Lasiodiplodia theobromae fungus (LTL1), which hydrolyses C8–C10 ME efficiently. LTL1 can perform hydrolysis over pH ranges from 3.0 to 9.0 and maintain thermotolerance up to 70 °C. It has high selectivity for monoesters over triesters and displays higher activity over commercially available lipases for C8–C10 ME to achieve 96.17% hydrolysis within 31 h. Structural analysis by protein X-ray crystallography revealed LTL1’s well-conserved lipase core domain, together with a partially resolved N-terminal subdomain and an inserted loop, which may suggest its hydrolytic preference for monoesters. In conclusion, our results suggest that LTL1 provides a tractable route towards to production of C8–C10 fatty acids from methyl esters for the synthesis of MCTs.Medium-chain triglycerides (MCTs) are an emerging choice to treat neurodegenerative disorders such as Alzheimer’s disease. They are triesters of glycerol and three medium-chain fatty acids, such as capric (C8) and caprylic (C10) acids. The availability of C8–C10 methyl esters (C8–C10 ME) from vegetable oil processes has presented an opportunity to use methyl esters as raw materials for the synthesis of MCTs. However, there are few reports on enzymes that can efficiently hydrolyse C8–C10 ME to industrial specifications. Here, we report the discovery and identification of a novel lipase from  Caprylic C8) and capric C10) triacylglycerols, commonly known as medium-chain triglycerides (MCTs), are widely used in food ingredients, nutritional supplements, and medical treatment for neurological diseases. A major therapeutic option is the use of MCTs in the treatment of Alzheimer’s disease (AD). AD is an age-associated and neurodegenerative condition that progressively reduces the individual’s cognitive memory and language abilities. Besides being a major health problem, AD has severe physical, financial, psychological, and social consequences for the patient and the patient’s family. Due to the ketogenic nature of MCTs , the utility of MCTs in the treatment of AD is currently being actively investigated 0 triacyl,3 and sl and caprIndustrial manufacturing of C8 and C10 MCTs largely involves the direct esterification of glycerol and medium-chain fatty acids under high pressure and temperature to form tricaprylin and tricaprin ,12.Candida antarctica [Rhizomucor miehei [Thermomyces lanuginosus [To obtain a steady source of C8 and C10 fatty acids, hydrolysis of C8-C10 ME can be carried out enzymatically with lipases and esterases. These enzymes catalyse the hydrolysis of ester bonds of tri-, di-, and monoglycerides into fatty acids and alcohol . Howevertarctica ,15, Rhizr miehei , and Theuginosus ,18 are nuginosus  had demoLasiodiplodia theobromae (L. theobromae) from our Wilmar International in-house culture collection. We first screened several microbial species for their growth on minimal media with tri- and mono-glycerides as a sole carbon and energy source. L. theobromae lipase 1 (LTL1) displayed the highest activity during zymographic analysis on chromogenic agar supplemented with tri- and mono-glycerides. Fed-batch fermentation was conducted to recombinantly express LTL1 in Pichia pastoris (P. pastoris) to characterise LTL1’s substrate, pH, and temperature specificities. Upon comparison, LTL1 outperformed various commercially available lipolytic enzymes and was able to match up against industrially relevant hydrolytic benchmarks for C8-C10 ME. Lastly, protein X-ray crystallographic studies were carried out to understand LTL1’s hydrolytic preference for monoesters. Taken together, LTL1 is a very promising candidate enzyme that can be used in the hydrolysis of medium-chain length monoesters for the industrial synthesis of MCTs.Here in this study, we present a novel lipase originally isolated from the filamentous fungal strain L. theobromae. This fungal strain was able to utilise olive oil as its sole carbon and energy source for growth (L. theobromae culture was separated with native-polyacrylamide gel electrophoresis (Native-PAGE) followed by zymographic analysis on chromogenic phenol red agar plate supplemented with monolaurin or tributyrin, as previously described [L. theobromae (NCBI Genome ID 54334) [L. theobromae; for brevity and clarity, this lipase will be referred to as LTL1 for the remainder of the study   to identhe study C. The re seriata D. P. pastoris codon-optimised LTL1 was cloned into a pPIC9K vector, under the control of methanol inducible AOX1 promoter, for extracellular expression in P. pastoris. Following the manufacturer’s protocol [Heterologous protein expression was carried out for the enzyme for functional characterisation: the gene encoding protocol , a glyceTo determine the substrate specificity of LTL1, we conducted hydrolysis assays against a panel of substrates including triesters  and monoesters . Among the group of triesters studied, lower hydrolytic activities were detected for olive oil and coconut oil, while higher hydrolytic activity was detected for tributyrin, which indicates the stronger preference for shorter-chain triesters. In contrast, the hydrolytic activities of LTL1 were much higher for monoesters compared to triesters A. ConsisTo determine the optimal pH and temperature profiles of LTL1, hydrolysis assays with tributyrin were conducted over a range of pH (from 3.0 to 10.0) and temperatures (from 20 to 90 °C). LTL1 hydrolytic activity was maintained throughout the pH range of 3.0 to 9.0 B, with pThermomyces lanuginosus lipase (TLL), Rhizomucor miehei lipase (RML), Candida antarctica lipase B (CALB), and Eversa 2.0. The C8-C10 ME used in this study was a mixture of 53% C8 ME and 46% C10 ME, respectively . These hydrolytic reactions were carried out at 37 °C, pH 7.2. We observed that LTL1 outperformed all commercial enzymes used in this study in the hydrolysis of vinyl laurate and C8-C10 ME, displaying activities of 11,000 and 3000 U/mg, respectively  of LTL1 was able to perform 96% of C8-C10 ME hydrolysis within 31 h, achieving an AV of 353 . The degree of hydrolysis is usually monitored by measuring the acid value (AV) of the hydrolysed product. In this context, a completely hydrolysed (100%) C8-C10 ME mixture would give an AV of 365. To determine if LTL1 can achieve the minimal 95% hydrolysis of C8-C10 ME, we performed the hydrolysis reactions and compared the performance of LTL1 against two commercial lipases, Eversa 2.0 and RML, respectively. Over two days, 100 parts per million (ppm) dosage (V of 353 . In the L. theobromae lipase B (UniprotKB: A0A5N5DNA6), respectively. These residues fold into lipase core domains  [C. antarctica  [A Dali search ,26 with D: 6IDY)  and C. aD: 5A71) , with reD: 5A71) . As showD: 5A71) .Reagents described in all experiments were sourced from Sigma-Aldrich (Singapore) unless stated otherwise. C8-C10 ME was obtained from Wilmar Oleochemicals Limited . Monoolein was obtained from Kao Chemicals, Japan. CALB , Eversa Transform 2.0 (Sigma-Aldrich SAE0065), and RML , TLL  were used as lipolytic enzyme standards.L. theobromae screened for lipolytic enzymes was obtained from an in-house culture collection. They were kept in glycerol stocks at −80 °C and maintained on YPD  agar (20 g/L) at 4 °C. When needed, L. theobromae was cultured in YPD liquid at 220 revolutions per minute (rpm), 30 °C . Escherichia coli DH5α used for vector propagation was obtained from Thermo Fisher, Singapore. They were kept in glycerol stocks at −80 °C and maintained on Lysogeny Broth (LB) agar  agar (15 g/L) at 4 °C. When needed, DH5α was cultured in LB liquid at 220 rpm, 37 °C.P. pastoris GS115 used for protein expression was obtained from Invitrogen, Singapore. They were kept in glycerol stocks at −80 °C and maintained on YPD agar 4 °C. When needed, GS115 was cultured in YPD liquid at 220 rpm, 30 °C.Liquid culture used to screen for lipolytic-producing enzyme contained minimal media  sulphate) , where aL. theobromae was separated in native conditions with Native-PAGE [Detection of lipase activity was performed with Zymography-LC-MS, as previously described . Brieflyive-PAGE . Native-ive-PAGE ,31, wherv/v formic acid  in distilled water as solvent A and 0.1% v/v formic acid in acetonitrile  as solvent B. The running gradient was as follows: 5% solvent B from 0 to 2 min, 5 to 70% solvent B from 2 to 21 min, 70 to 99% solvent B from 21 to 21.3 min, 99% solvent B from 21.3 to 25.5 min, 99 to 1% solvent B from 25.5 to 26 min, and 1% solvent B from 26 to 30 min. Data acquired on the mass spectrometer were operated in full scan mode followed by data-dependent tandem mass spectrometry (MS/MS) acquisition over a mass range of 300–1800 m/z.Digested peptides were resuspended in acetonitrile and analysed with an Ultimate 3000 High-Performance Liquid Chromatography system  coupled to Q Exactive Plus Orbitrap Mass Spectrometer , as previously described . Then, 5For peptide identification on the LC-MS, data analysis was performed with PEAKS Studio 7.5 (build 20150615) as previously described . SequencImages were visualised by open-source software Fiji (ImageJ) . The briP. pastoris GS115-pPIC9K-LTL1 was constructed by inserting a full-length codon optimised sequence of LTL1 for P. pastoris into the open reading frame of α-factor secretion signal sequence of expression vector pPIC9K  via the restriction sites SnaBI and EcoRI. Vector propagation was carried out in DH5α and selected with LB agar supplemented with kanamycin (50 µg/mL). Transformed colonies were screened for the correct inserts with colony polymerase chain reaction (PCR) and subsequently verified with Sanger Sequencing. Verified pPIC9K-LTL1 vector were linearised at SalI and transformed into P. pastoris GS115 competent cells by electroporation [LTL1 sequence with genomic DNA PCR, and subsequently maintained on BMGY  agar (20 g/L) plates.poration . TransfoLTL1 was prepared in BMGY medium. Fed-batch fermentation was conducted in 1 L bioreactor  containing 400 mL of basal salt medium  supplemented with 4.35 mL PTM (6 g/L copper (II) sulphate, 80 mg/L sodium iodide, 3 g/L manganese sulphate monohydrate, 200 mg/L sodium molybdate dihydrate, 20 mg/L boric acid, 500 mg/L cobalt chloride, 20 g/L zinc chloride, 65 g/L iron (II) sulphate heptahydrate, 200 mg/L biotin, and 5 mL/L sulphuric acid), as per the manufacturer’s protocol [v/v ammonium hydroxide. The airflow rate was set at 1 vvm (vessel volume per minute), the agitation speed was set at 1000 rpm, and the dissolved oxygen (DO) was maintained at greater than 30% saturation through a mixed supply of compressed air  and pure oxygen . The entire fermentation was conducted in three phases. Phase I was the glycerol batch phase, where vessel temperature was maintained at 30 °C until glycerol was fully consumed, indicated by a sharp spike of DO. Phase II was the glycerol fed-batch, 50% v/v glycerol with 12 mL/L of PTM was fed for an additional 4 h. Phase III was the methanol fed-batch, vessel temperature was lowered to 28 °C and pure methanol  with 12 mL/L of PTM was fed continuously to induce protein production for 96 h. The entire fermentation was periodically monitored for biomass (WCM), lipolytic activity, and protein concentration.The seed culture of GS115-pPIC9K-protocol . The pH Lipolytic enzymatic activity was measured using the appropriate tri- and mono-esters as substrate. One unit of activity was defined as the amount of enzyme catalysing the lipolysis of 1 µmol fatty acid per min .For pH specificity, hydrolysis was performed using 20 µL of LTL1, 120 µL distilled water, 60 µL of different 0.2 M buffer ranging from pH 3.0 to 10.0 and 100 µL of tributyrin in a 2 mL tube. The reaction was performed at 37 °C, 2000 rpm  for 15 min. The reactions were stopped by adding an equal volume of pure ethanol, and free fatty acid liberated was neutralised with 50 mM sodium hydroxide.For temperature specificity, hydrolysis was performed using 20 µL of LTL1, 120 µL distilled water, 60 µL 0.2 M Tris-HCl buffer (pH 7.2), and 100 µL of tributyrin in 2 mL tubes. The reaction was performed at temperatures ranging from 20 to 100 °C, 2000 rpm for 15 min. The reactions were stopped by adding an equal volume of pure ethanol and free fatty acid liberated was neutralised with 50 mM sodium hydroxide.For substrate specificity, hydrolysis was performed using 20 µL of LTL1, 120 µL distilled water, 60 µL 0.2 M Tris-HCl buffer (pH 7.2) and 100 µL of different substrates  in a 2 mL tube. The reaction was performed at 37 °C, 2000 rpm for 15 min. The reactions were stopped by adding an equal volume of pure ethanol, and free fatty acid liberated was neutralised with 50 mM sodium hydroxide.For comparison with commercial lipases, hydrolysis of vinyl laurate and C8-C10 ME were carried out with TLL, RML, CALB, and Eversa. The reaction was performed using 20 µL of enzyme, 120 µL water, 60 µL 0.2 M Tris-HCl buffer (pH 7.2), and 100 µL substrates in a 2 mL tube. The reaction was performed at 37 °C, 2000 rpm for 15 min. The reactions were stopped by adding an equal volume of pure ethanol, and free fatty acid liberated was neutralised with 50 mM sodium hydroxide.rcf for 30 min. The acid value of the top organic layer was determined with the AOCS Cd 3d-63 method [To screen LTL1 with industrial hydrolysis conditions, C8-C10 ME hydrolysis reactions were performed with 20 g methyl esters, 40 g distilled deionised water, and a corresponding dosage of lipases at 45 °C, 50 mbar of vacuum. After hydrolysis, the mixture was centrifuged at 7000 3 method .P. pastoris fermentation culture was centrifuged at 8000 rcf, 4 °C for 30 min and the supernatant was harvested, and the cell pellet was discarded. The supernatant was then filtered through a 0.22 μm PES (polyethersulfone) membrane  to remove any remnant microbial impurities. The supernatant was concentrated with Amicon Ultra-15 10 kDa Centrifugal Filter Unit  and buffer was exchanged to 10 mM Tris-HCl, pH 8.0 with 150 mM sodium chloride. The final concentration of LTL1 from fermentation was calculated to be approximately 17.48 g/L.The v/v 2-methyl-2,4-pentanediol, 100 mM sodium acetate, pH 4.6, 10 mM calcium chloride) and incubated at 20 °C. Crystals were cryoprotected with a 1:1 mixture of the same precipitant and saturated sodium malonate before being flash-frozen in liquid nitrogen. Diffraction data were collected at the MX1 beamlines of the Australian Synchrotron, part of ANSTO [Crystallisation of LTL1 was conducted using an NT8 drop setter for protein crystallisation  and 96-well 2-drop MRC sitting-drop vapor diffusion plates . Next, 100 nL of lipase solution (17.48 g/L) was mixed with 100 nL of a precipitant . Lastly, the protein X-ray crystallographic structure of LTL1 revealed the presence of the additional N-terminal subdomain and inserted loop covering the active site, which seems to suggest that steric effects could be part of the reason for LTL1’s preference for slimmer monoesters over bulkier triesters. Taken together, our results suggest that LTL1 provides a tractable route towards the production of C8–C10 fatty acids from methyl esters for the synthesis of MCTs. We believe that our newly discovered enzyme could be broadly applied as a greener choice to prepare the precursors for MCTs synthesis for therapeutic applications.A patent for the use of LTL1 for C8-C10 ME hydrolysis was filed in Singapore at the Intellectual Property Office of Singapore (application number 10201914033Y) on 31 December 2019 and published in the United States of America (publication number US2021198704A1 ) on 1 Ju


Holothuria fuscopunctata. As a selective inhibitor of intrinsic Xase (iXase), preclinical study showed it was a promising anticoagulant candidate without obvious bleeding risk. In this work, two bioanalytical methods based on the anti-iXase and activated partial thromboplastin time (APTT) prolongation activities were established and validated to determine dHG-5 concentrations in plasma and urine samples. After single subcutaneous administration of dHG-5 at 5, 9, and 16.2 mg/kg to rats, the time to peak concentration (maxT) was at about 1 h, and the peak concentration (maxC) was 2.70, 6.50, and 10.11 μg/mL, respectively. The plasma elimination half-life(1/2βT) was also about 1 h and dHG-5 could be almost completely absorbed after s.c. administration. Additionally, the pharmacodynamics of dHG-5 was positively correlated with its pharmacokinetics, as determined by rat plasma APTT and anti-iXase method, respectively. dHG-5 was mainly excreted by urine as the unchanged parent drug and about 60% was excreted within 48 h. The results suggested that dHG-5 could be almost completely absorbed after subcutaneous injection and the pharmacokinetics of dHG-5 are predictable. Studying pharmacokinetics of dHG-5 could provide valuable information for future clinical studies.dHG-5 (Mw 5.3 kD) is a depolymerized glycosaminoglycan from sea cucumber  Cardiovascular diseases are the main cause of a global health burden, with high morbidity and mortality ,2. TheirHolothuria fuscopunctata. dHG-5 was prepared from natural HG by β-eliminative depolymerization and it showed potent anti-iXase activity in vitro and an antithrombotic effect in venous thrombosis model with low bleeding risk 3-D-GlcA-β1,}n-3-D-GalNAc4S6S-β1,4- 3-D-GlcA-ol (n = 0–8) [nHG was extracted and purified from sea cucumber escribed . dHG-5 (escribed . It was n = 0–8) ,15 (SuppSprague-Dawley (SD) male rats, weighing 250–280 g, were purchased from Hunan SJA Laboratory Animal Co., Ltd., Changsha, China (License No. SCXK (Xiang) 2016-0002). Animals were maintained in an air-conditioned animal house at the temperature of 24 ± 2 °C, relative humidity of 40% to 60%. Animals were housed six per cage, eating and drinking ad libitum, and allowed to accommodate for about a week prior to experiment. Experiments were approved by the Animal Ethics Committee of Kunming Institute of Botany, Chinese Academy of Sciences.Preparation of Standard Samples: Stock solution of dHG-5 (1.92 mg/mL) was prepared with saline. The dHG-5 standard samples at 0.0625, 0.125, 0.5, 1, 8 and 16 µg/mL were prepared from stock solution which was diluted with drug-free rat plasma. Quality control (QC) samples at 0.131, 0.819, and 12.8 µg/mL were similarly prepared.2 solution, and the pretreated sample was ready for anti-iXase activity.Pretreatment of Plasma Samples: The rat plasma sample (200 μL) was centrifuged at 10,000 rpm for 10 min, then the supernatant (150 µL) was collected to another tube. In order to hydrolyze the plasma proteins that would interfere the detection, 5% alkaline protease solution (16.5 µL) and 1 M NaOH solution (3 µL) were added, mixed by vortex, and the mixed solution was sealed, and incubated at 50 °C for 15 h. After this, the sample was centrifuged at 13,000 rpm for 10 min, then 90 µL of the supernatant was collected. The supernatant was mixed with 10 µL of 0.4 M CaCl2+, human thrombin) were added in a 96-well microtiter plate and incubated at 37 °C for 2 min. Then 30 µL 50 nM FX (containing thrombin inhibitor) was added, and incubated at 37 °C for 1 min. Then the FXa activity which presents residual iXase activity, was detected by the addition of 30 μL SXa-11 (FXa chromogenic substrate). Optical density (OD) at 405 nm of the mixed solution was recorded by a Microplate Reader  with a kinetic method (read per 30 s for 2 min at 37 °C). The change rate in OD (ΔOD/min) of each sample was calculated, which represented the activity of iXase. ΔOD/min of the control was set as 100%, and the relative iXase activity of pretreated samples (% of control) was calculated. Finally, the concentration of dHG-5 in each sample was calculated according to the calibration curve introduced above.Bioanalysis: Anti-iXase activity was determined by chromogenic substrate method ,28,29,30Method Validation: Anti-iXase method was validated according to the standard guideline . LineariThe calibration curve was fitted by linear regression of the relative iXase activity (% of control) and logarithmic dHG-5 plasma concentration of a series of standard samples. The lowest plasma concentration of dHG-5 in the calibration curve was considered as the lower limit of quantification (LLOQ), whose accuracy and precision deviations were required to be within 20%.Accuracy and precision were evaluated using three QC concentrations. The accuracy was calculated as (mean measured concentration/labeled concentration) × 100%, and the precision was assessed by relative standard deviation (RSD).The dHG-5 levels in some rat plasma samples were beyond the highest concentration of the calibration curve, thereby validation of dilution reliability was conducted. Samples (dHG-5 at 64 µg/mL) were diluted 12.5-fold, the activity of diluted samples were tested, and the concentrations were calculated from the calibration curve.The stability of dHG-5 in rat plasma was verified by three QC concentrations at −20 °C for 30 d.All the experiments were repeated at least three times. For the above validation experiments, both the accuracy and precision of the tested concentrations should be within ±15%, and that of the concentration around LLOQ within ±20% is acceptable.g for 10 min to obtain the plasma, then stored at −20 °C before analysis. The plasma concentrations of dHG-5 were determined by anti-iXase method as described in 4.3.1. The selection of the most suitable compartment model was based on the minimum Akaike’s information criterion (AIC), and the pharmacokinetic parameters were analyzed from the concentration-time data using a two-compartment pharmacokinetic model by DAS 2.0 software . Absolute bioavailability of dHG-5 at different doses was acquired by the formula: F% = (AUCs.c. × dosei.v.)