MCFAs/mg CODTotal. Analyses of the microbial communities in the bioreactors suggest that lactic acid bacteria, Megasphaera, and Caproiciproducens were involved in MCFA and C4 production during DCDA hydrolysate metabolism.Dairy manure (DM) is an abundant agricultural residue that is largely composed of lignocellulosic biomass. The aim of this study was to investigate if carbon derived from DM fibers can be recovered as medium-chain fatty acids (MCFAs), which are mixed culture fermentation products of economic interest. DM fibers were subjected to combinations of physical, enzymatic, chemical, and thermochemical pretreatments to evaluate the possibility of producing carbohydrate-rich hydrolysates suitable for microbial fermentation by mixed cultures. Among the pretreatments tested, decrystalization dilute acid pretreatment (DCDA) produced the highest concentrations of glucose and xylose, and was selected for further experiments. Bioreactors fed DCDA hydrolysate were operated. Acetic acid and butyric acid comprised the majority of end products during operation of the bioreactors. MCFAs were transiently produced at a maximum concentration of 0.17 mg COD Raw dairy manure (DM) is an abundant organic waste stream produced at rates of hundreds of megatonnes per year . A commovia a cyclic pathway known as reverse beta-oxidation , which are five-to eight-carbon monocarboxylic acids that can supplement livestock feed , be usedxidation . In thisBatches of DM were obtained from a single dairy cow, immediately after excretion, at the Dairy Cattle Center housed at the University of Wisconsin-Madison. The manure was stored at 4°C until physical pretreatment. Two different schemes were used for the pretreatment of the lignocellulosic fraction of DM, a part of the solid fraction hereafter referred to as DM fibers. The first scheme (Scheme 1) involved physical, chemical, and enzymatic steps. The second scheme (Scheme 2) involved physical and thermochemical steps and did not require enzymatic hydrolysis . The twoCollected DM was dried at 101°C for 24 h. Dried DM was then milled to pass through a 1-mesh screen using a laboratory hammer mill . Five different chemical pretreatments were tested in 400-ml shaker-flasks using dried, milled DM at a solids loading of 10% (100 g/L) in working volumes of 150 ml of chemical liquor. The pretreatments were: 1) sulfite pretreatment to overcome recalcitrant lignocellulose (SPORL); 2) dilute acid (DA); 3) alkaline; 4) copper-alkaline-hydrogen peroxide (Cu-AHP); and 5) copper-alkaline-hydrogen peroxide with alkaline pre-extraction and batch addition of hydrogen peroxide (CAP); where DA and SPORL followed In a Certomat® BS-1 incubation shaking cabinet , enzymatic hydrolysis tests of dried, milled, chemically pretreated, re-dried, re-milled DM were carried out in 500-ml Erlenmeyer flasks at working volumes of 100 ml of buffer  and 10% solids loading. A temperature of 50°C and shaking rate of 180 revolutions per minute (RPM) were maintained during the experiments. An enzyme loading of 50 filter-paper-units/g cellulose was applied using commercially available Cellic® CTec 2, an enzyme complex cocktail primarily known for cellulase that also demonstrates xylanase, cellobiohydrolase, and β-glucosidase activities , by NovoIn a second pretreatment scheme , a two-pFermentation studies were carried out at mesophilic (35°C) conditions either in batch or flow-through continuously stirred tank reactors. All bioreactors were seeded with sludge from an acid-phase digester at the Nine Springs Wastewater Treatment Plant . Between inoculations, the sludge inoculum was stored at 4°C. A batch bioreactor was ran for 14 days at a working volume of 48 ml and was sealed with a rubber stopper. A long-term flow-through reactor was ran for 42 days. A short-term flow-through reactor was ran for 10 days. The flow-through bioreactors  with a working volume of ∼300 ml were mixed at 150 RPM with a magnetic stir bar, and operated with a residence time of 6 days by pumping out 1.9 ml of fermentation broth and replacing it with hydrolysate every hour. The bioreactors were sealed with a rubber stopper that included ports for sampling , influent flow, effluent flow, and pH control. A pH of 5.50 was maintained by automated addition of 5 M sodium hydroxide (NaOH). All bioreactors were operated at different times.DM fiber samples from Scheme 1 were submitted to the University of Wisconsin Soil and Forage Laboratory  for measurements of neutral detergent fiber , acid detergent fiber , lignin, total starch, and soluble crude protein (sCP) content  where NDSamples from all DCDA hydrolysates and bioreactors were centrifuged for 10 min at 10,000 relative centrifugal force, then filtered using 0.22-μm syringe filters  and used for quantification of lactic, acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids; D-glucose, xylose, ethanol, cellobiose, and sulfate and sodium ions. Samples used to quantify sCOD and soluble carbohydrates were obtained in the same way.2SO4 was used for the mobile phase with a flow rate of 0.50 ml min−1. A YSI 2700 Series Biochemistry Analyzer  was used to quantify L-lactic acid, ethanol, D-glucose, and xylose in aliquots of fermentation broths from all bioreactors using immobilized membranes of L-lactic acid oxidase, alcohol oxidase, glucose oxidase, and pyranose oxidase, respectively.Glucose, xylose, cellobiose, and acetic acid in DCDA hydrolysate and DL-lactic acid in fermentation broths of the two flow-through bioreactors were analyzed with high-performance liquid chromatography (HPLC) and quantified with an Agilent 1260 Infinity refractive index detector  using a 300 × 7.8 mm Bio-Rad Aminex HPX-87H column and a Cation-H guard column . A column temperature of 50°C was used and 0.02 N HThe concentrations of monocarboxylic acids  in fermentation broths were determined using gas chromatography with a flame ionization detector (GC-FID) using a Shimazdu 2010 GC System  equipped with an AOC-20i autosampler. The GC column was a high-performance capillary column with dimensions of 30 m × 0.53 mm × 1.00 μm  for underivatized acids and alcohols . Samplesvia ion chromatography . For sulfate, an IonPac AS11-HC 250 mm × 4 mm analytical column and an IonPac AG11-HC 50 mm × 4 mm guard column  were used in series with a 30 mM NaOH mobile phase at a flow rate of 4 ml min−1, and column temperature of 30°C. For sodium, an IonPac CS12A 250 mm × 4 mm analytical column and an IonPac CG21A 50 mm × 4 mm guard column  were used in series with a 20 mN methanesulfonic acid mobile phase at a flow rate of 4 ml min−1, and column temperature of 30°C.Sulfate and sodium concentrations were quantified https://www.biotech.wisc.edu/) with fusion set primers . Amplicons were sequenced on an Illumina MiSeq sequencer  using pair-end 300 base pair kits at UWBC. Microbial analyses of sequencing data were processed with the Quantitative Insights Into Microbial Ecology 2 (QIIME 2) pipeline (https://www.arb-silva.de/) was used for reference taxonomy . Extracted DNA was quantified using a Qubit 3.0 . The V3 and V4 hypervariable regions of the 16S rRNA gene were amplified by University of Wisconsin-Madison Biotechnology Center  to substrate ratio of 7:1 (volume basis) using DCDA hydrolysate  and C4 concentrations ranging from ∼13,000 to 27,000 mgCOD/L , respectively. On Day 12, the highest concentration of total MCFAs was observed at 7,900 mgCODMCFAs/L  was achieved wherein valeric acid (C5) and caproic acid (C6) concentrations were 5,700 ± 170 and 1,900 ± 760 mgCOD/L, respectively. An octanoic acid (C8) concentration of 700 ± 40 mgCOD/L was reached on Day 6. The lowest yields of both C2 and propionic acid (C3) also occurred on Day 12, which suggests a microbial competition between production of short-chain and medium-chain fatty acids  of the total COD in the bioreactor. Carbohydrate utilization was high, with both glucose and xylose consumption being greater than 93%, and the utilization of other soluble carbohydrates being greater than 50% throughout bioreactor operation (We operated a first DM bioreactor (DMB) for a total of 42 days, which was fed Batch 1 of DCDA hydrolysate . At Day ty acids . Lactic ty acids . Ethanolperation .Because the sampling during the first bioreactor operation was initiated after 6 days of operation, we set up a second reactor (DMB2), which was fed Batch 2 of DCDA hydrolysate , to inveTo determine the composition of the community that is enriched in the DCDA-hydrolysate fed bioreactors, we collected DNA samples for 16S rRNA gene amplicon sequencing. The most abundant ASVs  were affiliated with the phyla Firmicutes, Actinobacteria, Proteobacteria, and Epsilonbacteraeota throughout operation of both bioreactors . The abuLactobacillus, a Bacilli genus, into thirty-six ASVs, many of which were abundant in DMB2 even by Day 1 , which is a heterofermentative species (Enterococcus (ENTE01 and ENTE02), whose cultured representatives are described as homofermenative (Sporolactobacillus inulinis (SPOR01 and SPOR02). Cultured representatives of S. inulinis show that it is a sporeforming, heat-resistant, homofermentative lactic acid bacteria  were assigned to asphaera , a Firmioduction . Howeveroreactor . Anotherasphaera . After Mroducens , a Firmiroducens . Caproicubstrate  and is aubstrate . In bothperation . Given t stricto , ASVs as with C4 . Other stobacter , a genustobacter . Acidiprbacteria , correlaLactobacillus, Sporolactobacillus, Bifidobacterium, Megasphaera, Caproiciproducens, Clostridium sensu stricto 12, and Acetobacter  as a tool to explore the variation of response variables  with explanatory variables by multivariate multiple linear