Fluorescence enhancement of CQDs was observed after addition of sodium dodecyl benzene sulphonate, a major anionic surfactant in laundry powder. A linear relationship between fluorescence enhancement CQDs and the concentration of laundry powder was established. Thus, a hydrothermal method was developed to generate CQDs from waste biomass that may be used as a fluorescent probe to detect laundry powder.Surfactants are one of the major pollutants in laundry powder, which have an impact on the environment and human health. Carbon quantum dots (CQDs) are spherical zero-dimensional fluorescent nanoparticles with great potential for fluorescent probing, electrochemical biosensing and ion sensing. Herein, a bottom-up approach was developed for the synthesis of CQDs from biomass to detect laundry detergent and laundry powder. Waste chicken bones were used as carbon precursors after being dried, crushed and reacted with pure water at 180 °C for 4 h to generate CQDs, which exhibited a monodisperse quasi-spherical structure with an average particle size of 3.2 ± 0.2 nm. Functional groups, including -OH, C=O, C=C and C-O, were identified on the surface of the prepared CQDs. The optimal fluorescence excitation wavelength of the yellow-brown CQDs was 380 nm, with a corresponding emission peak at 465 nm. CQDs did not significantly increase cell death in multiple cell lines at concentrations of 200 µg·mL The consumption of laundry powder is showing a continuous upward trend with economic growth and the improvement of living standards. The main components of laundry powder are builders, including phosphates, surfactants, bleaches, enzymes and other ingredients . PhosphaSeveral methods were reported to detect laundry powder, including liquid chromatography tandem mass spectrometry to quantify enzymes in laundry powder , nuclearCarbon quantum dots (CQDs) are spherical zero-dimensional fluorescent nanoparticles with sizes below 10 nm . They arThe synthetic methods for CQDs can be classified into “top-down” and “bottom-up” strategies. Top-down synthesis starts from carbon nanotubes, carbon fibres, graphite rods, carbon ash and activated carbon. By contrast, bottom-up synthesis begins from carbon-containing compounds such as small organic molecules or oligomers ,21. HydrCQDs have been generated from lemon peel waste  and drieHerein, waste raw bones from chicken were used as carbon precursors to generate CQDs. The size, shape, functional groups, cytotoxicity, optical properties and stabilities of the prepared CQDs were measured. Finally, CQDs were found to exhibit increased fluorescence intensity after laundry detergent addition. Thus, we developed a method to generate CQDs, reuse waste biomass and detect laundry powder in a low-cost and environmentally friendly manner.CQDs were prepared from waste bones as described in the methods . Transmi−1 assigned to the stretching vibration of the -OH group [−1 corresponding to -CH3 [−1 assigned to the stretching vibration of C=O; (4) a characteristic peak at 1385 cm−1, which is the out-of-plane bending vibration of the olefin C-H.The X-ray diffractometer (XRD) profile of CQDs a indicatOH group ,36; (2)  to -CH3 ,38; (3) Full-scan X-ray photoelectron spectrometry (XPS) analysis of CQDs c indicatCQDs in aqueous solution were yellow-brown and transparent, and they emitted light blue fluorescence under 365 nm UV light irradiation a. PhotolThe highest fluorescence intensity of CQDs was observed when the pH of the solution was 7.21 (pure water). The fluorescence intensity of CQDs was slightly decreased when the pH was 2, 4, 6 or 8 compared to 7.21. The fluorescence intensity of CQDs was relatively stable when the pH of CQD solutions was between 2 and 6. When the pH was >8, the fluorescence intensity was sharply decreased a.−1. The highest fluorescence intensity of CQDs was observed in salt-free conditions  compared with non-treatment controls . The cell survival rates after treatment with 1.25 to 20 µg·mLcontrols a, and cefor 48 h b. The reFlow cytometry was used to analyse the relative fluorescence intensity of the prepared CQDs. Forward scatter (FSC) represents the size of the prepared CQDs a. Approx−1 Mg2+, Na+, K+, Ca2+, Zn+, Al3+, Cu2+ or vitamin C , or a nonionic surfactant NP40, which are absent in laundry powder. The fluorescence intensity of CQDs did not increase in solutions with SDS or NP40 compared to those without b. Moreovitamin C . There witamin C c. Thus, First, the fluorescence stability of the reaction system between CQDs and Liby was analysed under different conditions. When the pH of the reaction system was between 2 and 10, Liby-induced fluorescence enhancement of CQDs was relatively stable a. Additi0) of CQD solutions and