/(AUCi.v. × doses.c.) × 100. The statistical significance of results was determined by GraphPad Prism 7.00 software using one-way analysis of variance (ANOVA) followed by Dunn’s multiple comparisons test. p values of less than 0.05 were considered significant, and p values of more than 0.05 were considered not significant.Rats were weighed and randomly assigned into six groups (n = 5/group). For s.c. administration, three animal groups were dosed with dHG-5 at 5 mg/kg, 9 mg/kg and 16.2 mg/kg. For i.v. injection, another three groups were dosed with dHG-5 at 5.2 mg/kg, 9 mg/kg, and 16.2 mg/kg. After single dose s.c. administration, blood (0.45 mL) was collected from the ocular vein and anticoagulated with 50 μL of 3.8% sodium citrate solution at 0, 0.25, 0.5, 1, 2, 4, 6, and 8 h. Similarly, for single dose i.v. administration, blood collection was conducted before and at 0.033, 0.083, 0.167, 0.333, 0.75, 1, 1.5, 2, 4, 6, and 8 h after dosing. Each blood sample was immediately centrifuged at 3000× g for 15 min to obtain the plasma for APTT assay. The APTT of rat plasma samples was detected with a coagulometer  as previously reported [Rats were randomly divided into six groups (n = 5/group). Animals were grouped and treated with dHG-5 as described in 4.3.2. After i.v. (or s.c.) administration of dHG-5, blood (0.27 mL) was drawn from the ocular vein and anticoagulated with 30 µL of 3.8% sodium citrate solution at 0, 0.083, 0.167, 0.333, 0.75, 1, 1.5, 2, 4, 6, and 8 h . Each blood sample was then centrifuged at 1800× reported . Moreove1H/13C dual probe in FT mode at 298 K.Metabolization of dHG-5 was analyzed by HPLC and NMR method. After administration of 5.2 mg/kg (i.v.) or 5 mg/kg (s.c.) dHG-5 to rats (n = 5), the urine sample during 0–48 h was collected and concentrated. Then, the sample was purified with a FPA98 strong ion-exchange chromatography (1.5 × 15 cm), sequentially eluted with NaCl solution at 0, 0.5, 0.8, 1.0, 1.2, 1.5, and 2.0 M at 2-fold bed volume (BV). Each eluted fraction was desalted by a Sephadex G-25 column, and analyzed by HPLC with a Superdex peptide 10/300 G L column and a differential refractive index detector (RID), eluted by 0.2 M NaCl eluent at the flow rate of 0.4 mL/min. The eluates contained dHG-5 were lyophilized into powders and analyzed by NMR with a Bruker Advance 600 MHz spectrometer and a Preparation of standard samples: Mother solution of dHG-5 at 0.96 mg/mL was prepared with drug-free rat urine which was collected for 24 h from normal rats. A series of rat urine standard samples at 7.5, 15, 30, 60, 120, and 240 µg/mL were obtained by diluting the mother solution with drug-free rat urine. Likewise, rat urine QC samples  were prepared.Pretreatment of Urine Samples: The rat urine sample was centrifuged  to obtain the supernatant, then filtering the supernatant through a 0.45 µm microporous membrane, the filtrate was collected as the pretreated sample for APTT prolongation detection.2 reagent (50 µL). The prolongation of APTT (ΔAPTT) of each sample was defined by subtracting the APTT of control. The calibration curve ΔAPTT-dHG-5 urine concentration was introduced above, and the dHG-5 concentrations in urine samples were calculated accordingly.Bioanalysis: A 5 µL pretreated sample (drug-free rat urine sample as control) and 45 µL of human coagulation control plasma were pipetted into a blood clotting cup, then incubated at 37 °C for 2 min. Then, 50 µL of APTT reagent was added and incubated at 37 °C for 3 min. The clotting time was recorded after the addition of CaClMethod validation: Similar to the validation of anti-iXase method as described in 4.3.1, APTT prolongation method was validated in linearity, accuracy, precision and stability.Ten rats were randomly allocated to two group (n = 5/group), in which dHG-5 was administrated at 5.2 mg/kg (i.v.) or 5 mg/kg (s.c.). After dosing, the rats were housed in separate metabolic cages for urine collection, and urine during 0–12, 12–24, 24–48 h was collected. Urine was centrifuged at 4000 rpm for 5 min, and the supernatant was filtered via a 0.45-µm microporous membrane. The pretreated urine sample was then frozen at −20 °C until APTT prolongation detection.maxC, AUC, and rat plasma APTT values increased dose-dependently, indicating that the PK/PD of dHG-5 was well correlated. In addition, dHG-5 was mainly excreted by urine as the unchanged parent drug, and about 60% of it was excreted within 48 h. These results suggested that dHG-5 has predictable pharmacokinetic characteristics, and that s.c. administration should be an appropriate delivery method for its possible clinical application in the future. Together, these results provide important insights into the PK/PD of dHG-5, implying the potential druggability of dHG-5.In this work, two biological methods were developed, validated, and then applied to study the pharmacokinetics of dHG-5 in rats. To our best knowledge, the pharmacokinetics of dHG-5 studied by the anti-iXase method and the excretion of dHG-5 studied by the APTT prolongation method are reported for the first time. The results showed that, by s.c. administration of dHG-5 to rats, the absolute bioavailability of dHG-5 was high (above 90%), suggesting that dHG-5 was almost completely absorbed.


Background: Acute lung injury (ALI) is characterized by dysfunction of the alveolar epithelial membrane caused by acute inflammation and tissue injury. Qingwenzhike (QWZK) prescription has been demonstrated to be effective against respiratory viral infections in clinical practices, including coronavirus disease 2019 (COVID-19) infection. So far, the chemical compositions, protective effects on ALI, and possible anti-inflammatory mechanisms remain unknown.Methods: In this study, the compositions of QWZK were determined via the linear ion trap/electrostatic field orbital trap tandem high-resolution mass spectrometry (UHPLC-LTQ-Orbitrap MS). To test the protective effects of QWZK on ALI, an ALI model induced by lipopolysaccharide (LPS) in rats was used. The effects of QWZK on the LPS-induced ALI were evaluated by pathological changes and the number and classification of white blood cell (WBC) in bronchoalveolar lavage fluid (BALF). To investigate the possible underlying mechanisms, the contents of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein (MCP-1), interleukin-1β (IL-1β), interleukin-18 (IL-18), and immunoregulatory-related factors interferon-γ (IFN-γ) were detected by ELISA. Furthermore, the expression of Toll-like receptor 4 (TLR4), p-IKKα/β, IKKα, IKKβ, p-IκBα, IκBα, p-NF-κB, nuclear factor-κB (NF-κB), NOD-like receptor family pyrin domain containing 3 (NLRP3), cleaved caspase-1, pro-caspase-1, apoptosis-associated speck-like protein containing CARD (ASC), and β-actin were tested by Western blot.Results: A total of 99 compounds were identified in QWZK, including 33 flavonoids, 23 phenolic acids, 3 alkaloids, 3 coumarins, 20 triterpenoids, 5 anthraquinones, and 12 others. ALI rats induced by LPS exhibited significant increase in neutrophile, significant decrease in lymphocyte, and evidently thicker alveolar wall than control animals. QWZK reversed the changes in WBC count and alveolar wall to normal level on the model of ALI induced by LPS. ELISA results revealed that QWZK significantly reduced the overexpression of proinflammatory factors IL-6, TNF-α, MCP-1, IL-1β, IL-18, and IFN-γ induced by LPS. Western blot results demonstrated that QWZK significantly downregulated the overexpression of TLR4, p-IKKα/β, p-IκBα, p-NF-κB, NLRP3, cleaved caspase-1, and ASC induced by LPS, which suggested that QWZK inhibited TLR4/NF-κB signaling pathway and NLRP3 inflammasomes.Conclusions: The chemical compositions of QWZK were first identified. It was demonstrated that QWZK showed protective effects on ALI induced by LPS. The possible underlying mechanisms of QWZK on ALI induced by LPS was via inhibiting TLR4/NF-kB signaling pathway and NLRP3 inflammasome activation. This work suggested that QWZK is a potential therapeutic candidate for the treatments of ALI and pulmonary inflammation. The coronavirus disease 2019 (COVID-19) pandemic causes tremendous catastrophe worldwide. During the development of COVID-19 infection. Acute lung injury (ALI) is a critical step and causes high mortality . In ALI,rickettsia, and parasites. Due to the limitation in availability of high levels of bio-safety labs for antiviral studies, lipopolysaccharide (LPS) has been used extensively in studies on inflammatory diseases. LPS is a major microbial mediator in Gram-negative bacterial infection  and pneumonia induced by various microbial pathogens, such as bacteria, viruses, fungi, nfection , and Tolnfection . Meanwhinfection . Thus, Tnfection . It has nfection . So TLR4nfection .Current clinical therapeutic drug for ALI is corticosteroids (CS). On the one hand, it exerts a wide spectrum of bioactivities including anti-inflammatory, antioxidant, pulmonary