regressions . As expltobacter .Lactobacillus and lactic acid , sulfonation- (SPORL), and alkaline-based  chemical pretreatments prior to enzymatic hydrolysis. Weak acids can increase the exposure of cellulose in lignocellulosic biomass primarily through lignin penetration, pre-hydrolysation of cellulose, and hemicellulose solubilization , all of The DCDA pretreatment (Scheme 2) was found to be advtangeous over chemical-enzymatic pretreatments (Scheme 1) due to its higher biomass conversion rates, which we interpret as the efficiency of DM fiber breakdown, and resultant hydrolysate concentrations of glucose and xylose , which sConceptually, DM handling facilities could integrate Scheme 2 alongside anaerobic digesters (ADs). Raw DM could first undergo separation, with the liquid fraction directed to an AD and the solid fraction directed to Scheme 2. Alternatively, Scheme 2 could be applied post-AD, receiving digester effluent. In both scenarios, the solid fraction that is directed out of Scheme 2 .The economic viability of the DM-to-MCFA pathway would increase with increases in MCFA selectivity and productivity during DCDA hydrolysate fermentation, which mostly produced C2 and C4 rather than MCFAs. Such improvement would involve engineering strategies to select for specific organisms. We referenced the strategy of membrane electrolysis, which selected for hydrogen . Anotherhydrogen . Yet anoinulinus  and for ens spp. , a synthBifidobacterium and Lactobacillus as the main lactic acid producing organisms and Megasphaera as the main lactic acid utilizer and MCFA producer. Later in reactor operation, C4 and C2 were major end products of DCDA hydrolysate metabolism. The statistical analyses supported the hypothesis that under the later operation conditions Lactobacillus and Sporolactobacillus were the main lactic acid producing bacteria and that Clostridium senso stricto 12 and Caproiciproducens were the primary C4 producers, who may have co-utilized lactic acid and C2. After the demonstration that MCFA can be produced from DM fibers presented here, future studies could be directed to identifying areas for optimization and analyzing the economic viability of using DM fibers for MCFA production. Additionally, a more in-depth understanding of the metabolism of the identified microbial community members may aid in further refining their role in MCFA production from DCDA hydrolysates.In this study, we demonstrated a novel bioprocessing pathway in which DM was converted into SCFA and MCFA. A comparison of pretreatment processes for DM indicated that the thermochemical DCDA process yielded the highest combined glucose and xylose concentrations and did not require enzymatic treatment to produce carbohydrate-rich hydrolysates. Mixed fermentations of DCDA hydrolysate involved the near complete utilization of glucose and xylose, with lactic acid emerging as the key intermediate in the transformation of sugars to SCFA and MCFA. Statistical analyses such as GLS and RDA supported a hypothesis that the microbial community network was based on the co-existence of abundant lactic acid producing bacteria and lactic acid utilizing bacteria. In early stages of reactor operation, this co-existence lead to accumulation of MCFA with


Vaccinium virgatum Aiton; Kinisato 35 Gou) leaves have recently attracted increasing attention as a useful material for the prevention of lifestyle diseases. Here, we examined the effects of the hot water extract of blueberry leaves (BLEx) on lipogenesis and uric acid production in 3T3-L1 adipocytes. The results showed that BLEx suppressed lipid accumulation and the mRNA expression of differentiation markers in 3T3-L1 adipocytes. A fractionation study showed that the highly polymerized proanthocyanidin-rich fraction was responsible for this effect. Upon maturation to adipocytes, 3T3-L1 cells produced uric acid and tumor necrosis factor-α, and hypoxia stimulated the production of uric acid and xanthine oxidoreductase activity. BLEx suppressed the production of uric acid under these conditions. Although BLEx inhibited the enzymatic activity of xanthine oxidase, this activity was observed in several fractions containing catechin, epicatechin, chlorogenic acid, rutin, and low molecular weight proanthocyanidins. Taken together, these results indicate that BLEx contains various compounds with the ability to suppress lipid accumulation and uric acid production in adipocytes.Blueberry ( Blueberry leaf infusion has been used as a folk medicine for lifestyle-related diseases in Europe, even though scientific evidence proving its benefits has not been fully established. Actually, blueberry (Vaccinium corybosum L.) leaf extract had the ability to inhibit inflammatory response in mouse macrophage-like cells /[total free flavan-3-ol], where n represents the degree of polymerization of detected flavan-3-ol by thiolysis. Average percentage of A-type bonds = [sum of (thiolysis compounds containing A-type bonds · n)/[total free flavan-3-ol]/mDP × 100. Average percentage of cinchonain I unit = [sum of (thiolysis compounds containing cinchonain I unit · n)]/[total free flavan-3-ol]/mDP × 100.Polymerized degree of proanthocyanidins: The degree of polymerization of proanthocyanidins was estimated by thiolysis . Fr. 4–8v/v) methanol and extracted by ultrasonication  for 15 min at 37 °C and passed through a 0.22 μm membrane filter . Each sample underwent HPLC analysis  equipped with an Inertsil ODS3 (Shimadzu) . The mobile phase conditions were as follows: solvent A, 100% (v/v) ethanol; solvent B, 20 mM potassium phosphate (pH 2.4); column temperature, 40 °C; detection at 280 nm; flow rate, 1.0 mL/min. The binary gradient was as follows: 85%–68% B (0–12 min), 68% B (12–15 min), 68–55% B (15–20 min), 55–85% B (20 min), and 85% B (20–29 min). Standards of chlorogenic acid, catechin, epicatechin, rutin, and caffeic acid were used to identify and quantify the peaks. Data are shown as the mg of each polyphenol/g weight of each fraction.Polyphenol analysis was performed using HPLC, according to our previous report . SamplesAfter the differentiation and maturation of 3T3-L1 cells, the cells were treated with BLEx or its fractionated samples for 24 h. The cells were then washed twice with Hank’s buffered saline containing 0.2% bovine serum albumin. The cells were then stimulated with 1 μM norepinephrine for 90 min to release fatty acids into the media. The fatty acids were measured using a commercial kit  according to the manufacturer’s protocol.g for 5 min at 4 °C. The samples were filtered through a 0.45 μm syringe filter before analysis . HPLC separation was conducted using the Infinity II LC system (Agilent Technologies) equipped with an Intersil ODS-2 column  and maintained at 40 °C. The mobile phase was 98% of 74 mM phosphate buffer (pH 2.2), and 2% methanol and uric acid was detected by measuring the absorbance at 284 nm.Uric acid in the culture supernatant was measured using HPLC. The supernatant was treated with 60% perchloric acid to remove proteins and was centrifuged at 10,000× 4P2O7 with protease inhibitor cocktail  on ice for 30 min. The lysate was centrifuged at 12,000× g for 30 min at 4 °C. Samples (50 μL) were mixed with 50 μL of 100 μM pterin in a 96-well black plate (Thermo Fischer) to initiate the enzyme reaction. Isoxantopterin production was monitored for 30 min based on fluorescent intensity at Ex 355 nm/Em 405 nm using the Varioskan Flash (Thermo). XOR activity was calculated as pmol isoxantopterin production/min/mg protein.XOR activity was measured as described in a previous report . BrieflyThe direct inhibitory effect of BLEx and its fractions on XOD activity was evaluated as the conversion of xanthine to uric acid under XOD . Uric acid, xanthine, and allopurinol (Fujifilm Wako) were dissolved in 0.1 M phosphate buffer . Next, 50 μL PB, 50 μL samples , and 100 μL of 1 mM xanthine were added to a 96-well plate . Finally, XOD was added to a final concentration at 0.1 mU and reacted for 3 min at 37 °C. The production of uric acid was measured as the absorbance at 293 nm using SpectraMax .t-test or one-way analysis of variance (ANOVA) and post hoc tests (Tukey–Kramer multiple comparisons). Differences were considered significant at p < 0.05.Statistical analysis was performed using Statistical Analysis for Mac ver.3.0 . The data were analyzed using Student’s Blueberry leaves are a novel material for the prevention of various lifestyle-related diseases. Here, we evaluated the effect of blueberry leaf hot water extract (BLEx) on adipogenesis and uric acid production in adipocytes. This study revealed that BLEx has the ability to inhibit adipogenesis of 3T3-L1 cells in terms of lipid accumulation and mRNA expression of adipocyte maturation markers. The proanthocyanidin fraction with a mean degree of polymerization of 7.55 (Fr. 6) was responsible for this effect but failed to show lipolytic activity. Maturated 3T3-L1 adipocytes prominently produced uric acid with high XOD activity, which was further increased by hypoxic stress and TNF-α. BLEx prevented the action of hypoxia and TNF-α. Interestingly, allopurinol, a representative inhibitor of XOD, inhibited uric acid production, but not adipogenesis. This implies that adipogenesis and an increase in uric acid during adipocyte differentiation and maturation are independent events. Fr. 6 of BLEx was responsible for the inhibiting adipogenesis, but not for uric acid production and XOD activity. Further, proanthocyanidins, with a relatively low mean degree of polymerization (Fr. 4 and 5), as well as other polyphenolic compounds (Fr. 2 and 3) may contribute to the reduction in uric acid synthesis.