the fluorescence intensity (F) of CQD solutions with different concentrations (C) of Liby was measured. The results’ linear regression equation (r2 = 0.9925) was (F − F0)/F0 = 0.0327C − 0.0014, and the limit of detection for Liby was 0.17 μg·mL−1 . This indicated that CQDs could be used to detect laundry powder in water samples from different sources. Finally, the recovery rates for Liby in water samples from three sources were measured to evaluate the validity of the assay using prepared CQDs. The recovery rates of Liby in the three water samples were 87.2–104.2%, 85.2–94% and 95.3–101.6%, respectively . The limIt has been widely reported that CQDs can be produced from various kinds of biomass, including plants and their derivatives such as corn powder  and greeThe optimal fluorescence excitation wavelength of the CQDs was 380 nm, and the corresponding emission peak was 465 nm. Therefore, emission of blue-green light was visible by the naked eyes under 380 nm UV light irradiation. The optical properties of prepared CQDs are quite similar with some published literature ,42,45,46Considering that laundry powder contains more than 20 components  , an F97 Fluorescence Spectrophotometer , a JEM- 2100 transmission electron microscope , a D8 Advance X-ray diffractometer , a Nicolet IS50 Fourier transform infrared spectrometer , an ESCALAB 250Xi X-ray photoelectron spectrometer (Thermo Scientific), a PHS 3W pH meter , a Varioskan LUX microplate reader (Thermo Scientific), an EVOS FL Auto 2 microscope (Thermo Scientific), and an LSR Fortessa Flow cytometer  were used in this research.2, FeCl3, MgCl2, ZnCl2, KCl, AlCl3, CuCl2 and NaCl , Dulbecco’s Modified Eagles Medium , a Cell Counting Kit-8 , fetal bovine serum , sodium dodecyl benzene sulphonate , Liby laundry powder , Sodium dodecyl sulfate , NP40 .Reagents and materials were from the following sources: pure water , HCl , vitamin C, CaCl−1 CQD solutions were prepared using pure water.Waste raw bones from chicken were obtained from the cafeteria of Shenyang Pharmaceutical University, washed, dried in a drying oven at 60 °C for 2 h, and crushed. A 1 g sample of crushed chicken skeleton powder was reacted with 50 mL pure water in a 100 mL reaction kettle. The reaction was performed in the oven at 180 °C for 4 h . After c−1) was diluted to 3 mL for fluorescence intensity, excitation and emission spectra measurement by a fluorescence spectrophotometer. Liby, NaCl, Mg2+, Na+, K+, Ca2+, Zn+, Al3+, Cu2+ or vitamin C was added to the solution at the indicated concentration. All experiments were performed in triplicate.A 100 μL sample of CQD solution (0.1 mg·mLHEK 293T cells were transfected with packaging vectors and an empty plasmid. The lentivirus was collected from the supernatant after 48 h. The concentration of lentivirus was quantified.−1) was diluted to 1 mL with or without 2.5 μg·mL−1 SDBS or Liby. The relative fluorescence intensity of CQDs was measured by flow cytometry with a 405 nm laser and a 450/40 filter.A 100 μL sample of CQD solution , and primary mouse embryonic fibroblasts (MEFs) were purified from mouse embryos at E14.5. A green fluorescent protein (GFP)-encoding vector was transfected into HCT-116 cells and stable expression of GFP was established. All cells were maintained in DMEM high glucose medium with 10% FBS and 100 U/mL penicillin and streptomycin at 37 °C under 5% CO−1 CQD solutions for 48 h. The cell survival rate was measured by CCK-8 assay, as described previously [Cells were seeded in 96-well plates and cultured in an incubator for 24 h. Cells were treated with 0, 1.25, 2.5, 5, 10 and 20 μg·mLeviously . The optt-test (p < 0.05 was considered significant). Each group included three biological replicates.For cytotoxicity tests, data were expressed as mean ± standard deviation (SD), and the difference between control and CQDs-treated groups was analysed using an unpaired two-tailed Student’s


Lactobacillus reuteri MJM60668 on lipid metabolism and lipogenesis. C57BL/6 mice were fed a high-fat diet (HFD) and orally administrated with MJM60668. Our results showed that mice treated with MJM60668 significantly decreased liver weight and liver/body weight ratio, without affecting food intake. Serum levels of ALT, AST, TG, TCHO, and IL-1β in mice fed with MJM60668 were decreased compared to the HFD group. Investigation of gene and protein expression on the lipogenesis and lipid metabolism showed that the expression of ACC, FAS, and SREBP was decreased, and PPARα and CPT was increased. Furthermore, an increase of adiponectin in serum was shown in our experiment. Moreover, serum IL-1β level was also significantly decreased in the treated mice. These results suggested that MJM60668 can strongly