vasodilator, and antiedematous effects; on the other hand, side effects of CS are evident, such as immunosuppression, osteoporosis, and peptic ulcers . AlthougTraditional Chinese medicine (TCM) has a long history in clinical practices in China. During the COVID-19 pandemic, TCM has been widely used in China and has been demonstrated to show convincing effects. Qingwenzhike (QWZK) is a TCM preparation derived from recombination of ancient Chinese classical prescriptions, including Maxingshigan decoction , SheganmEscherichia coli O55:B5, abs47014848, Absin, Shanghai, China) and dexamethasone  were purchased from Absin and Sigma-Aldrich.Ephedrae Herba (No. 20200103), Gypsum Fibrosum (No. 20200327), Rhei Radix Et Rhizoma (No. 20200107), Belamcandae Rhizoma (No. 20200412), Asteris Radix Et Rhizoma (No. 20200310), Farfarae Flos (No. 20200411), Citri Reticulatae Pericarpium (No. 20200511), Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumin (No. 20200717), Poria (No. 20200904), Armeniacae Semen Amarum (No. 20200414), Cicadae Periostracum (No. 20200813), Fritillariae Thunbergii Bulbus (No. 20200907), Taraxaci Herba (No. 20200528), and Platycodonis Radix (No. 20200816) were provided by Beijing Bencaofangyuan Pharmaceutical Co., Ltd. . The standards including alanine, caffeic acid, quercetin, β-sitosterol, chrysophanol, amygdalin, and hesperidin were purchased from National Institutes for Food and Drug Control. Lipopolysaccharides  were deposited in the Beijing Research Institute of Chinese Medicine, Beijing University of Chinese Medicine.The preparation methods of the QWZK powder were as follows: the crude drugs of QWZK accurately weighed 1.43 kg. These drugs were soaked in 14.30 L  pure water for 30 min and was then boiled for 2 h. Subsequently, they were boiled in 11.40 L  pure water for 1 h twice.The extracts were filtered through three-layer gauze, then combined and concentrated to 66.50 ml in a rotary evaporator at 75°C. The concentrate was vacuum dried, and 0.446 kg dry powder was obtained. The extract rate (%) = extract dry powder/total quality of crude drugs. Therefore, the extract rate of QWZK powder was 31.20%. The powder was used in the follow-up experiments.According to the body surface area (BSA) scaling for converting the dose of a test drug from human clinical trials to animal species , the tesLiquid chromatography was performed using a Dionex Utimate 3000 UHPLC Plus Focused Ultra High-Performance Liquid Chromatography System . Chromatographic separation was achieved through a ACQUITY UPLC C18 column  at a flowrate of 0.3 ml/ min, defended by a high-pressure column prefilter (2 mm)  at 35°C. Mass spectrometric detection was performed with an LTQ-Oribitrap XL linear ion trap tandem electrostatic field orbital trap mass spectrometer  in positive and negative ion modes, which was equipped with an electrospray ion source in MRM modes.QWZK powder was accurately weighed 1.00 g and added into 10.00 ml methanol, ultrasonically treated for 45 min using an ultrasonic cleaning instrument , and the solution was filtrated through 0.22 μm microporous membrane. The standards such as alanine, caffeic acid, quercetin, β-sitosterol, chrysophanol, amygdalin, and hesperidin were weighed in precision, dissolved in methanol with a standard solution of 1 mg/ml, and filtered through 0.22 μm microporous membrane. Samples or strands were separated on an ACQUITY UPLC C18 column  at 35°C. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile solution (B). The gradient elution conditions were as follows: 0–6 min (90–60% A), 6–9 min (60–40% A), 9–42 min (40–20% A), and 42–60 min (20–90% A). The flowrate was 0.3 ml/min, and the injection volume was 3.0 μl.Electrospray ionization mass spectrometry (ESI-MSP) analyses were performed on an LTQ-Oribitrap XL linear ion trap tandem electrostatic field orbital trap mass spectrometer . Samples of QWZK were detected in positive ion detection mode, and the spray and capillary voltages were set to 4.0 KV and 35.0 V, respectively. The tube lens voltage was 110 V, and the source temperature was set to 350°C. Nitrogen (purity >99.99%) was used as both the sheath gas (40 arb) and auxiliary gas (20 arb). Then, samples were analyzed in negative ion detection mode, with the spray and capillary voltages set to 3.0 kV and 35.0 V, respectively. The tube lens was set to 110 V, and the source temperature was set to 350°C. Nitrogen (purity >99.99%) was used as both the sheath gas (30 arb) and auxiliary gas (10 arb). Data-dependent acquisition (ddms3) of high-resolution Fourier transform  and CID fragmentation were used for positive and negative ion data acquisition. The compositions of QWZK were authenticated by referring to the retention time of each chemical component, high-resolution precise molecular weight, and MSn multilevel fragment information detected by LC-MS and combined with the extraction of ion flow map and standard product information and related literature.n = 10/group): the control group was treated by saline only, the LPS group was treated by LPS  only, the dexamethasone group was treated by dexamethasone and LPS, and the QWZK groups were pretreated with QWZK followed by LPS. QWZK was administrated with 3, 6, and 12 g/kg via intragastric (i.g.) administration once per day for 7 consecutive days. LPS was injected intraperitoneally (i.p.) after final injection of medication for 1 h. At 4-h intervals, the left lung was lavaged with cool phosphate-buffered saline (PBS) to collect the bronchoalveolar lavage fluid (BALF); the middle lobe of the right lung was fixed with 4% paraformaldehyde (PFA), and the upper lobe, lower lobe, and accessory lobes were stored at −80°C for protein expression tests.Adult Wistar rats  were purchased from the Vital River Laboratories (SYXK 2016-0006). All the animals were housed in an environment with temperature of 23 ± 1°C, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 h. All animal experimental procedures were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Beijing University of Chinese Medicine. After acclimation for 7 days for 7 days, the rats were randomly assigned into 6 groups  were dewaxed and soaked. Then, they were stained with hematoxylin aqueous solution and eosin staining solution, respectively. Finally, they were dehydrated and rendered transparent and sealed with neutral gum. Sections were observed under a microscope and photographed. A 20-fold field was selected for the statistics of alveolar wall area (%). The airway wall area was detected and calculated with Image-Pro Plus software according to references . We usedTo investigate the expression of proteins by Western blot analyses, animal tissue samples were lysed in a protein cell lysis buffer . The protein concentration of the samples was determined using a bicinchoninic acid (BCA) protein assay kit . The samples were boiled for 10 min, and proteins were separated by electrophoresis using a 10% or 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel. After the transfer of protein to a polyvinylidene difluoride membrane , the membrane was incubated in blocking buffer [5% non-fat dairy milk in Tween-20 Tris-buffered saline (TBST)] for 2 h at ambient temperature and probed with various antibodies in a blocking buffer overnight at 4°C. The membrane was washed four times with 0.1% TBST, probed with a secondary antibody in the blocking buffer for 2 h at ambient temperature and then washed again with TBST. The membranes were detected with an enhanced chemiluminescence kit . The primary antibody included TLR4 , IKKα , IKKβ , p-IKKα/β , IκBα , p-IκBα , NF-κB p65 , p-NF-κB p65 , NLRP3 , pro-caspase-1 + p10 + p12 , ASC/TMS1 , and β-actin . The secondary antibodies include goat antirabbit IgG H&L , goat antimouse IgG H&L .The contents of IL-6, TNF-α, IFN-γ, MCP-1, IL-1β, and IL-18 were determined by ELISA method. The lung tissues were crushed with balls at 0–4°C and centrifuged at 12,000 rpm for 20 min. Protein concentration was determined by BCA protein assay kit . The levels of IL-6, TNF-α, IFN-γ, MCP-1, IL-1β, and IL-18 in lung tissues were measured by using commercially procured ELISA assay kits, including IL-6 ELISA kits , TNF-α ELISA kits , IFN-γ ELISA kits , MCP-1/CCL2 ELISA kits , IL-1β ELISA kits , and IL-18 ELISA kits .British Journal of Pharmacology on experimental design and analysis in pharmacology . All data were presented as means ± standard error of mean (SEM). All rights reserved based on at least three independent experiments and analyzed on GraphPad Prism 8.0 . Statistical analysis was undertaken for studies where each group rats were at least n = 8. Statistical data conforming to a Gaussian distribution was performed either with one-way analyses of variance (ANOVAs) followed by Fisher’s least significant difference (homogeneity of variances) and Tamhane T2 (heterogeneity of variance) post-hoc test using SPSS version 25 for windows . Mann–Whitney U test was applied to data analysis of abnormal distribution. p < 0.05 was considered statistically significant.The data and statistical analysis comply with the In this study, the chemical compositions of QWZK were analyzed by UHPLC-LTQ-Orbitrap MS. There were 21 compounds identified in the negative spectrum and 78 compounds were identified in the positive spectrum. A total of 99 compounds were identified, including 33 flavonoids, 23 phenolic acids, 3 alkaloids, 3 coumarins, 20 triterpenoids, 5 anthraquinones, and 12 others . The lis3 cells/μl) of leukocyte in BALF of the rats in LPS group was significantly increased than that in the control group (1.63 ± 0.26 × 103 cells/μl), and the number of leukocyte in BALF of rats in the dexamethasone group and QWZK 3 g/ kg group, QWZK 6 g/ kg group, and QWZK 12 g/ kg group were 1.24 ± 0.11 × 103 cells/μl, 1.94 ± 0.35 × 103 cells/μl, 1.38 ± 0.41 × 103 cells/μl, and 1.69 ± 0.30 × 103 cells/μl, respectively, which were declined significantly than that in the LPS group (3 cells/ μl) and proportion (84.17 ± 2.27%) of neutrophils in BALF of LPS group were enhanced significantly compared with those of control group . The dexamethasone and QWZK 3 g/ kg, QWZK 6 g/ kg, and QWZK 12 g/kg treatments all decreased the