Dinophysis, yet the comparative efficacy of their detection methods has not been systematically determined. Here, we examined DSTs in spiked and naturally contaminated shellfish–Sydney Rock Oysters (Saccostrea glomerata), Pacific Oysters , Blue Mussels  and Pipis (Plebidonax deltoides/Donax deltoides), using LC-MS/MS and LC-MS in 4 laboratories, and 5 rapid test kits (quantitative Enzyme-Linked Immunosorbent Assay (ELISA) and Protein Phosphatase Inhibition Assay (PP2A), and qualitative Lateral Flow Assay (LFA)). We found all toxins in all species could be recovered by all laboratories using LC-MS/MS (Liquid Chromatography—tandem Mass Spectrometry) and LC-MS (Liquid Chromatography—Mass Spectrometry); however, DST recovery at low and mid-level concentrations (<0.1 mg/kg) was variable (0–150%), while recovery at high-level concentrations (>0.86 mg/kg) was higher (60–262%). While no clear differences were observed between shellfish, all kits delivered an unacceptably high level (25–100%) of falsely compliant results for spiked samples. The LFA and the PP2A kits performed satisfactorily for naturally contaminated pipis . There were correlations between spiked DSTs and quantitative methods was highest for LC-MS (r2 = 0.86) and the PP2A kit (r2 = 0.72). Overall, our results do not support the use of any DST rapid test kit as a stand-alone quality assurance measure at this time.Rapid methods for the detection of biotoxins in shellfish can assist the seafood industry and safeguard public health. Diarrhetic Shellfish Toxins (DSTs) are produced by species of the dinoflagellate genus  Marine biotoxins are toxic chemical compounds produced by certain microalgae, which can bioaccumulate in shellfish and other marine organisms, and cause poisoning to seafood consumers. As well as seafood related illnesses, marine biotoxin contamination can lead to damaged public perceptions of seafood, direct economic losses and a restriction in the growth of the shellfish industry.Dinophysis and Phalacroma, and more rarely benthic Prorocentrum, and can bioaccumulate in shellfish and cause Diarrhetic Shellfish Poisoning (DSP). With approximately 11,000 human poisonings reported globally over the period 1985–2018 [Diarrhetic Shellfish Toxins (DSTs) are produced by dinoflagellates of the planktonic genus DSTs are a group of heat stable, polyether toxins consisting of okadaic acid (OA) and its isomer 19-epi-okadaic acid; the OA congeners dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2); and the 7-acyl derivatives of OA, DTX-1 and DTX-2 that are collectively known as DTX-3. Together, they are referred to as the OA group toxins or the ‘okadaates’ (OAs). While OA, DTX-1 and DTX-2 only differ slightly in their molecular structure, the DTX-3 (group) includes a wide range of derivatives esterified with saturated and unsaturated fatty acids, products of metabolic transformations that occur in the shellfish .Patinopecten yessoensis). This contamination was linked to toxins produced by Dinophysis fortii. Following this event, further toxic episodes occurred in Japan, Spain and France, with several thousands of cases of human poisonings occurring over the 1970s and 1980s, and leading to the development of many regional monitoring programs. This monitoring has seen a gradual increase in reported DSP episodes in countries including Chile, Argentina, Mexico, the east coast of North America, Scandinavia, Ireland, Great Britain, Spain, Portugal, Italy, Greece, India, Thailand, Australia and New Zealand [DSP was first described after a large toxin event occurred in Japan in 1976 ,6, where Zealand ,7,8,9.Dinophysis is common in Australian waters, with 36 species reported [D. acuminata Claparede and Lachmann, D. acuta Ehrenberg, D. caudata Saville-Kent, D. fortii Pavillard, D. norvegica Claparede and Lachmann and D. tripos Gourret. There have been three serious DSP events in Australia. The first episode was caused by contamination of Pipis (Plebidonax deltoides) in New South Wales in 1997 (NSW) by D. acuminata [reported ,11,12. Tcuminata . One huncuminata . The fincuminata . While nDetection methods for DSTs using liquid chromatography with tandem mass spectrometry (LC-MS/MS and LC-MS) ,16 and iIn a comprehensive review by McLeod et al.  of the cTM, Eurofins/AbraxisTM and EuroProximaTM; a quantitative PP2A kit by Eurofins/AbraxisTM, and a qualitative LFA kit by NeogenTM.To date, these rapid detection kits have not been tested on various shellfish matrices in a systematic manner, nor a comparison made across multiple analytical laboratories to assess LC-MS/MS or LC-MS detection of DSTs in shellfish. With this in mind, the present study aimed to undertake a comparative study to detect DSTs in differing shellfish matrices using commonly implemented protocols for LC-MS/MS or LC-MS in several different laboratories, as well as compare five commercially available rapid test kits for the detection of DSTs in these same shellfish tissues. The rapid test kits included three quantitative ELISA kits by BeaconSaccostrea glomerata) (SRO), Pacific Oysters  (PO), Blue Mussels  (MUS) and Pipis (Plebidonax deltoides/Donax deltoides) (PIPI)) screened before spiking began (see Methods). Of the triplicate SROs spiked with OA at 0.02 mg/kg, Laboratory 1 detected OA in all three samples , Laboratory 2 and 4 reported concentrations below the detection limit for all samples (<0.01 mg/kg and <0.025 mg/kg respectively), and Laboratory 3 detected OA in all three samples . In summary, two out of the four laboratories detected OA at this low level, with recoveries between ~50–100% , Laboratory 2 did not detect OA in SRO or PO; however, it was detected in both MUS and PIPI , and Laboratory 3 did not detect OA in PO or MUS, but detected it in SRO and PIPI . Laboratory 4 did not detect OA at this concentration (less than detection limit <0.025 mg/kg). Laboratory 4, however, did detect OA in one PIPI sample at 0.03 mg/kg (>spike concentration). In summary, OA was detected in all matrices at this concentration, although not all laboratories detected toxins in all four matrices. Recovery across all laboratories ranged from ~50–150% .For the shellfish spiked with DTX-1 at 0.04 mg/kg, Laboratory 1 recovered this analogue in all matrices , with one PIPI sample returning a concentration of 0.01 OA mg/kg. Laboratory 2 detected DTX-1 in all matrices , also with a detection of OA in PIPI at 0.02 mg/kg. Laboratory 3 detected DTX-1 in all matrices , while Laboratory 4 did not detected this toxin in MUS  . In summFor all shellfish spiked with DTX-2 at 0.01 mg/kg, Laboratory 1 did not recover this analogue in SRO or PIPI, and was only detected it in PO and MUS (both at 0.01 mg/kg). No toxin at this concentration was recovered from either Laboratory 2 nor Laboratory 3, while Laboratory 4 was unable to detect this toxin (below the limit of reporting <0.025 mg/kg) . In summWhen shellfish were spiked with all toxins , laboratory recovery of total toxin per sample for each laboratory was as follows: Laboratory 1: 53–75%; Laboratory 2: 35–88%; Laboratory 3: 13–41%; and Laboratory 4: 0–88% . More spIn our final analysis to determine the recovery of CRM (OA/DTX-1/DTX-2), all laboratories detected all toxin analogues. Individual toxin recoveries ranged from 88 to 131% for Laboratory 1, 79–81% for Laboratory 2, 83–95% for Laboratory 3 and 101–262% for Laboratory 4 . HoweverPrior to rapid test kit screening, OA, DTX-1 and DTX-2 analysis by LC-MS for wild harvest Pipis resulted in a OA toxin range of 0.1 to 0.3 mg/kg . After hydrolysis, no DTX-1 or DTX-2 was detected in any samples. Three batches comprising 10 replicates of each OA toxin concentration of 0.1, 0.2 and 0.3 mg/kg were subsequently screened using each rapid test kit.2 = 0.86) (Using LC-MS (Laboratory 3), all control shellfish samples (no toxin added) returned a ‘not detected’ result . For OA  = 0.86) . SubsequThe Neogen kit returned negative readings for the eight negative control samples across all species-specific shellfish matrices. However, 23 out of 46 samples (50%) of spiked samples  returned a negative result when they contained okadaic acid . Within 2 = 0.72 (The Abraxis PP2A returned 25% (2/8) false positive results, that is, they returned concentrations of toxin within the kit’s working (range 0.06 to 0.35 mg/kg), when the samples contained no okadaic acid. Of those shellfish that were spiked, 29% (13/45) of samples returned values that were outside the working range (8 samples below 0.06 mg/kg and 5 samples above 0.35 mg/kg), with 27% (12/45) samples being underestimated and 44% (20/45) returning a concentration which was equal to, or greater than, the spiked toxin concentration . When sa2 = 0.72 . This ki2 = 0.72 .2 = 0.05  false positives and 43% (20/46) of spiked samples below the limit of quantification. Of the samples that were spiked (and results above the quantification limit), 22% (10/46) were underestimated, while 35% (16/46) were equal to, or greater than, the spiked toxin concentration . When sa2 = 0.05 . This ki2 = 0.05 .2 = 0.08  false positives and 59% (27/46) of spiked samples below the working range. Of the samples that were spiked (and results within the working range), 24% (11/46) were underestimated and 17% (8/46) were equal to, or greater than, the spiked toxin concentration . Again, 2 = 0.08 . Subsequ2 = 0.08 .2 = 0.01  false positives and 65% (30/46) of spiked samples returning results outside the limit of quantification (<0.04 mg/kg). Of the samples that were spiked (and results reported were above the limit of quantification), 33% (15/46) were underestimated, while only 2% (1/46) were equal to, or greater than, the spiked toxin concentration . When ei2 = 0.01 . This ki2 = 0.01 .The repeatability/reliability of all kits was high . The only exception to this was the Abraxis ELISA kit. From the naturally contaminated Pipi batch with the highest toxin concentration (0.3 OA mg/kg), the repeatability of this kit was low at 0.02 (based on a relatively low number of samples however) .Dinophysis blooms and their impacts remain one of the most problematic HABs worldwide, especially in Mediterranean and European waters [Toxic n waters . Positivn waters ,27. UsinIn the present study, we spiked four different shellfish matrices  with fixed volumes of relevant CRM to determine the ability of laboratories to quantify DSTs in shellfish using LC-MS/MS and LC-MS. We found that all spiked analogues, OA, DTX-1, and DTX-2, were recovered in all shellfish species across