inhibit lipogenesis, enhance fatty acid oxidation, and suppress inflammation. Additionally, supplementation of MJM60668 increased the proportion of Akkermansiaceae and Lachnospiracea, confirming a potential improvement of gut microbiota, which is related to mucus barrier and decrease of triglycerides levels.Non-alcoholic fatty liver disease (NALFD) is a disease characterized by liver steatosis. The liver is a key organ involved in the metabolism of fat, protein, and carbohydrate, enzyme activation, and storage of glycogen, which is closely related to the intestine by the bidirectional relation of the gut-liver axis. Abnormal intestinal microbiota composition can affect energy metabolism and lipogenesis. In this experiment, we investigated the beneficial effect of  Non-alcoholic fatty liver disease (NAFLD), a worldwide disease of interest with a dramatic escalation, is affecting around one-third of the population . It has NAFLD is characterized by fat accumulation and inflammation in the hepatocytes, increasing levels of TNF-α stimulating chronic liver inflammation, progressing to steatosis and fibrosis of the liver parenchyma, leading to cirrhosis, hepatocellular carcinoma, and liver failure as an end-stage ,6. AdipoLactobacillus and Bifidobacterium genera are the most common probiotics. They show bile acid resistance associated with the activation of glycolysis [Intestinal microbiota are a group of millions of diverse microorganisms that consistently interact with the host  with theycolysis . TherefoLactobacillus spp. is a large group of Gram-positive bacteria with an important role in food fermentation. They can be found in the gastrointestinal tract of mammals [Lactobacillus can help prevent and treat enteric infections, antibiotics diarrhea, enterocolitis, colorectal cancer, and inflammatory disease by improving gastrointestinal dysbiosis [Lactobacillus reuteri is a heterofermentative species that can grow in oxygen-limited atmospheres and easily colonize the gastrointestinal tract and confer excellent probiotic properties, surviving in different pH levels and secreting antimicrobial intermediaries [L. reuteri can promote health in humans [Lactobacillus reuteri can reduce fat accumulation, steatosis [L. reuteri strain has shown a positive effect in LPS-induced intestinal tight junction protein destruction, decreasing intestinal and inhibiting intestinal and hepatic inflammatory signals in piglets [ mammals ,17. Studysbiosis . Lactobaediaries ,20. Diffn humans . A previteatosis , and fibteatosis  in high- piglets . Additio piglets .L. reuteri MJM60668 on NAFLD in a high-fat induced mice model because this strain showed strong inhibitory activity to lipid accumulation in HepG2 hepatic cells, as well as good probiotic characteristics. In addition, the underlying mechanism was also investigated through anti-adipogenesis and anti-inflammatory pathways.Therefore, in the present study, we studied the effect of Strain MJM60668 was isolated from fecal samples of baby infants and stored at the lactobacillus library in our laboratory. During the screening of the useful Lactobacillus project in our laboratory, strain MJM60668 showed good probiotic characteristics and thus was selected for further study. To identify lactic acid bacteria (LAB), 16S rDNA sequencing was performed to identify species. Genomic DNA was isolated from MJM60668 using an Exgene cell SV DNA isolation kit . The 16S rDNA sequence was amplified with universal primers 27F and 1492R and sequenced. Obtained sequences were blasted against the Ezbiocloud database . Finally, 16S rDNA sequences of closely related strains were aligned and a phylogenetic tree was constructed by the neighbor-joining method using MEGA X software .2. Cells were seeded and treated with samples for 6 h following previously published methods [6 cells/well in 2 mL of medium, incubated for 24 h, and then starved for 24 h in an FBS-free culture medium. HepG2 cells were then treated with or without fatty solution  containing probiotics via the insert Transwell membrane , which was seeded with 108 to 109 CFU/mL MJM60668. Simvastatin (1 µM) was used as a positive control.To assess the anti-lipidemic activity, HepG2 cells obtained from Korean Cell Line Bank (KCLB) were cultured in DMEM media  supplemented with 10% FBS and 1% antibiotics  at 37 °C in humidifier chamber containing CO methods ,28, withThe MTT -2,5 diphenyl tetrazolium bromide) assay was used to determine the toxicity of MJM60668 to HepG2 cells. After 6-h treatment with samples, transwell membranes were removed and 20 µL MTT (5 mg/mL) was added to each well followed by incubation for an additional 4 h. Finally, the medium was removed and then 200 µL DMSO was added to each