number and proportion of neutrophils in BALF , and QWZK treatment increased compared with that of the LPS group , QWZK 3 g/kg (73.39 ± 3.74 pg/mg), QWZK 6 g/kg (61.22 ± 18.07 pg/mg), and QWZK 12 g/kg (70.25 ± 11.02 pg/ mg) administration, respectively, which was in contrast with that of the LPS group (83.17 ± 4.82 pg/ mg). Similarly, LPS induced IFN-γ production, and dexamethasone (24.28 ± 9.38 pg/mg), QWZK 3 g/kg (46.43 ± 7.64 pg/mg), QWZK 6 g/kg (26.63 ± 10.98 pg/mg), and QWZK 12 g/kg (59.34 ± 6.64 pg/mg) treatments could reverse the IFN-γ level to normal .Among them, the QWZK 6 g/kg group showed the optimal effect on the downregulation of proinflammatory cytokines, and the inhibition effect on some cytokines, such as TNF-α, IL-18, and IFN-γ, were better than dexamethasone.As is well-known, the TLR4/NF-κB signaling pathway is involved in regulating proinflammatory factors . Further, we investigated the effect of QWZK on TLR4/NF-κB signaling pathway. As shown in via activation of NLRP3 inflammasomes. Western blot results showed that the expression of NLRP3, cleaved caspase-1 and ASC increased significantly in the rats of the LPS group compared with those in control group. All of dexamethasone, QWZK 3 g/kg, QWZK 6 g/kg, and QWZK 12 g/kg treatments could downregulate NLRP3, cleaved caspase-1, and ASC expression  and iNOS, and NF-κB activation , tumor necrosis factors (TNFs), interleukins (ILs), and chemokines . The repTo investigate the underlying mechanisms of protective effects of QWZK on ALI induced by LPS, TLR4/NF-κB signaling pathway was studied. It is well known that LPS can activate the TLR4/NF-κB signaling pathway and initiate the transcription of its downstream inflammatory cytokines IL-6, TNFα, IL-1, and chemokines . Our stuThe NLRP3 inflammasome is critical for host immune defenses against bacterial, viral, and fungal infections. The activation of NLRP3 inflammasome needs a priming signal. For example, ligands for TLRs or cytokine receptors could activate the transcription factor NF-κB . NF-κB cvia inhibiting TLR4/NF-kB pathway and NLRP3 inflammasome activation and then downregulated the expression of IL-6, TNF-α, MCP-1, IL-1β, IL-18, and IFN-γ (Generally, our study has verified that LPS could activate the TLR4/NF-kB pathway and NLRP3 inflammasome activation; upregulate the level of some proinflammatory cytokines, chemokines, and lymphokine; and ultimately lead to ALI . QWZK cand IFN-γ . In presnd IFN-γ . The posvia inhibiting TLR4/NF-kB signaling pathway and NLRP3 inflammasome activation. Our work suggested that QWZK might be a potential therapeutic candidate for the treatment of ALI and pulmonary inflammation.The chemical compositions of QWZK were first identified. It was demonstrated that QWZK showed protective effects on LPS-induced ALI. The possible underlying mechanisms of QWZK on ALI induced by LPS was


Selenomonas ruminantium subsp. lactilytica (NBRC No. 103574) with different main species of hydrocarbon chains at the sn-1 position and shorter fatty acids at the sn-2 position than animal plasmalogens. Optimum culture conditions of S. ruminantium for high-yield production of plasmalogens, such as pH and the concentration of caproic acid, were investigated under anaerobic conditions using a 2-L scale jar fermenter. The obtained plasmalogen mainly consisted of the ethanolamine plasmalogen (PlsEtn). The molar ratios of PlsEtn species obtained from S. ruminantium, at sn-1/sn-2 positions, were p16:1/14:0 (68.4%), p16:1/16:1 (29.2%), p16:1/16:0 (0.7%), p16:1/15:0 (0.3%), and p17:1/14:0 (0.3%). Subsequently, duodenal infusion of the emulsion carrying the lipid extracted from S. ruminantium was carried out in lymph duct-cannulated rats. In the lymphatic plasmalogen of rats, the level of PlsEtns with molar ratios p16:1/14:0 and p16:1/16:1, the main species of plasmalogens from S. ruminantium, increased gradually until 3–4 h after lipid injection and then gradually decreased. In addition, the level of PlsEtns with p16:1/20:4 and p16:1/22:6 rapidly increased, peaking at 1–1.5 h and 1.5–2 h after lipid injection, respectively. The increase in the number of PlsEtns with p16:1/20:4 and p16:1/22:6 suggested that 20:4 and 22:6, the main fatty acids at the sn-2 position in the rat lymphatic plasmalogen, were preferentially re-esterified at the sn-2 position, regardless of the types of hydrocarbon chains at the sn-1 position. Thus, we showed that bacterial PlsEtns with “unnatural” structures against rats could be absorbed into the lymph. Our findings provide insights into the association between the chemical structure of plasmalogens and their biological functions in humans.Plasmalogens, functional glycerophospholipids with biological roles in the human body, are associated with various diseases. Although a variety of saturated and/or unsaturated fatty acids in plasmalogens are presumed to have different functions in the human body, there are limited reports validating such functions of plasmalogens. In this study, we focused on the bacterial plasmalogen derived from  They are classified into three subclasses: diacyl, alkyl, and alkenyl, based on the aliphatic hydrocarbon chain at the sn-1 position and an ester bond at the sn-2 position of the glycerol backbone (sn-1 and -2 positions consist of saturated and/or unsaturated fatty acids (FAs) with various carbon chains. It is well known that plasmalogens are present in human and animal tissues  structural components of the cell membrane , 2) mainmalogens . It is nmalogens . For insmalogens . Thus, uSelenomonas ruminantium, Ruminococcus albus, Ruminococcus flavefaciens, Bacteroides succinogenes, Borrelia sp., and Clostridium butyricum, which are classified as rumen bacteria  fraction obtained from ethanol–ether extraction of the cultivated cells  2SO4, 0.001% MnCl2·4H2O, 0.001% CoCl2·6H2O, 0.001% CaCl2·2H2O, and 0.0001% resazurin. After air in the 988TYG medium was partially replaced with N2, the medium was autoclaved, and then, 0.2 ml (for 100 ml medium) or 4.2 ml (for 2.1 L medium) of filter-sterilized L-cysteine hydrochloride solution (250 mg/ml) was aseptically added to the medium using syringes. 103574)  was obtaAfter cultivation, the cells were harvested by centrifugation  and washed thrice with distilled water. The microbial cells or porcine brain were lyophilized using an FD-1000 vacuum freeze dryer  at −80°C for 2 days. Lipids were extracted with chloroform/methanol/1% KCl solution , followed by extraction with chloroform twice. All extract solutions were collected, evaporated to dryness, and dissolved in methanol. This fraction was analyzed by ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) and subjected to further experiments.S. ruminantium and porcine brain in a 1 ml emulsified solution contained 1.83 and 2.77 µmol PlsEtn , respectively. The FA composition and molecular species composition of plasmalogens in the PlsEtn are shown in The extracted lipids (25 g/L) from Male Wistar/ST rats , aged 9 weeks, were fed a standard diet (AIN 93G formula) for a 3-day acclimation period.After overnight fasting, a vinyl catheter and a silicone catheter were implanted in the thoracic lymph duct and the duodenum, respectively, as described previously . After tThe lymph was collected from the thoracic duct lymph over time, that is, at 0.5 h intervals during the first 2 h and at 1-h intervals during the next 7 h, following the administration of the test solution. The collected lymph was frozen immediately and kept at –80°C until subsequent analyses.This study was approved by the Iwate University Animal Committee , and animals were maintained in accordance with the Iwate University guideline for the care and use of laboratory animals.sn-1 position . “p” indicates the carbon chain of sn-1 in plasmalogens with a vinyl-ether linkage. PlsCho was identified by three fragments and was quantified by phosphocholine as a fragment ion at m/z 184 after the separation of each PlsCho molecule by UPLC. In addition, the presence of plasmalogens was confirmed by the disappearance of the peak after treatment with acid. The parent and fragment ions of the plasmalogen molecular species measured in this study are shown in Total lipids in the lymphatic fluid were extracted by the Bligh & Dyer method . All plaSynthetic p18:0–18:1 and p18:0–20:4 of PlsCho and PlsEtn,  were used to generate a standard curve to quantify individual species of ether glycerophospholipids. Total PlsEtn and PlsCho were taken as the sum of all molecular species in each class measured by UPLC-MS/MS. The lipid composition in the lipid extracts from bacteria and porcine brain was measured by thin layer chromatography on chromarods with flame ionization detection (FID) using an Iatroscan MK-6 instrument  .p < 0.05 were taken to be statistically significant.Data are expressed as mean ± SEM (standard error of the mean). The results were analyzed by one-way analysis of variance followed by Dunnett multiple comparisons. Data analysis was performed by the add-in software Statcel 3 . Differences with S. ruminantium. The effects from 0.001 to 0.04% caproic acid in the glucose medium  were examined at 37 °C and pH 7.0 for 24 h. Although there was no effect on the cell growth at different caproic acid concentrations (0.001–0.04%)  , PlsEtn 1–0.04%) . Subsequm 6 to 8 , but Plsm 6 to 8 . The maxm 6 to 8 . Furtherinantium .S. ruminantium, the lymphatic output of plasmalogens in rats administered with bacterial plasmalogens from S. ruminantium or porcine brain was analyzed using the UPLC-MS/MS method and compared. In the lipid extract from porcine brain, the molar ratios of PlsEtn species were p18:1/18:1 (21.4%), p18:1/20:4 (13.7%), and p18:0/22:6 (12%)  . Phospha:6 (12%) . In addiS. ruminantium containing the PlsEtn, the total PlsEtn output in the lymph was not significantly increased compared to that prior to the administration of the lipid emulsion containing plasmalogens   . Howeverat 1–4 h . The mol6:1/22:6 . The amop to 7 h . The molncreased .sn-1 position was barely detected in the lymph, and the level of PlsEtn containing p16:0, p18:0, and p18:1 at the sn-1 position increased, particularly that of 18:0   .S. ruminantium and porcine brain were 6.2 ± 0.5 