all laboratories, but the results were not consistent across all samples. For example, low and mid-concentration toxin recovery was variable both within and between laboratories (0–150%), while high concentration toxin recovery, which included CRM, was higher, between 60–262%. Two false positives were reported in Pipi samples in which OA was detected at 0.01 and 0.02 mg/kg , and one anomalously high concentration of 2.8 mg/kg was reported from CRM that was submitted at a concentration of 1.07 mg/kg . These rFinally, we cannot completely discount that there may have been some very low toxin concentrations in these samples which were not detected by the original LC-MS screening. Lab 3, in fact, had the highest level of detection  across all the labs used in this study.In a single laboratory validation study to detect and quantify six lipophilic toxins  in Greenshell mussel, Pacific Oyster, cockle and scallop roe, McNabb et al.  reportedDinophysis species isolated from bloom samples [In another study to compare the mouse bioassay (MBA) to electrospray ionization (ESI) LC-MS/MS for the quantification of lipophilic toxins in ~200 samples of shellfish, Suzuki and Quilliam  similarl samples .There are, however, disadvantages to using LC-MS/MS and LC-MS for the detection of toxins in shellfish. LC-MS/MS (and LC-MS) is expensive, particularly for farmers in low-risk areas who have a regulatory requirement to undertake marine biotoxin testing using LC-MS/MS at regular intervals . The cost is also high for farmers in remote areas, where transport of samples to specialized laboratories is expensive. The LC-MS/MS and LC-MS method is also complex, requiring expert analyst training in dedicated laboratories for sophisticated instrument maintenance and performance. Time delays are another concern, and it can take between 2–7 days to obtain results from a contract laboratory, potentially causing a loss in harvest time and profits to shellfish farmers, and risk to consumers. Finally, high quality and expensive reference material is required to calibrate the method. Despite these disadvantages, and in the absence of a more reliable, sensitive and rapid test, there remains an international acceptance that LC-MS/MS and LC-MS continue to be the standard operating procedure , for the determination of lipophilic marine biotoxins in mollusks .In the search for an inexpensive and reliable alternative method to LC-MS/MS or LC-MS that could be used for screening purposes to serve as an early warning for the shellfish industry, we compared five Rapid test kits against the LC-MS/MS and LC-MS methods. Fifty-five shellfish samples  were screened with four quantitative  and one qualitative (Neogen LFA) rapid test kit to detect OA in Sydney Rock Oysters, Pacific Oysters, Blue Mussels and Pipis. Okadaic acid was the only DST analogue to be tested with these kits for multiple reasons: (i) It has been the dominant analogue detected in Australian shellfish to date; (ii) The cost of purchasing sufficient CRM for spiking all other analogues to detection levels is high; and (iii) Rapid test kit results are reported as µg OA eq/kg, and a spike of varying DST analogues will not reveal individual analogue concentrations . Furthermore, each kit reports a level of cross reactivity to the various analogues, and while in most cases this is 100% for OA, it varies for DTX-1 and DTX-2 between kits. For example, if three samples were individually spiked with the same concentration of okadaic acid, DTX-1 and DTX-2, the concentration of okadaic acid from the Abraxis ELISA kit would read as double the concentrations of the other two compounds. This is because DTX-1 (50%) and DTX-2 (50%) only give half of the response that okadaic acid does with this technology.With this in mind, all quantitative kits should theoretically provide a comparable concentration of OA to that obtained using the LC-MS method. Regression analyses showed the correlations between the ELISA Rapid test kits and LC-MS in our study were all very low (0.002–0.19), while the correlation between the PP2A Abraxis kit and LC-MS was moderate to high (0.72) . The obsMytilus edulis) were examined for total DST toxin content including esters and DTX-3. The ELISA showed matrix effects on hydrolyzed samples, which had both high and low levels of toxins, while the PP2A adequately detected both low and high DST concentrations in mussel samples. While the Okatest was recommended in preference to the ELISA, it was concluded to be a specific assay (could not detect other regulated DSTs), and therefore could not replace LC-MS/MS or LC-MS. Subsequent to these findings, three further studies—a single laboratory validation and an interlaboratory study on the PP2A Okatest [After the development of the first ELISA method by Dubois et al. , a compa Okatest ,35, and  Okatest  were undIn 2015, Jawaid et al. reported on the development and validation of a new rapid test kit, the Neogen LFA, this time a qualitative test strip/reader for the OA group toxins in shellfish . This vaSince the development of the LFA technology, two additional studies generated rapid test kit comparisons ,36. The In the present study, all four quantitative kits showed varying levels of over/underestimation (many at the regulatory limit). Many results were outside the working range or limit of these kits. This ranged from 29% of samples using the Abraxis PP2A to 65% with the EuroProxima ELISA . Two kitOverall, considering the highly varied, and sometimes erroneous results, along with other factors such as method cost, preparation time, test complexity, and extra equipment required, the disadvantages of using the currently available rapid test kits are considerable (Saccostrea glomerata), Pacific Oysters , Blue Mussels  and Pipis (Plebidonax deltoides/Donax deltoides) were sourced from the Sydney Fish Markets on 6/6/2019. From here on, these matrices are referred to as SRO, PO, MUS and PIPI, respectively. These were stored at 4–8 °C and transported immediately to the laboratory for processing. All shellfish were washed thoroughly with fresh water, shucked (if necessary) and tissue was removed. Stock material of each species was made by pooling the tissue of 3–6 individuals (for each spike treatment) of that species, homogenizing and spiking with fixed volumes of relevant standards (see below) and homogenizing again. Subsamples of this species-specific tissue homogenate were then accurately weighed (~3 g) and aliquoted into 5 mL polypropylene Bacto sample jars (Model No. SCP5014UU) and frozen at −20 °C until they were dispatched to contract laboratories for toxin determination by LC-MS/MS and LC-MS.Sample preparation was based on the standard operating procedure for the determination of lipophilic marine biotoxins in molluscs by LC-MS/MS and LC-MS. Specifically, raw samples of Sydney Rock Oysters (Mytilus edulis) and the dinoflagellate Prorocentrum lima, with toxin levels of okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) at 1.07 ± 0.08 µg/g, 1.07 ± 0.11 µg/g and 0.86 ± 0.08 µg/g, respectively (positive control); (ii) CRM-OA-d which contained ~0.5 mL of a solution of OA in methanol at a concentration of 8.4 ± 0.4 µg/mL; (iii) CRM-DTX-1-b which contained ~0.5 mL of a solution of dinophysistoxin 1 (DTX-1) in methanol at a concentration of 7.8 ± 0.5 µg/mL; and (iv) CRM-DTX-2-b which contained ~0.5 mL of a solution of dinophysistoxin-2 (DTX-2) in methanol at a concentration of 3.8 ± 0.2 µg/mL.Certified reference materials (CRMs) were purchased from the National Research Council Canada (NRC) for shellfish spiking and quality control testing. These included: (i) CRM DSP-Mus-c which is a thermally sterilized homogenate (4.0 ± 0.75 g) of mussel tissue (A subsample (3 g) of each pooled, species-specific matrix  was first analyzed by LC-MS at Laboratory 3 (see below) to ensure each matrix contained no DSTs before the experiment began (limit of detection (LOD) = 0.006–0.007 mg/kg for analogues OA, DTX-1 and ± DTX-2) .Spiking of each species-specific homogenate with a range of DST concentrations then followed for both LC-MS/MS and LC-MS. These concentrations were chosen based on the capability of most laboratories to achieve a limit of reporting (LOR) of ~0.01 mg/kg . In brieNext, a second species-specific homogenate was spiked with a combination of all three toxins: 35 µL/3 g OA for SRO and PO which is 10 × LOR (0.1 mg/kg) or 7.2 µL/3g OA for MUS and PIPI which is equivalent to 2 × LOR (0.02 mg/kg); 17.6 µL/3 g DTX-1 which is 5 × LOR (0.05 mg/kg) into all shellfish species; and 16 µg/3 g DTX-2 which is 2 × LOR (0.02 mg/kg) again into all shellfish species. These combination-spiked samples were then aliquoted (~3 g) and sent to each laboratory to test toxin profile detection capability and also any matrix effect .Furthermore, to test the reproducibility/repeatability of each laboratory, a third batch of the SRO homogenate was spiked with OA  and three replicate aliquots of this stock material (3 g) were dispatched to each laboratory. Finally, one sample (~3 g) of the CRM DSP-Mus-c was sent to each laboratory as a positive control. In total, 19 samples (randomly numbered 1–19) were dispatched frozen to each of four laboratories .Four commercial and/or government analytical laboratories with experience in conducting LC-MS/MS and LC-MS of marine biotoxins in shellfish were engaged to determine DSTs in spiked shellfish, identified only as Laboratories 1–4. The aim of this part of the study was to determine an inter-laboratory comparison of standardized samples, in order to obtain a baseline result using currently mandated seafood safety procedures in Australia . The LC-Raw samples of SRO, PO, MUS and PIPI (same species as above), were sourced from the Sydney Fish Markets on 29/4/2020. These were stored at 4–8 °C and transported immediately to the University of Technology Sydney laboratory for processing. Again, all shellfish were washed thoroughly with fresh water, shucked and tissue was removed. Bulk material of each species was then made by pooling the tissue of individuals of that species up to 90 g, homogenizing and separating into 3 batches for downstream processing. The first batch served as unspiked controls and were first examined by LC-MS at Laboratory 3 (see above) to ensure each matrix was clear of toxins before the experiment began. The second batch was spiked with CRM-OA-d at ~12 µL/g (0.1 OA eq. mg/kg), which is half the regulatory limit, and the third batch was spiked at ~24 µL/g, which is equal to the regulatory limit. Once prepared all batches were returned to the freezer (−20 °C) until further processing.Additionally, during Oct/Nov 2019, DSTs were detected in wild harvest Pipis from Sydney Fish Markets (~400 mg/kg), and a recall was immediately actioned. A batch of these naturally contaminated Pipis were obtained and prepared as positive controls: Sample 4A—14/11/19 Stockton 4–6 km; 4B—7/11/19 Stockton 4 km; 4C—31/10/19 Stockton 2–4 km; and 4D—Sydney Fish Market Stockton recall Nov 2019. Once the OA toxin concentration was determined using LC-MS for these environmentally contaminated samples, samples with toxin level closest to the regulatory level (0.2 mg OA eq/kg) were chosen, and 10 replicates of these positive controls were run on each kit to test the reliability/repeatability of each kit.A subsample (3 g) of each pooled, species-specific matrix was first examined by LC-MS (Laboratory 3) to ensure each matrix was clear of toxins before the experiment began (unspiked controls). All remaining batches (spiked and positive controls) were then subsampled and prepared according to the rapid test kit protocols for each kit or for LC-MS analysis. Duplicate samples of each treatment/shellfish were tested using both LC-MS and the five test kits.A list of DST rapid test kits were screened, their method details including their limit of quantification or working range, amount of tissue required, cost, time for analysis etc., are summarized in ® syringe filter into another clean 15 mL tube. Eighty µL of filtered extract was then transferred to a clean glass vial, followed by 100 µL of 2.5 M NaOH, before being capped tightly and mixed using a vortex on full speed for 30 s. The sample vial was then transferred to a heater block set at 76 °C for 40 min, after which time the sample was cooled on ice. At room temperature, 100 µL of 2.5 M HCl was added to the sample extract, mixed by hand for 30 s, before 100 µL transferred into a DSP buffer A vial (provided). The sample was again vigorously mixed, before 100 µL was transferred to a microwell plate. A DSP strip was then placed into the microwell plate for 15 min before being immediately placed into the AccuScan® PRO 2.0 scanner for result interpretation.Neogen Reveal 2.1 DSP Test strips  and DSP hydrolysis packs (Lot: 9555-09) were stored at room temperature until experiments began. Each shellfish sample (2 g) was defrosted to room temperature (20–25 °C), then transferred to the extraction bag provided before being homogenized with 8 mL analytical grade methanol . The sample extract was then poured from each extraction bag (from opposite side of mesh divider) into a 15 mL falcon tube, prior to filtration using a 0.45 µm sterile Minisartg for 10 min at 4 °C and 640 µL of the methanolic extract removed and transferred to a clean 15 mL falcon tube. The extract was then mixed with 100 µL of 2.5 N NaOH, sealed, and placed in a water bath at 76 ± 2 °C for 40 min. After removal from the water bath, 80 µL of 2.5 N HCl was added to each sample, followed by 20 mL buffer solution.The Eurofins/Abraxis Okadaic Acid (PP2A) Microtiter Plate kit Product No. 520025, Lot No. 19/1259, Eurofins Abraxis, Warminster, PA, USA) was stored at 4 °C prior to use. Upon opening, the solutions were prepared as per the manufacturer’s protocols and allowed to reach room temperature before analysis began. Each shellfish subsample (5 g) was defrosted and 25 mL methanol  added before homogenization in a tube shaker for 2 min. The sample was then centrifuged at 2000× For the test protocol, a volume of 50 µL of each OA standard  and each shellfish sample was added to the 96 well-plate provided. To each of these wells, 70 µL phosphatase solution was added. The plate was then tapped gently to ensure mixing, before being covered with parafilm and incubated for 20 min at 30 ± 2 °C. Immediately after this incubation period, 90 µL of chromogenic substrate was added to each well, and again, the plate was tapped gently to ensure mixing. The plate was then incubated (covered) for a further 30 min at 30 °C ± 2 °C, after which 70 µL of stop solution was added to each well. Absorbance was immediately read at 405 nm using a Tecan Infinite M1000 PRO plate reader.y-axis and the concentration of okadaic acid in a logarithmic x-axis. The OA concentration contained in the sample (Cs) was then calculated using the following equation: For data analysis, a standard curve was obtained by plotting the absorbance values in a linear ® syringe filter and the extract diluted 1:50 into 10% methanol/10 mM PBS  .The Beacon Okadaic Acid (ELISA) Plate kit  was stored at 4 °C and all reagents brought to room temperature before use. Each shellfish sample (1 g) was defrosted and 2 mL 80% methanol /water was added before homogenization and transfer to a clean 15 mL falcon tube. A further 8 mL of 80% methanol/water was then added, before vortexing for 5 min followed by centrifugation at 3000 rpm for 5 min. The supernatant was then filtered into a clean 15 mL tube through a 0.45 µm sterile MinisartFor the test procedure, 50 µL of enzyme conjugate was added into each test well, followed by 100 µL of each OA calibrator  or shellfish sample, and 50 µL of antibody. Wells were then mixed for 30 s using gentle shaking, followed by incubation at room temperature for 30 min. The content of the well plates were then decanted, and well plates were washed four times using Milli-Q water, and inverting the plate onto absorbent paper between each wash. After the final wash, 100 µL of substrate was added to each well, before incubation for 30 min at room temperature. Finally, 100 µL of stop solution was added to each well and absorbance read at 450 nm using the Tecan Infinite M1000 PRO plate reader.y-axis versus the concentration of okadaic acid in a logarithmic x-axis. The OA concentration (ppb) contained in the sample (Cs) was then calculated using Equation (1) above. Finally, to obtain the final DST (mg/kg) in each sample, a factor of ×500 to account for the dilution during the shellfish extraction step was applied.For quantitative interpretation of the absorbance readings, a standard curve was then constructed by plotting the absorbance of the calibrators (standards) on the g and the supernatant was transferred to a clean 15 mL falcon tube. A further 2 mL methanol/Milli-Q was added to the shellfish residue, the sample centrifuged again for 10 min at 3000× g, and the supernatant added to the first portion. The final volume was brought up to 10 mL with methanol/Milli-Q, before filtration into a clean 15 mL tube through a 0.45 µm sterile Minisart® syringe filter. For the hydrolysis step, 500 µL of each sample extract was added to a 2 mL glass vial, and 100 µL of 1.25 N NaOH added. The sample was then vortexed for 15–20 s before incubation on a heat block at 80 °C for 40 min. Each sample was then cooled and 100 µL of 1.25 N HCl added and vortexed for 15–20 s. Finally, 10 µL of the hydrolyzed extract was mixed with 990 µL of 1× sample diluent (1:100 dilution) in a 2 mL glass vial with a cap and vortexed again.The Eurofins/Abraxis Okadaic Acid (DSP) ELISA, Microtiter Plate  was stored at 4 °C and brought to room temperature before use. All solutions were prepared as per the manufacturer’s protocols. Each shellfish subsample (1 g) was defrosted and 6 mL methanol /Milli-Q water (80/20) added before homogenization for 2 min. Each sample was then centrifuged for 10 min at 3000× For the assay procedure, a volume of 100 µL of each OA standard  and shellfish sample was added to each strip well and placed into the well plate provided. To each of these, 50 µL of enzyme conjugate and 50 µL of antibody solution was added. The plate was then covered with parafilm, rotated carefully to mix and left to incubate for 60 min at room temperature, after which the covering was removed and the contents were decanted by inverting the plate onto a paper towel. Each well was then thoroughly washed three times using the diluted wash buffer (~25 µL for each wash/each well), blotting after each step. Following the final washing step, 150 µL of substrate solution was added to each well, before covering with parafilm, rotating gently to mix, and incubating at room temperature for 30 min. Finally, 100 µL of stop solution was added to each well plate prior to immediate absorbance reading at 450 nm using the Tecan Infinite M1000 PRO plate reader.y-axis versus the concentration of okadaic acid in a logarithmic x-axis. The OA concentration (ppb) contained in the sample (Cs) was then calculated using Equation (1) above. Finally, to account for hydrolysis sample extraction, hydrolysis and dilutions during the hydrolysis step, all results were multiplied × 1400 to obtain the DSP concentration (ppb) before conversion to mg/kg.Kit performance was evaluated by calculating %B/Bo for each standard by dividing the absorbance value for each standard by the Zero standard mean absorbance. A standard curve was then constructed by plotting the %B/Bo for each standard on the g for 10 min. The clear supernatant was then filtered using a 0.45 µm sterile Minisart® syringe filter into a clean 15 mL falcon tube and the sample subsequently diluted 1:50 with the sample dilution buffer provided.The EuroProxima Okadaic Acid ELISA  was stored at 4 °C before use and subsequently brought to room temperature before use. Reagents were prepared as specified in the manufacturer’s protocol. To begin, 1 mL of water was added to each 1 g of shellfish, the sample vortexed for 1 min, and a further 2 mL of 100% methanol  was added. The sample was again vortexed for 1 min followed by centrifugation at 2000× For the assay procedure, 100 µL of the zero standard (0 ng/mL) was pipetted into the first well, and 50 µL thereafter of each OA standard  and shellfish samples into the 96 well-plate provided. Following on, 25 µL of enzyme conjugate and 25 µL of antibody was added to each well, except A1. The plate was then sealed with parafilm and gently shaken for 1 min before incubation at room temperature for 30 min. Parafilm was subsequently removed, the well contents discarded onto absorbent paper, and all wells were washed three times with a rinsing buffer. After the final rinse, 100 µL of substrate solution was added to each well, mixed thoroughly and left to incubate for 15 min in the dark prior to 100 mL of stop solution being added. Absorbance was read at 450 nm using the Tecan Infinite M1000 PRO plate reader.x-axis. The OA concentration (ng/mL) contained in the sample (Cs) was then calculated using Equation (1) above, but this time where x was the OA concentration in the sample (Cs) and y the % max OD of the sample. Finally, to obtain OA equivalents in the final shellfish, a factor of × 200 (and/1000) was applied.For data interpretation, the mean optical density (OD) value of the wells A1 and A2 were subtracted from the individual OD reading from each of the standards and samples. The OD values of the six standards and samples are then divided by the OD value of the zero standard (well no. B1) and multiplied by 100. The zero standard is then equal to 100% (maximum OD) and the other OD values are % of the maximal OD. A calibration curve was then constructed with the values  plotted on the y-axis versus the concentration of okadaic acid (ng/mL) in a logarithmic Toxin recovery from samples analyzed using LC-MS/MS and LC-MS were assessed in four ways. 1. Where sample replication was available, mean (±SD) toxin recoveries were calculated and compared to the spiked concentration and LOR, and finally compared across laboratories. 