well to dissolve formazan crystals, the metabolite of MTT. The absorbance of each well was measured at a wavelength of 570 nm using a microplate reader . The viability of the non-treatment group was set to 100%.w/v) to obtain a stock solution. The working solution was obtained by diluting the stock solution at 2:3 with distilled water. It was filtered with a 0.2 µm syringe filter before use.Intracellular lipid accumulation was measured by Oil Red O staining. Briefly, 0.2 g of Oil red O powder  was dissolved in 40 mL 2-propanol  before staining with the Oil red O working solution. After 30 min, cells were washed twice with distilled water and photographed under a microscope. Finally, the dye retained in HepG2 cells was eluted by adding 100% isopropanol and quantified at a wavelength of 510 nm with a microplate reader .D-lactate is generated by D-lactate dehydrogenase under hypoxic or anaerobic conditions and is only produced in low quantity in animals under normal conditions because D-lactate is a specific indicator of bacterial fermentation. D-lactate is also produced in the human colon under normal conditions by the fermentative process of lactic acid produced by Gut microorganisms, however, D-lactate increasing in the gastrointestinal tract can develop D-lactic acidosis which is responsible for triggering multiple problems in the liver and kidneys making these organs unable to remove acid from the body .2O, 0.05 mL supplied buffer (pH 10), 0.01 mL of NAD+ solution, and 0.002 mL of D-glutamate-pyruvate transaminase (D-GPT) and incubated at room temperature for 3 min. After, the absorbance of the solution was measured at 340 nm wavelength . Next, 0.002 mL of 2000 U/mL D-lactate dehydrogenase (D-LDH) was added to the above reaction mixture, and absorbance was measured for 5 min until the D-LDH reaction stopped. Finally, the solution absorbance was measured at 340 nm wavelength . Concentrations of D-lactate were calculated according to the manufacturer’s equation.D-Lactate production of MJM60668 was determined using a D-lactate enzyme test kit . LGG was used as a control group. Then, 0.01 mL of LAB supernatant culture was mixed with 0.15 Hw/v) sodium salt of Sodium taurodeoxycholate hydrate. The plates were incubated at 37 °C for 2 and 5 days. After, the diameters of the deconjugated bile acid precipitation zones  were measured. LAB strains placed on MRS agar medium plates without supplementation of the conjugated bile acids were used as a control and LGG was a positive control group.To this, MJM60668 was placed on MRS agar plates supplemented with 0.5%  recommended by European Food Safety Authority  . The minThe test method was referred to the American Society for Microbiology guidelines. Hemolytic activity of the MJM60668 and LGG was tested by streaking to 5% sheep blood in tryptic soy agar  and incubated at 37 °C for 24 h. Hemolytic activity was determined by the measurement of the photochromic properties around the colonies under transmitted light.Decarboxylase medium with or without supplemented 1% amino acid  was used to determine biogenic amine production of MJM60668 following previous study . The comMucin degradation activity was tested by using 0.3% porcine gastric mucin in an agarose medium with or without glucose supplementation. Then, 10 μL of MJM60668 was added to the medium and cultured at 37 °C, for 72 h. After that, the medium was dyed with 0.1% amido black in acetic acid (3.5 M) for half-hour and washed with acetic acid (1.2 M) until the clearance zone was detected in the positive control. The determination of mucin degradation activity was conducted via measurement clearance zone around the colony.Salmonella gallirarum KCTC 2931, Escherichia coli K99, Escherichia coli O1 KCTC 2441, Escherichia coli 0138, Escherichia coli ATCC25922, Salmonella chloreraesuis KCTC 2932, Salmonella typhi KCTC 2514, and Pseudomonas aeruginosa KCCM 11802 using the agar well diffusion method. Then, 100 μL cell-free culture medium of MJM60668 or LGG was added to 8 mm wells of agar plates containing individual pathogenic strains and incubated at 37 °C for 1 day. Finally, antimicrobial activity was determined by measuring the diameters of inhibition zones except 8 mm of well. The assay was conducted in triplicates.MJM60668 was tested with eight pathogenic strains including 9 CFU/mL were initially subjected to oral stress solution  at 37 °C for 10 min. After centrifugation at 1800× g for 5 min, the supernatant was removed and the cells were resuspended to gastric stress solution  for a half-hour, then treated with gastric stress solution at pH 2 for a half-hour. After centrifugation, the gastric solution was removed and the intestinal solution  was added for 2 