nmol and 33.0 ± 6.5 nmol, respectively. The absorption percentage of total PlsEtn from S. ruminantium into the lymph was approximately one-fourth compared to that derived from the porcine brain (0.33 ± 0.03% for S. ruminantium and 1.19 ± 0.23% for porcine brain).The total lymphatic outputs of PlsEtn for 7 h after the administration of plasmalogens derived from S. ruminantium containing PlsEtn, the lymphatic output of total PlsCho was decreased and then gradually recovered by half of the initial value at 6–7 h  administration are as follows: 41.0%, p16:0; 13.5%, p16:1; 32.6%, p18:0; and 12.9%, p18:1. Moreover, only the p16:1 ratio was maintained until after 6–7 h. With regard to FAs at the sn-2 position, PlsCho with 16:0, 18:2, 20:4, 22:4, 22:5, and 22:6 was decreased after administration of porcine plasmalogens, whereas those with 18:1 and 20:5 was increased .S. ruminantium in the present study show differences when compared to results of other previous experiments. A previous study showed that glucose media containing volatile FAs or lactate media containing biotin were appropriate for the growth of S. ruminantium (S. ruminantium grew well in the glucose medium and not so much in the lactate medium (data not shown). In addition, the production of serine plasmalogens (PlsSer) was not detected in our study, although it is reported that PlsEtn and PlsSer are the main plasmalogens produced in S. ruminantium  and then absorbed by the intestinal mucosal cells as free FAs and lyso-phospholipids (Lyso-PLs) in the small intestinal lumen  . Lyso-phficantly . The ratS. ruminantium (0.33 ± 0.03%) into the lymph for 7 h after administration was lower than that of PlsEtn derived from porcine brain (1.19 ± 0.23%). This might be a result of the LPLAT activity for PlsEtn with p16:1 being weaker than that for PlsEtn with p18:0 and p16:0 during re-esterification. Moreover, bacterial lipids consist of relatively short-chain FAs with less than 16 carbons such as 14:0 and 16:1, while pig brain lipids consist of long-chain FAs with more than 18 carbons including 20:4 and 22:6 , according to the composition analysis using the GC and LC-MS/MS method . This finding is expected to not only lead the effective utilization of bacterial resources as food but also clarify the relationship between the chemical structure of plasmalogens and their biological functions in humans.In this study, we focused on


Alicyclobacillus acidoterrestris, Bacillus coagulans and Bacillus cereus at two stages of orange juice production. To simulate fruit disinfection before processing, the orange peel was artificially inoculated with each of the bacterial spores and submitted to UV-C light (97.8–100.1 W/m2) with treatment times between 3 s and 10 min. The obtained product, the orange juice, was also tested by exposing the artificially inoculated juice to UV-C light (100.9–107.9 W/m2) between 5 and 60 min. A three-minute treatment (18.0 kJ/m2) reduced spore numbers on orange peel around 2 log units, while more than 45 min (278.8 kJ/m2) were needed to achieve the same reduction in orange juice for all evaluated bacterial spores. As raw fruits are the main source of bacterial spores in fruit juices, reducing bacterial spores on fruit peels could help fruit juice processors to enhance the microbiological quality and safety of fruit juices.Spore-forming bacteria are a great concern for fruit juice processors as they can resist the thermal pasteurization and the high hydrostatic pressure treatments that fruit juices receive during their processing, thus reducing their microbiological quality and safety. In this context, our objective was to evaluate the efficacy of Ultraviolet-C (UV-C) light at 254 nm on reducing bacterial spores of  Escherichia coli O157:H7 and Salmonella the main pathogens implicated [The microbial population of fruit and vegetable juices depends on the agricultural practices used to obtain the raw materials, the employees’ health and hygiene and the source of agricultural water, as well as on the production practices and the hygienic conditions of the processing plant. The utilization of fruit that has fallen to the ground can introduce additional microorganisms due to their contact with the soil. Traditionally, foodborne disease outbreaks have not been associated with fruit juices mainly because of their low pH values (pH < 4.5)  and the plicated . On the plicated . Although thermal pasteurization is widely used during fruit and vegetable juice processing to ensure its microbial safety and quality, thermal treatments are detrimental for juice organoleptic and nutritional quality. Therefore, some non-thermal pasteurization methods have been proposed during the last decades, including HPP, pulsed electric fields, ultrasound and ultraviolet light . CurrentAlicyclobacillus is a thermoacidophilic spore-forming bacterium that can spoil acidic juices as it is able to grow at a temperature range from 20–70 °C and at a pH from 2.5–6.0 [Alicyclobacillus contamination. Fruit juice spoilage caused by Alicyclobacillus species is difficult to detect by fruit juice processors as it does not produce gas or any other visible changes in the product. Sometimes, consumers’ complaints are the only way for companies to become aware of the problem. Alicyclobacillus causes off-flavours in the odour and taste of the spoiled product that has been described as medicinal, disinfectant-like or antiseptic due to the production of guaiacol and other chemical compounds, such as the halophenols 2,6-dichlorophenol and 2,6-dibromophenol [Alicyclobacillus acidoterrestris is the main species implicated in juice spoilage [A. acidoterrestris and also the most important source of contamination of acidic products [ 2.5–6.0 , it has  2.5–6.0 , 78% of mophenol . Alicyclspoilage . Soil isproducts . StudiesBacillus coagulans and Bacillus cereus. B. coagulans causes flat sour-type spoilage in heat-processed acid beverages, tomato juice and acidified vegetable products because they form heat-resistant spores that can grow at low pH. This organism is frequently isolated from spoiled canned vegetables acidified to pH 4.0–4.5 and has been considered the primary cause of economically important spoilage in thermally processed tomatoes and tomato products [B. coagulans is a non-pathogenic microorganism, it might cause a food safety hazard due to its ability to increase the pH of acidic foods to a level that can allow the germination of surviving Clostridium botulinum spores [Other spore-forming bacteria genera have been isolated from fresh fruits and their juices, such as products ,14. Althm spores ,15.B. cereus is a ubiquitous bacterium, the soil is the main source of its spores. Therefore, its presence in most raw foods is inevitable. Moreover, the spores have strong adhesion properties, so additional contamination may occur due to biofilms formed on the surface of processing equipment. This bacterium has been isolated in almost every category of foodstuffs [B. cereus was detected in 56.7% of the studied unpasteurized juices. In another study, B. cereus was observed in 64.9% of the samples of unpasteurized street-vended fruit juices [B. cereus is not particularly tolerant to acid, orange juice has been implicated in B. cereus foodborne poisoning [Though odstuffs , includiodstuffs , B. ceret juices . Althougoisoning . A. acidoterrestris population. Short-wave ultraviolet-C (UV-C) light radiation in the range of 200–280 nm, which mainly breaks down DNA molecules resulting in a germicidal effect on bacteria, virus, protozoa, fungi and algae [A. acidoterrestris spores in white grape, apple, ‘Cantaloupe’ melon and orange juices with treatments ranging from 8 to 20 min at UV-C intensities among 1.31–14 W/m2 [A. acidoterrestris population of spores depended on the UV dose and fruit juice type, observing a higher reduction of bacterial spores in white grape juice (5.5 log CFU/mL reduction) when compared to apple juice (2.1 log CFU/mL) at the same UV-C intensity (1.31 mW/cm2). The difference in UV-C treatment effectiveness was given by the lower absorption coefficient of the tested grape juice (5.82 cm−1) when compared to apple juice (12 cm−1). The reduction of B. cereus and B. coagulans spores by UV-C treatment has been reported in synthetic media [As current techniques used for juice production do not control spore-forming bacteria, other strategies and treatments are under study. For example, Roig-Sagués et al.  combinednd algae , has beend algae . Howevernd algae . Juice tnd algae , and it –14 W/m2 ,25,26,27Alicyclobacillus spp. and other spore-forming bacteria are soil-borne, their control should start with proper cleaning of the fruits at the beginning of processing [A. acidoterrestris, B. coagulans and B. cereus at two stages of orange juice production. Specifically, the application of this technique on the whole fruit (orange peel) and after obtaining the juice (orange juice) is studied. As the efficacy of UV-C treatment on fruit juices depends on the transmittance of the juice and considering that ocessing . TherefoAlicyclobacillus acidoterrestris , Bacillus cereus  and Bacillus coagulans , were provided by the Spanish Type Culture Collection (CECT). In addition, another A. acidoterrestris strain, TA-1.183, isolated from a pasteurized apple juice sample in our laboratory, was used. This strain was identified by 16S rDNA gene sequencing and included in the Food Technology Department of the University of Lleida collection. All the strains were stored in 30% glycerol at −20 °C. The strains used in this investigation, A. acidoterrestris strains were streaked onto Yeast Starch Glucose Agar ((YSG), Scharlau, Spain) plates and incubated at 45 °C for 24 h. The Bacillus spp. strains were streaked onto nutrient agar ((NA), Biokar, France) plates and incubated at 30 °C for 24 h. To obtain the vegetative cultures of the A. acidoterrestris strains, a single colony was inoculated into 10 mL of Bacillus acidoterrestris broth ((BAT broth), Scharlau) and incubated at 45 °C for 24 h. For the Bacillus spp., a single colony was inoculated into 10 mL of Tryptic Soy Broth ((TSB), Biokar) and incubated at 30 °C for 24 h. The A. acidoterrestris