2. To determine each analogue recovery using LC-MS/MS and LC-MS, toxin results from each shellfish species were compared to the spiked toxin concentration, and then compared across laboratories. 3. For shellfish that were spiked with a combination of OA analogues, the results were compared to both spiked concentration and the ML (0.2 mg/kg OA), as well as across laboratories. 4. Finally, the recovery of toxins in certified reference material CRM (DSP-Mus-c) were compared across laboratories.To examine the performance of the rapid test kits, firstly, we assessed the performance of the qualitative Neogen kit by comparison to the spiked toxin concentration in each sample . Secondly, the performance and recovery of all quantitative methods (including LC-MS) were compared  to the spiked concentration of each sample. For those samples spiked at, or above, the ML adopted by the Food Standards Australia New Zealand (0.2 OA mg/kg), we also determined whether they were “falsely compliant” or “falsely non-complaint” with the ML. These terms refer to the comparison of the results obtained to the maximum regulatory limit. For example, if a sample was spiked above the regulatory limit but resulted in a concentration below the regulatory limit, it was referred to as “falsely compliant”. Conversely, if a sample was spiked below the regulatory limit but returned a concentration above the regulatory limit, it was referred to as “falsely non-compliant”. Thirdly, a comparison across species-specific matrices was undertaken to assess the suitability of rapid test kits across a range of shellfish species. Finally, the reliability or repeatability of each kit was assessed  from the replicate positive controls  across all quantitative kits.


Bifidobacterium genus abundance. The observed benefits are enhanced by pasteurization. Pasteurized products also had a significant effect at the host level, improving inflammation-induced intestinal epithelial barrier disruption and increasing IL-10 and IL-1β compared to the control condition. Our data support the potential health benefits of water kefir and demonstrate that pasteurization, performed to prolong shelf life and stability of the product, also enhanced these benefits.Fermentation is an ancient food preservation process, and fermented products have been traditionally consumed in different cultures worldwide over the years. The interplay between human gut microbiota, diet and host health is widely recognized. Diet is one of the main factors modulating gut microbiota potentially with beneficial effects on human health. Fermented dairy products have received much attention, but other sources of probiotic delivery through food received far less attention. In this research, a combination of in vitro tools mimicking colonic fermentation and the intestinal epithelium have been applied to study the effect of different pasteurized and non-pasteurized water kefir products on gut microbiota, epithelial barrier function and immunomodulation. Water kefir increased beneficial short-chain fatty acid production at the microbial level, reduced detrimental proteolytic fermentation compounds and increased  Gut microbes play a key role in human health, including the development of the immune system ,2,3 and Fermented products have been linked to health benefits, potentially through gut microbiota modulation. Indeed, fermented products can be considered a source of probiotic microorganisms, defined as live microorganisms that confer a health benefit on the host .Among fermented food products, water kefir, is made by fermentation of simple carbohydrate solutions  and dried fruits like figs or dates, generating a beverage with a blond to yellowish color, acidic and slightly alcoholic . The oriSimilar to milk kefir, the fermentation of water kefir is carried out by a distinct starter cultures attached to predominantly polysaccharide matrix called the kefir grain or tibicos. However, differently to milk kefir, the polysaccharide matrix is mostly composed of dextran produced by a variety of lactic acid bacteria species ,18. ThesMultiple potential health-benefits have been associated with milk kefir, mainly derived from in vitro and animal studies. These effects include immunomodulatory, cholesterol-lowering, anti-allergic, anti-inflammatory, antitumoral, antimicrobial activity (reviewed in ) and effCandida albicans, Salmonella typhi, Shigella sonnei, Staphylococcus aureus and Escherichia coli  and at 0 h, 24 h and 48 h for community structure analysis .g. A Fastprep-24 device  was used for homogenization (two cycles of 40 s at 4 m/s). Quality control PCR was conducted using Taq DNA Polymerase with the Fermentas PCR Kit according to the manufacturers’ instructions . The DNA quality was verified by electrophoresis on a 2% (w/v) agarose gel for 30 min at 100 V.The DNA was extracted from a pellet of bacterial cells originated from a 1 mL sample after centrifugation for 5 min at 7700× Microbial community composition was assessed at the start of the experiment and at 24 and 48 h of incubation. Samples were sent out to LGC Genomics GmbH  for next-generation 16S rRNA gene amplicon sequencing of the V3–V4 region. Library preparation and sequencing were performed using an Illumina MiSeq platform with v3 chemistry. The 341F (5′-CCTACGGGNGGCWGCAG-3′) and 785R (5′-GACTACHVGGGTATCTAAKCC-3′) primers were used as previously described  with theSaccharomyces cerevisiae strain ySR128 was included. The standard curves for all runs had efficiencies between 90 and 105%. Samples were analyzed in technical triplicates and melt curve peaks were checked in each run. Results are reported as log units (18S rRNA gene copies/mL).Subsequently, the qPCR for total yeast quantification were performed using a QuantStudio 5 Real-Time PCR system , using the primers and conditions described in Bacterial cell densities were measured using a BD FacsVerse flow cytometer and live cells were distinguished from damaged cells using SYBR Green /propidium iodide  staining, as previously described . Proport4, O2, H2, N2), a Rt-Q-bond pre-column and column , and a thermal conductivity detector. SCFA  and BCFA  were determined as described previously [Metabolic parameters were quantified at t0, t24 and t48. In addition, lactate was measured at 6 h (t6). pH measurements were performed using a Senseline pH meter F410 . Gas formation was measured using a pressure meter to which a needle was connected . Gas phase composition was analyzed using a compact GC , equipped with a Molsieve 5A pre-column and Porabond column (for CHeviously . Lactatev/v) heat-inactivated (HI) fetal bovine serum (FBS). THP1-Blue™ were obtained from InvivoGen (Belgium) and consisted in stably transfected cell line with a reporter construct expressing a secreted alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factor nuclear factor kappa B (NF-κB). THP1-Blue were maintained in Roswell Park Memorial Institute (RPMI)1640 medium containing glucose and glutamine, supplemented with HEPES, sodium pyruvate and 10% (v/v) HI-FBS. Cells were incubated at 37 °C in a humidified atmosphere of air/CO2 .Routine maintenance of cells was performed as described . Briefly4 cells/cm2), until a functional cell monolayer with a transepithelial electrical resistance (TEER) above 500 ohm Ω cm2 was obtained. 48 h before the start of the co-culture, THP1-Blue™ cells were seeded in 24-well plates and stimulated for 48 h with phorbol 12-myristate 13-acetate (PMA) at a concentration of 100 ng/mL to induce macrophage-like phenotype in THP1 cells [The Caco-2/THP-1 co-culture was done as previously described . Caco-2 P1 cells . After P®® Excel®® 2016 MSO (16.0.11901.20070) . Reads occurring only 5 times in all samples were removed, as they were supposedly artefacts or bacteria that were not having any biological impact. For the most abundant OTUs, the sequences retrieved from 3% dissimilarity level fasta file obtained in mothur were classified through the RDP web interface using the RDP SeqMatch tool. The database search was restricted to type strains with only near-full-length and good quality sequences. The sequences were blasted in NCBI against the 16S rRNA gene sequences, selecting only type material, with optimization of the BLAST algorithm for highly similar sequences (accession date: December 2018) [Bioinformatics analysis of amplicon data was performed as previously described . Brieflyer 2018) ,43,45. ATo get a general overview of treatments on microbiota, 16S rRNA raw gene sequences were analyzed using Calypso software Version 8.84. Data were normalized by total sum (TSS) and transformed by square root (Hellinger Transformation). Taxa with less than 0.01% abundance or less than 100 reads were removed.t-tests were performed to determine statistical significance between different conditions versus the control.To take interindividual differences into account, microbial levels at family and OTU for each treatment were normalized by subtracting the value of the control condition for every donor separately. Besides donor effect, the effects of the different treatment parameters were calculated following a similar calculation. In brief, values from pasteurized samples were subtracted from values obtained from non-pasteurized samples per donor to evaluate the pasteurization effect and values from K1 sample were subtracted from the corresponding K2 sample per