacter, rapidly propagated and became the dominant bacteria under this condition. Then, a large amount of AA was metabolised continuously, which is a recognized as a good antibacterial substance. Therefore, AJ achieved high-efficiency and long-acting antifungal activity through this synergistic effect between AS and BM.Based on the above analysis, an antifungal mode called ‘two-step inhibition’ of AJ was proposed, as shown in Fig. Additional file 1.:Table S1. Summary of qPCR primers. Table S2. Cycling protocols of qPCR. Fig. S1. IC50 of AJ against B. cinerea. Broken, horizontal and vertical lines represent the fitting line, half of the colony diameter of CK and the corresponding dosages, respectively. Black and red circles represent the observed value and the IC50, respectively. Fig. S2. Increase part of the inhibitory rate of AJ. Blue and green bar represents the inhibitory rate of AS(T2) and BM (T3) respectively. And the red bar represents the higher part of inhibitory rate of AJ compared to the AS and BM.