strains, the cells grown at 45 °C for 24 h on BAT broth were spread onto YSG agar and incubated at 45 °C for seven days until at least 80% of the cells sporulated, according to Baysal et al. [Bacillus spp., the cells grown at 30 °C for 24 h on TSB were spread onto NA supplemented with MnSO4·7H2O (50 mg/mL) (NAMS) and incubated at 30 °C for five days until at least 80% of the cells sporulated, according to Beuchat et al. [g, 15 min 20 °C, Hettich-Universal 320 R, Tuttlingen, Germany) and resuspended in sterile deionized water. The washing procedure was repeated four times. The final pellets were resuspended in sterile deionized water and stored at 4 °C until use. The viability of A. acidoterrestris spore suspensions was determined by dilution in saline peptone solution ((SP), 1.0 g/L peptone (Biokar) and 8.5 g/L NaCl ), activated by heat shock (80 °C for 12 min in a water bath), serially diluted in SP, spread onto a YSG agar plates and incubated at 45 °C for 24 h. The viability of B. cereus and B. coagulans spore suspensions was determined by dilution in SP, activated by heat shock (80 °C for 10 min in a water bath), serially diluted in SP, spread onto an NA plates and incubated at 30 °C for 24 h.To induce sporulation of the l et al. . For thet et al. . BrieflyA. acidoterrestris in the orange juice was confirmed according to IFU Method No. 12:2019; meanwhile, the absence of Bacillus spp. spores was confirmed by plating a sample of juice after a heat shock. The pH values of the juice were measured by a pH meter . The soluble-solid content (SSC) was determined using a handheld refractometer , and the results were expressed in °Brix. To measure the titratable acidity (TA), 10 mL of orange juice was diluted with 10 mL of deionized water and titrated with a sodium hydroxide  solution to pH 8.1. The results were calculated as g of citric acid per L of juice. The UV light transmittance at 254 nm was measured using a spectrophotometer . The sample’s transmittance as percent transmittance, which is related to absorbance (A) by Equation (1), was evaluated using a quartz cuvette with path lengths of 1.0 cm.The oranges were purchased from a local supermarket, and the pasteurized orange juice was provided by a juice industry located in Reus (Spain). The absence of 2/plug) were cut using a cork borer. Each peel plug was spot inoculated with 0.05 mL of spore suspension (6 × 106 CFU/mL) to obtain a population of 5.0 log CFU/cm2. Peel plugs were kept at room temperature for 2 h before UV-C treatment. Orange juice was inoculated with the spore suspension to obtain an initial concentration of 5.0 log CFU/mL. The bacterial spore population was checked as described in Prior to orange peel inoculation, the surface wax was washed off by immersing the fruit in hot water (70 °C) for 30 s and dried at room temperature, according to Yoo et al. . After tUV-C treatments were carried out in laboratory scale equipment manufactured by LAB-UVC-Gama . The camera (61.8 × 27.7 × 20 cm) was equipped with three monochromatic UV-C lamps emitting at 254 nm . The samples were positioned at a 12 cm distance from the lamps. The intensity of UV-C during the treatments was monitored using a radiometer UV-sensor Easy H1  placed at the same distance from the lamps as the samples. Before the experiment started, UV-C lamps were switched on for 30 min to ensure light stability. 2) for 3, 6, 9, 12, 18, 24, 27, 60 and 180 s, and 3, 5 and 10 min. At each evaluated time, a pool of five peel plugs was placed in a sterile filter bag  by triplicate. The inoculated orange juice was placed in 12-well plates . Each well contained one millilitre of juice, with a depth of 0.4 cm. Orange juice was exposed to UV 254 nm radiation (intensity 100.9–107.9 W/m2) for 5, 10, 15, 20, 25, 30, 45 and 60 min, and three samples were taken at each time. The UV-C dose (kJ/m2) was calculated as the intensity measured (W/m2) × time (s)/1000 (Orange inoculated peel plugs were placed in a petri dish and treated by UV 254 nm radiation (intensity ranged from 97.8–100.1 W/m(s)/1000 . The assA. acidoterrestris CECT 7094T and A. acidoterrestris TA-1.183 were plated on YSG agar and incubated at 45 °C for 24 h. B. cereus and B. coagulans samples were spread on NA plates and incubated at 30 °C for 24 h. After the UV 254 nm treatments, each orange peel sample was diluted with 5 mL of buffered peptone water ((BPW), Biokar) and homogenised in a blender  for 90 s (250 impacts/min). The homogenates of orange peel plugs and the samples of orange juice were activated by heat shock (as described in 0) to avoid small differences in the initial concentrations between experiments. Survival curves were obtained by plotting the logarithm of the survival fraction (N/N0) versus treatment doses, expressed in kJ/m2. N is the spore count after UV-C exposition, and N0 is the initial number of spores before treatment. The GInaFiT software was used for modelling the fit of the survival curves [Inactivation data were expressed as log , and d is the applied UV 254 nm dose (kJ/m2). The Log-linear regression, a model typically used to assess the effect of a wide array of processing factors on microbial inactivation, including UV 254 nm treatment, is described by Equation (2) :(2)log Nd is the applied UV dose (kJ/m2), δ (kJ/m2) is a scale parameter indicating the UV 254 nm dose for the first decimal reduction, and p (dimensionless) is a shape parameter describing concavity or convexity of the curve.The Weibull model is described by Equation (3) : (3)log resN represents the residual cell concentration where the tail starts, d is the applied UV dose (kJ/m2), δ (kJ/m2) is a scale parameter indicating the UV 254 nm dose for the first decimal reduction, and p (dimensionless) is a shape parameter.The Weibull plus tail model is described by Equation (4) :(4)log Nf is the fraction of initially major subpopulation, maxK1 and maxK2 are the first-order inactivation rate constants for initially major and minor population (m2/kJ), and d is the applied UV 254 nm dose (kJ/m2).The Biphasic model is described by Equation (5) :(5)log N2 (R2-adj). RMSE measures the average deviation between the observed and the fitted values. Therefore, the model with the smallest RMSE was considered to be the best fit for the inactivation curve. Model performance and comparison were performed using the root mean square error (RMSE) and the adjusted correlation coefficient RA. acidoterrestris CECT 7094T, A. acidoterrestris TA-1.183, B. cereus and B. coagulans spores on orange peel was 5.07 ± 0.11, 4.92 ± 0.03, 4.80 ± 0.10 and 5.01 ± 0.09 log CFU/cm2, respectively. The reduction curves of the four bacterial spores artificially inoculated on orange peel and exposed to UV 254 nm light for 0 to 10 min were assessed ; meanwhile, no significant differences in spore reduction were observed when the UV 254 nm dose increased from 18.0 to 59.9 kJ/m2, with spore reductions ranging from 2.06 ± 0.37 to 2.28 ± 0.21 log. A. acidoterrestris TA-1.183 spores also showed a significant reduction after exposure to a UV 254 nm dose of 0.3 kJ/m2 (0.94 ± 0.13 log reduction). When the treatment time was increased from 5.9 to 59.9 kJ/m2, A. acidoterrestris TA-1.183 spore counts on the orange peel were not significantly different with spore reductions of 1.96 ± 0.1, 1.88 ± 0.35, 2.05 ± 0.15 and 2.08 ± 0.23 log after 5.9, 18.0, 29.9 and 59.9 kJ/m2, respectively. B. cereus spores reached a significant reduction after 0.3 kJ/m2 (0.84 ± 0.16 log reduction), whereas 0.9 kJ/m2 of a UV 254 nm exposition were needed to obtain a significant reduction on B. coagulans spores on the peel (0.93 ± 0.13 log reduction). B. cereus spores on the peel reached 1.92 ± 0.12 log reduction after the exposition to 18.0 kJ/m2, a value that was not significantly different to the reductions observed after UV 254 nm doses of 29.9 and 59.9 kJ/m2 . Similar results were obtained for B. coagulans, spore inactivation counts were not significantly different in a range of 18.0–59.9 kJ/m2, with reductions ranging from 1.86 ± 0.40–2.05 ± 0.37 log. The initial population of assessed . Table 1The orange juice used in the spore inactivation trials had a pH value of 3.31 ± 0.01. TA was 11.2 ± 0.1 g citric acid/L, and SSC was 11.77 ± 0.06 °Brix. The juice UV light transmittance measured at 254 nm was 0.15%.A. acidoterrestris CECT 7094T, A. acidoterrestris TA-1.183, B. cereus and B. coagulans spores was 5.23 ± 0.25, 5.13 ± 0.03, 4.21 ± 0.30 and 4.76 ± 0.46 log CFU/mL, respectively. After the higher exposition to UV 254 nm light (370.6 kJ/m2), reductions were 2.76 ± 0.42, 2.22 ± 0.45, 2.46 ± 0.05 and 2.42 ± 0.42 log for A. acidoterrestris CECT 7094T, A. acidoterrestris TA-1.183, B. cereus and B. coagulans spores, respectively. A. acidoterrestris CECT 7094T spores in the orange juice showed a significant reduction after a UV 254 nm exposition of 94.4 kJ/m2 (0.48 ± 0.09 log reduction). The number of spores in the juice decreased drastically after 157.2 kJ/m2 UV 254 nm treatment (1.25 ± 0.18 log reduction). A significant reduction of A. acidoterrestris TA-1.183 spores in the juice was observed after UV 254 nm treatment of 125.9 kJ/m2 (0.66 ± 0.21 log reduction). The spore counts decreased with a higher exposition time, with reductions of 1.17 ± 0.28 and 1.74 ± 0.25 log after UV 254 nm doses of 185.8 and 278.8 kJ/m2, respectively. After a UV 254 nm exposition of 125.9 kJ/m2, B. cereus spore population was significantly reduced by 0.69 ± 0.34 log units, whereas B. coagulans spore numbers were reduced by 0.33 ± 0.11 log after 94.4 kJ/m2. In all of the evaluated bacterial spores, the increased dose of UV 254 nm decreased the spore population in juice until the maximum evaluated time (60 min) equivalent to 370.6 kJ/m2. No changes in juice taste or colour were observed after the longest time tested (60 min).2-adj of the Weibull model, the Weibull plus tail model and the Biphasic model estimating bacterial spore reduction on orange peel after UV-C at 254 nm treatment. The UV-resistance parameters obtained from fitting the evaluated inactivation models are shown in In order to analyse the goodness of fit, 2-adj values of 0.717–0.887 , followed by B. cereus (δ = 0.27 ± 0.18), A. acidoterrestris