donor to assess type of kefir effect. The obtained values for the different conditions were averaged over the three donor and t-tests were used. A General Linear Model was used to determine the effect of donor, pasteurization, kefir culture and water used for kefir preparation as factors.For each microbial metabolic marker  and qPCR data, changes in production or counts between 0 and 6 h (Δ6 h), 6 and 24 h (Δ24 h), and 24 and 48 h (Δ48 h) were calculated. As each variable showed a different trend on maximum changes depending on time, selected values of maximum differences were used for visual representation. After testing for normality (Shapiro Wilk test), one-way ANOVA test was used to determine significance, with Tukey’s multiple comparison test as post-hoc. Arithmetic mean and standard error of the mean (SEM) were used as descriptive statistics. Data were also analyzed after normality test, using two-way ANOVA, including repeated measures on different time points, and checking the interaction between treatment and time. In this case, Dunnett’s multiple comparison tests was used to determine the significance between different treatments and the control condition. To compare the overall changes of kefir treatments versus the control, paired Clustvis software was used to perform the Principal Component Analysis (PCA) plots on microbial metabolites. Briefly, unit variance scaling is applied to rows and SVD with imputation was used to calculate principal components, represented in the X (PC1) and Y (PC2) axis. Prediction ellipses were drawn at 95% of confidence interval.p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively.For cell culture assays, treatment samples were compared to the control samples using two-way ANOVA with Sidak’s multiple comparisons test. Significant differences are represented by (*), (**), (***) and (****) representing Statistical analysis was performed in GraphPad Prism version 8.2.0 (435) for Windows  or Minitab version 18.1 . All formal hypotheses were conducted on the 5% significance level (α = 0.05).Fecal samples of the healthy donors were collected according to the ethical approval of the University Hospital Ghent (reference number B670201836585).In general, water kefir, pasteurization and donor had a significant effect on most of the microbial metabolic markers, while type of water used to prepare the kefir only affected lactate, ammonia and BCFA .6 h 33.7 ± 3 mM) compared to the control (Δ6 h 14.9 ± 0.8 mM) (6 h) but was not significant at longer incubation times (Δ24 h). Changes in total SCFA were primarily caused by the major metabolite acetate, which showed the same trend described above (6 h 10.2 ± 1.4 mM) compared to the control condition (Δ6 h 3.9 ± 0.3 mM) (24 h 4.2 ± 0.5 mM) compared to control (Δ24 h 2.7 ± 0.5 mM) (p < 0.04). BCFA did not differ at Δ6 h or Δ24 h. Consistently with observed changes in SCFA, a decrease in pH (Δ6 h −0.5 ± 0.09) and increased levels of lactate (Δ6 h 3 ± 0.6 mM) and gas production (Δ6 h 25.1 ± 2.9 kPa) were observed in kefir-exposed reactors (24 h 289.4.7 ± 18.5 mg/L), compared to the control condition (Δ24 h 380.4 ± 5.3 mg/L)  A. This eed above . Propion 0.3 mM) B. Butyrareactors C,E. As a.3 mg/L) F.p < 0.001, When analyzing the pasteurization effect on microbial metabolic products, acetate and propionate were higher in pasteurized samples than in non-pasteurized ones at 6 h (K1 and K2 (6 h 48.3 ± 1.7 mM), propionate (Δ6 h 13.8 ± 2 mM) and butyrate (Δ24 h 6.8 ± 0.5 mM) and reduced proteolytic fermentation markers BCFA (Δ24 h 0.4 ± 0.1 mM) and ammonia (Δ24 h 144.1 ± 4.5 mg/L), compared to control condition   compared to V (0.5 ± 0.1 mM)  and lact 0.9 mM) .p = 0.457).In our setup, the main effects on microbial communities in vitro were time and donor . OverallBifidobacteriaceae, Leuconostocaceae and Sphingomonadaceae  analysis at family level, kefir-treated reactors were enriched (LDA > 3) in nadaceae B compareby kefir D. At OTUby kefir E.Bifidobacteriaceae compared to non-pasteurized samples, which contrarily showed a higher abundance of Sphingomonadaceae and Esysipelatoclostridiaceae   .p < 0.0001). The highest levels of EtOH were quantified on colonic incubations exposed to non-pasteurized products , with the highest values observed for K1. Pasteurized samples showed a consistent decrease in EtOH levels  compared to non-pasteurized ones (p < 0.001).In the control condition, EtOH levels were lower  than in water kefir-treated reactors  (zed ones A (p < 0.10 units), while non-pasteurized products significantly increased 18S RNA copies . Pasteurization significantly reduced 18S RNA copies, especially in kefir K1, which shows similar levels to control (4.4 ± 0.2 copies/mL log10 units), while pasteurized K2 still had higher levels at 24 h and 48 h compared to the control condition (5.5 ± 0.06 copies/mL log10 units). The amount of yeast in non-pasteurized product decreased over time.Quantification of 18S RNA copies showed similar values for control condition at the different time points (4.6 ± 0.2 copies/mL logp = 0.019) percentages of TEER (89 ± 2%) compared to the initial values than the control condition (81 ± 6%). Pasteurization increased the effect of kefir products on TEER enhancement (p = 0.014), with values of 92 ± 3% and 86 ± 3% for pasteurized and non-pasteurized products, respectively, with pasteurized K2 showing the highest increase on TEER values (93 ± 4%)  .630)  .p < 0.001) and IL1β   .The effect of different parameters, including inoculum, pasteurization and water on epithelial barrier integrity, NF-κB activation and immunomodulatory activity in each donor is presented in Modulation of composition and metabolic activity of gut microbiota from three healthy donors by different kefir products was evaluated using a short-term simulation of the colonic fermentation process.Despite significant interindividual differences in basal microbiota, kefir supplementation showed a consistent potential to modulate microbial activity in vitro, likely by adding fermentative substrates and microorganisms to the background colonic media. Overall, all products increased short-chain fatty acids and lactate and reduced ammonia production, with the highest effect observed for pasteurized K2, which increased health-promoting propionate and butyrate. Major SCFAs, such as acetate, propionate and butyrate, are important metabolites for intestinal homeostasis, metabolic control, and immune programming ,47,48,49Bifidobacteriaceae family being consistently increased by water kefir. Bifidobacterium spp. are early colonizers of the human gut and are associated with a healthy condition [Bifidobacteriaceae populations have been described in different pathological conditions such as allergy or inflammatory bowel disease [Bifidobacteriaceae in our setup likely explain the increase of acetate and lactate production, which can cross-feed commensal butyrate or propionate-producing anaerobes [Bifidobacterium spp might be linked to the stimulation of resident bifidobacterial populations. This observation also suggests that the thermal degradation of water kefir yeast and bacterial microbial populations makes available different carbon sources that promote Bifidobacterium growth, such yeast glycans [Bifidobacterium spp. have been reported to selectively uptake carbohydrates from glycans. Significant increases in the relative abundance of Actinobacteria have been reported in a parallel-group, randomized, controlled clinical trial setting with patients with metabolic syndrome receiving milk kefir compared to unfermented milk during 12 weeks [Bifidobacterium adolescentis/faecalis was significantly induced by water kefir, especially by pasteurized samples, extending the paraprobiotic/postbiotic application of water kefir products and suggesting a species-specific bifidogenic effect depending on background host microbiota. In parallel to Bifidobacterium spp. stimulation, kefir induced a decrease in the abundance of Dorea genus, a commensal member of the gut microbiota, which is over-represented in individuals with asthma, type 2 diabetes [Linked to microbial metabolic activity, changes in ecosystem structure were also detected, with the ondition . Reduced disease ,55,56. Wnaerobes . This in glycans ,59. Quan12 weeks , a trait12 weeks . Levan i12 weeks . In our diabetes  and obesdiabetes .Multiple mechanisms have been previously described for the modulation of gut microbiota by probiotics or paraprobiotics to beneficially affect host health, including microbe-microbe interaction of kefir strains with intestinal microorganisms, microbe-host interaction with intestinal epithelium, modulation of mucus production or epithelial barrier function, local or distal immunomodulation or effects on gut-organs communication  . We demoSaccharomyces strains also showed a stimulatory effect on IL-10 production by human peripheral blood mononuclear cells [Consistently, pasteurized kefirs enhanced intestinal barrier function and promoted anti-inflammatory IL-10 production. Microbial metabolites, like butyrate, acetate or propionate, but also yeast-derived molecules could be involved in the upregulation of IL-10 observed in our model. IL-10 is a central cytokine in maintaining mucosal tolerance, signaling different lymphocyte T subpopulations, downregulating TNFα, and influencing selective colonization of the gut ,68 and iar cells . The samar cells .Pasteurized water kefir also induced activation of NF-κB and secretion of IL-1β, both usually considered pro-inflammatory markers. NF-κB regulates inflammation, immune responses, cell proliferation, survival, and apoptosis, and it is considered a key signaling molecule in maintaining epithelial barrier and immune homeostasis , while dMost of the research involving pre and probiotic properties of kefir is based on milk kefir products, while this research provides novel information on potential modulatory effect of water kefir on human gut microbiota. Our study using an artificial colon set up suggests beneficial effects of water kefir consumption through SCFA production, bifidobacteria induction and epithelial barrier enhancement, while limitations on translatability and interindividual variability will require validation in clinical settings. Intriguingly, the observed benefits are enhanced by pasteurization, supporting water kefir derived products as paraprobiotics/postbiotics with potential health benefits and the added value of extended shelf life and stability of the product.