CECT 7094T (δ = 0.54 ± 0.26) and finally B. coagulans, which needed the highest dose (δ = 1.58 ± 0.77).The Weibull model accurately described all the inactivation curves, with R17–0.887 . All ther than 1 , which w2-adj values of 0.838–0.900 were observed, which means that it adequately characterized the bacterial spore inactivation curves. Similar to the Weibull model, all the curves showed p values < 1 according to the upward concavity. In comparison to the Weibull model, higher doses of radiation were needed to achieve the first decimal reduction. δ values were 0.44 ± 0.16, 0.45 ± 0.19, 0.75 ± 0.25 and 1.58 ± 0.78 for A. acidoterrestris TA-1.183, B. cereus, A. acidoterrestris CECT 7094T and B. coagulans, respectively. Regarding the Weibull plus tail model, RA. acidoterrestris CECT 7094T, A. acidoterrestris TA-1.183, B. cereus and B. coagulans with R2-adj values of 0.845, 0.782, 0.821 and 0.849, respectively. From the total spore population on the orange peel, a high proportion of spores sensitive to UV 254 nm were observed for the four bacteria studied, as f values ranged between 0.94 and 0.95. The sensitive population of A. acidoterrestris TA-1.183 showed the highest speed of decrease (maxk1 = 3.44 ± 0.55 m2/kJ); meanwhile, the sensitive population of B. coagulans showed the lowest speed of decrease (maxk1 = 2.09 ± 0.33 m2/kJ). The inactivation rate of the resistant population was similar for all the bacteria tested.The Biphasic model was appropriate for representing survival data of A. acidoterrestris strains and B. cereus, as it was the model that showed the lowest RMSE. However, the Biphasic model showed the best performance characterizing B. coagulans spore inactivation curves. The goodness of fit of the three evaluated models for the four bacterial spore inactivation curves on orange peel can be evaluated throughout the comparison of RMSE values. The Weibull plus tail model showed the best performance when modelling inactivation curves of spore inactivation of both The statistical indices of the Log-linear model and the Weibull model estimating bacterial spore reduction in orange juice after UV-C exposure are shown in 2-adj values of 0.850–0.932. The inactivation rate of bacterial spores in orange juice ranged from 0.01–0.02 m2/kJ for the four bacteria tested.The Log-linear model accurately described all the inactivation curves of the studied spores in orange juice, with RA. acidoterrestris CECT 7094T, A. acidoterrestris TA-1.183, B. cereus and B. coagulans, as it exhibited a high R2-adj value 0.933, 0.904, 0.863 and 0.933, respectively. The observed p values were higher than 1 for all spore inactivation curves, showing a downward concavity. A. acidoterrestris CECT 7094T needed the lower dose for the first decimal reduction (δ = 147.5 ± 14.59), followed by A. acidoterrestris CECT 7094T (δ = 172.05 ± 19.07), B. cereus (δ = 201.38 ± 17.08) and finally B. coagulans, that needed the highest dose (δ = 212.98 ± 12.45).The Weibull model was appropriate for representing the survival data of A. acidoterrestris TA-1.183, whilst the Weibull model showed the lowest RMSE values for A. acidoterrestris CECT 7094T, B. cereus and B. coagulans.In order to analyse the goodness of fit of the two predictive microbial models evaluated, the corresponding values of RMSE were compared. The Log-linear model showed the lowest RMSE value for A. acidoterrestris, B. coagulans and B. cereus on orange peel and in orange juice has been evaluated to simulate UV-C treatment of whole oranges at their arrival to the food industry and UV-C treatment of the final product (juice).Spore-forming bacteria are one of the main concerns for fruit juice processors, as spores can survive thermal and HPP treatments that are usually applied in the food industry. The main source of bacterial spores in the fruit juice industry is the field soil where fruits are cultivated. Therefore, cleaning and disinfecting fruits before juice processing could help to reduce bacterial spores in the juice. Once bacterial spores reach the juice, alternative methods to thermal and HPP treatments are needed. UV-C radiation has been shown previously to reduce bacterial spores in some fruit juices. In this study, the efficacy of UV-C radiation at 254 nm to eliminate the spores of 2) could be enough to significantly reduce the spore numbers on the orange peel (around 2 log units). Incrementing treatment time did not significantly enhance UV 254 nm treatment efficacy. Little information about microorganism’s inactivation on a fruit surface by UV-C light is found in the literature. Gündüz et al. [2) on citrus fruit inoculated with Penicillium spp. spores by spot method. Maximum reductions of 2.75 log CFU/orange of Penicillium digitatum were obtained at the UV-C dose of 3.17 kJ/m2. However, previous studies suggested that fungi are more resistant to UV-C light than bacterial spores due to their size [According to the data obtained, a three-minute treatment  were needed to significantly reduce the initial spore counts in orange juice, and more than 45 min (278.8 kJ/m2) was needed to achieve a spore reduction higher than 2 log units. Tremarin et al. [A. acidoterrestris spores reduction in apple juice, 5 log after 8 min of exposure to UV-C (13.44 W/m2). This difference could be attributed to the higher transmittance of apple juice (27%) compared to the transmittance of the orange juice used in this experiment (0.15%). On ‘Cantaloupe’ melon juice, 20 min of exposure at the same UV-C radiation intensity (13.44 W/m2) were required for a 4.7 log unit reduction of the A. acidoterrestris spores [A. acidoterrestris spores in grape juice was achieved after 15 min of UV-C treatment (1.31 W/m2), whereas spores in apple juice were only reduced by 2.1 log reduction under the same treatment conditions. In this case, the difference in efficacy of UV between the juices was also attributed to the lower absorption coefficient of grape juice when compared to apple juice. Higher reductions of the A. acidoterrestris spores in apple juice were observed by Sauceda-Gálvez et al. [A. acidoterrestris spores to UV-C (4.2–12.6 kJ/m2) in orange juice (2% transmittance) reduced their population until 3 log units. Lower A. acidoterrestris spores reduction after orange juice (2% transmittance) treatment with UV-C light were reported by Ferreira et al. [2 treatment. For all bacterial spores studied, 15–20 min UV 254 nm treatment , according to the Biphasic model. The A. acidoterrestris CECT 7094T and B. cereus spores showed intermediate resistance to UV 254 nm; meanwhile, the B. coagulans spores were the most resistant with the highest δ values from the Weibull and the Weibull plus tails models and the lowest speed of decrease of the sensitive population (maxk1), according to the Biphasic model. Spore inactivation curves after the UV 254 nm treatment of all tested species on orange peel showed tails, which suggests varying levels of resistance, possibly caused by mixed populations or aggregation of microorganisms. However, according to the A. acidoterrestris TA-1.183 spores or with downward concavity for the rest of bacterial spores tested, according to the p values of the Weibull model. Similar to the results observed with orange peel, the B. coagulans spores in orange juice were the most resistant to UV-C and showed the highest δ values, according to the Weibull model. However, in orange juice, the most sensitive populations were the A. acidoterrestris CECT 7094T spores, with the lowest δ values and the highest speed of decrease according to the Log-linear model. In orange juice, the spore inactivation curves did not show shoulders or tails. The obtained curves were almost linear for the A. acidoterrestris spore inactivation by UV-C light in white grape juice fitted to the Log-linear plus tail and Weibull models, while the inactivation data in apple juice only fitted Log-linear plus tail model [2 and R2-adj. The A. acidoterrestris spore inactivation curves in apple juice obtained by Tremarin et al. [B. cereus and B. coagulans spore survival curves after UV-C treatment in a buffer with an absorption coefficient of 11.1 cm−1 fitted a Log-linear plus shoulder model [maxK values among the species, but the shoulder length widely changed, and therefore, the UV-C resistance was due to changes in shoulder length. Similar to the results obtained in the present study, Gayán et al. [B. coagulans when compared to B. cereus. In the case of non-sporulated bacteria, Fenoglio et al. [Escherichia coli, Lactobacillus plantarum and Saccharomyces cerevisae UV-C inactivation curves could be appropriately represented by the Weibull, Coroller and Biphasic models. As it has been observed in the present study and as it has been previously described, the inactivation of microorganisms with UV-C treatment can show different shapes, from first-order kinetics  to sigmoil model . Howevern et al. ,42 folloer model . The autn et al.  reportedo et al.  observedA. acidoterrestris spores subjected to 12.6 kJ/m2 UV-C light treatment showed visible changes, such as morphological distortions, central depression and expressive roughness, when observed with a scanning electron microscopy.According to Setlow , bacteriAs it has been demonstrated, UV-C light at 254 nm is capable of reducing the bacterial spores tested in both orange peel and orange juice. However, in fruit juices, UV-C efficacy depends on the juice characteristics (mainly light transmittance) because of the low-penetration capacity of UV-C light. Therefore, establishing a standard treatment for juices may be difficult, as light penetration is matrix-dependent . To the best of our knowledge, this is the first report about bacterial spore inactivation on a fruit peel using UV-C light, and we have demonstrated that it is more effective in reducing bacterial spores on orange peels than in orange juice. As raw fruits are the main source of bacterial spores in fruit juices, reducing bacterial spores on fruit peels could, consequently, reduce their entrance into the processing plants. Therefore, implementing a UV-C treatment for the whole fruit before processing could improve the safety and quality of fruit juices.