Bupleurum subsp. paniculatum (Brot.) H.Wolff essential oil from Portugal. The oil obtained by hydrodistillation and characterized by GC-MS, showed high amounts of monoterpene hydrocarbons, namely α-pinene (29.0–36.0%), β–pinene (26.1–30.7%) and limonene (10.5–13.5%). The antifungal potential was assessed, according to CLSI guidelines, against several clinical and collection strains. The essential oil showed a broad fungicidal effect being more potent against Cryptococcus neoformans and dermatophytes. Moreover, a significant germ tube inhibition was observed in Candida albicans as well as a disruption of mature biofilms, thus pointing out an effect of the oil against relevant virulent factors. Furthermore, fungal ultrastructural modifications were detected through transmission electron microscopy, highlighting the nefarious effect of the oil. Of relevance, the oil also evidenced anti-inflammatory activity through nitric oxide inhibition in macrophages activated with lipopolysaccharide. In addition, the essential oil’s bioactive concentrations did not present toxicity towards macrophages. Overall, the present study confirmed the bioactive potential of B. rigidum subsp. paniculatum essential oil, thus paving the way for the development of effective drugs presenting concomitantly antifungal and anti-inflammatory properties.Fungal infections remain a major health concern with aromatic plants and their metabolites standing out as promising antifungal agents. The present study aims to assess, for the first time, the antifungal and anti-inflammatory potential of  Candida albicans, nevertheless other Candida species are becoming important etiological agents such as C. krusei, C. parapsilosis and C. tropicalis . Voucher specimens were deposited at the herbarium of the Department of Life Sciences of the University of Coimbra (COI).Aerial parts of two samples of The essential oils were obtained by hydrodistillation (3 h) using a Clevenger-type apparatus as described in the European Pharmacopoeia . Their cA Hewlett-Packard 6890  chromatograph with a single injector, two flame ionization detectors and a HP GC ChemStation Rev. A.05.04 data handling system was used for GC analysis. Two columns: polydimethylsiloxane (SPB-1) and polyethylene glycol (SupelcoWax-10) with 30 m × 0.20 mm and a film thickness 0.20 µm were used and GC parameters were as follows: oven temperature: 70–220 °C (3 °C/min), 220 °C (15 min); injector temperature: 250 °C; carrier gas: helium, with a linear velocity of 30 cm/s; splitting ratio 1:40; detectors temperature: 250 °C. A Hewlett-Packard 6890 gas chromatograph (SPB-1 column), interfaced with an Hewlett-Packard mass selective detector 5973  with a HP Enhanced ChemStation software, version A.03.00  was used for GC–MS analysis. MS parameters: source temperature: 230 °C; MS quadruple temperature: 150 °C; ionization energy: 70 eV; ionization current: 60 µA; scan range: 35–350 units; scans per second: 4.51; interface temperature: 250 °C.Retention indices and mass spectra were used to identify the compounds with the first being compared to samples from the Laboratory of Pharmacognosy database of the Faculty of Pharmacy, University of Coimbra and the later with that of reference spectra from a home-made library or from literature data. GC peak area was used to calculate relative amounts of the identified compounds.Candida albicans ATCC 10231, C. guillermondii MAT23, C. krusei H9, C. parapsilosis ATCC 90018, C. tropicalis ATCC 13803, and Cryptococcus neoformans CECT 1078; dermatophytes: Epidermophyton floccosum FF9, Microsporum canis FF1, M. gypseum CECT 2908, Trichophyton mentagrophytes FF7, T. mentagrophytes var. interdigitale CECT 2958, T. rubrum CECT 2794, and T. verrucosum CECT 2992 and Aspergillus strains including A. flavus F44, A. fumigatus ATCC 46645 and A. niger ATCC 16404.Collection and clinical strains were used to assess the antifungal effect of the essential oil, including yeasts: The fungal isolates were identified by standard microbiological methods and stored at −80 °C on Sabouraud broth with 20% glycerol, being inoculated on Sabouraud dextrose agar (SDA) to ensure optimal growth and purity, prior to each assay.To assess the effect of the essential oil on fungal growth, two parameters were assessed: the minimal inhibitory concentration (MIC) and the minimal lethal concentration (MLC). A broth macrodilution method was used according to the Clinical and Laboratory Standards Institute (CLSI) reference protocols M27-A3 and M38-A2, for yeasts and filamentous fungi, respectively. Serial dilutions of the oil (up to 0.036 µg/mL) were tested as well as a negative control (medium without fungi) and a positive control (inoculated medium with DMSO 1%).C. albicans and T. rubrum were selected to perform the transmission electron microscopy studies. Cultures untreated (DMSO 1%) or treated with the essential oil (MIC) were used. Fungi cultures were collected and centrifuged at 3000 rpm for 5 min. Pellet cells were fixed using 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 h and post-fixed in 1% osmium tetroxide during 1 h. Following rinsing, samples were embedded in 2% molten agar, dehydrated in ethanol (30–100%), and impregnated and included in epoxy resin . Ultrathin sections (70 nm) were obtained and stained with uranyl acetate 2%, for 5 min followed by lead citrate 0.2% for 5 min. Observations were carried out on a FEI Tecnai G2 Spirit Bio Twin at 100 kV.C. albicans ATCC 10231, from overnight cultures on SDA, were prepared in NYP medium   . The susC. albicans density was adjusted to 1 × 106 CFU/mL and 100 μL of this suspension added to 96-well microtiter plates and incubated for 24 h at 37 °C for biofilm adhesion and formation, followed by 24 h at 37 °C in the presence or absence of the essential oils.Serial dilutions of the essential oil (1125–72 µg/mL) and fluconazole (200–1 µg/mL) were used to assess their effect on mature biofilms, as reported elsewhere . C. albiBiofilm viability was determined using the XTT/menadione metabolic assay. Briefly, menadione (10 mM in acetone) was added to a XTT solution (1 mg/mL in PBS) to a final concentration of 4 µM. A volume of 100 µL of this solution was added to each well and incubated for 2 h at 37 °C in the dark. The absorbance was measured at 490 nm and cell viability expressed as [(AbsT/AbsC) × 100], where AbsT represents the absorbance of the treated cells and AbsC the absorbance of control cells .Biofilm biomass was assessed using the crystal violet assay. Following the treatment period, performed as referred above, the culture medium was removed and cells were fixed with methanol 99% for 15 min. The supernatant was discarded, and the wells air dried. Then, 100 µL of crystal violet (CV) solution (0.02%) was added to each well and left to stain the biofilm for 15 min. After CV removal, the wells were washed twice with sterile water to remove excessive reagent. Then, 150 µL of acetic acid 33% were added to release the stain from the cells and the supernatant was transferred to a 96-well microtiter plate. The absorbance was then read at 620 nm using a microtiter plate reader. Biomass decrease was determined as [(AbsT/AbsC) × 100], where AbsT represents the absorbance of each treated well and AbsC the absorbance in the control wells. Negative and positive controls comprising biofilm-free and oil-free wells, respectively, were also included.v/v) non-inactivated fetal bovine serum, 3.02 g/L sodium bicarbonate, 100 µg/mL streptomycin and 100 U/mL penicillin at 37 °C in a humidified atmosphere with 5% CO2. Morphological appearance of macrophages was microscopically monitored during the assays.The mouse macrophage cell line, Raw 264.7 (ATCC number: TIB-71) was kindly supplied by Dr. Otilia Vieira . The cells were cultured on endotoxin-free Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (6 cells/well) were cultured in a 48-well microplate and left to stabilize for 12 h. The cells were then incubated for 24 h in the culture medium alone (control) or with different concentrations of the essential oils (144–576 µg/mL).The production of nitric oxide (NO) was measured by the accumulation of nitrites in the culture medium, using the colorimetric Griess reaction. Briefly, 170 µL of culture supernatants were added to an equal volume of Griess reagent [0.1% (w/v) N-(1-naphthyl)-ethylenediamine dihydrochloride and 1% (w/v) sulphanilamide containing 5% (w/v) H3PO4] and maintained for 30 min, in the dark. Absorbance was measured using an ELISA automatic microplate reader  at 550 nm and the nitrite concentration determined from a regression analysis prepared with serial dilutions of sodium nitrite.To evaluate the anti-inflammatory potential of the oils, the in vitro model of lipopolysaccharide (LPS)-stimulated macrophages was used. The cells (0.6 × 102. Supernatants were centrifuged (1000× g during 5 min) to recover viable cells. To dissolve formazan crystals formed in adherent cells in the microplates, 300 µL of acidified isopropanol (0.04 M HCl in isopropanol) were added to each cell and recovered to the respective tube containing the pellet formed after centrifugation. Quantification of formazan was performed using an ELISA automatic microplate reader  at 570 nm.Cell respiration, an indicator of cell viability, was performed using a colorimetric assay with 3--2,5-diphenyl tetrazolium bromide (MTT). The cells were cultured and treated as referred above. After cells treatments, 43 µL of MTT solution  were added to each well and the microplates were further incubated at 37 °C for 15 min, in a humidified atmosphere with 5% COThe results presented as mean ± SD are from experiments performed at least in triplicate. The statistical test one-way ANOVA followed by Dunnett’s Multiple Comparison Test were performed using GraphPad Prism, version 6 .