h. LAB, which was treated only with PBS for the entire assay duration, was used as the control. Cells after each step were diluted, plated on MRS agar plates, and incubated for 48 h. The cell viability of LAB was calculated based on the number of colonies grown on MRS agar plates.MJM60668 and LGG (positive control) with a density of 10To assess the effectiveness of MJM60668 to adhere to the gastrointestinal tract, the cell adhesion assay was performed.2. HT-29 cells were seeded at concentrations of 2 × 105 cells/well in 24 well plates and incubated for 24 h to get polarized monolayer and 80% confluence.HT-29 cells were purchased from Korean Cell Line Bank (KCLB) and cultured RPMI 1640 , 100 U/mL penicillin and 100 mg/mL of streptomycin , was incubated at 37 °C on 5% CO8 CFU/mL) and incubated for 2 h at 37 °C in a 5% CO2 incubator. After incubation, monolayers were washed three times with PBS and treated with 1000 µL of Trypsin  for 5 min. Then, 1 mL aliquot of homogenate was serially diluted and plated on MRS agar and incubated at 37 °C for 48 h. The percentage of bacteria adhered to the plate was calculated by dividing the remaining bacteria grown on MRS agar by the initial inoculation bacteria. All experiments were performed in triplicate.HT-29 monolayers were washed three times with PBS, the medium was replaced with antibiotic-free RPMI 1640. Each well was inoculated with bacteria  were randomly assigned into cages with four mice in each of them in a room with 12-h light cycle (lights on at 6:00 am and off at 6:00 pm) at 22 ± 2 °C and 55 ± 5 of humidity and with water and feed freely supplied for 1-week prior experiment. All protocols for animal experiments were approved by the Institutional Animal Care and Committee (IACUC) of Myongji University (MJIACUC-2021003) and thus conducted in accordance with the NIH guide for the care and Use of Laboratory Animals. 8), and MJM60668 (109) groups. Control group mice were fed a normal rodent diet from Raon Bio . Groups HFD, Silymarin, MJM60668 (108), and MJM60668 (109) were fed a high-fat diet (HFD) composed of 45% fat, 35% carbohydrates, and 20% proteins  to increase adipocyte infiltration in the liver and induce steatohepatitis (NASH) [After acclimatization for 1 week, mice were divided into five groups (n = 12 mice per each group): Control, HFD, Silymarin, MJM60668 (10s (NASH) .8) mice were treated with L. reuteri MJM60668 at 1 × 108 CFU/mice/day. Group MJM60668 (109) mice were treated with L. reuteri MJM60668 at 1 × 109 CFU/mice/day. All treatments were administrated orally once daily for 12 weeks. LABs were cultured freshly daily, washed, and resuspended in saline (0.85% NaCl) before use.Animals in control and HFD groups were treated orally with saline solution. Group Silymarin mice were treated with silymarin (50 mg/kg) . Group MJM60668 , mice were taken out and immediately euthanized. After 12 weeks of treatment, mice were euthanized by cervical dislocation after anesthesia induction with 3% isoflurane. Organs were then taken for analysis.g for 15 min at 4 °C. The serum was separated and stored at −80 °C. Separated plasma samples were analyzed with FUJIFILM DRI-CHEM NX500i to measure the contents of Aspartate transferase (AST), Alanine Aminotransferase (ALT), Creatinine (CREA), Uric Acid (UA), Blood Urea Nitrogen (BUN), Total Triglycerides (TG), Gamma-glutamyl Transferase (GGT), Albumin (ALB), Total Cholesterol (TCHO), and High-Density Cholesterol (HDL-C).Blood samples were collected from the heart at 4 and 12 weeks of treatment. Collected samples were immediately moved onto ice. After 1 h, samples were centrifuged at 2000× After mice were euthanized, the liver, kidney, and intestine were immediately removed and rinsed. Liver and kidney weights were recorded. The liver was sectioned into three parts: median and left lobe, right lobe, and caudate lobe.The caudate lobe and right kidney were fixed in 4% paraformaldehyde  for 24 h. They were then paraffin-embedded, sliced into 5 µm thick sections, and stained with hemoxylin-eosin (H&E). Histological changes were examined at 200× magnification using a computer image analysis system . Each image was analyzed and graded according to percentages of inflammation and vacuolation of hepatocytes to three levels: mild (0–25%), moderate (26–50%), and severe (>51%) as described before ,35.Frozen median and left lateral lobe of liver samples were ground with liquid nitrogen to powder. RIPA lysis buffer supplemented with 1% protease and phosphatase inhibitors was added to each sample and incubated on ice for 30 min. After that, samples were centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was collected and transferred to a new tube. Total protein was measured with a BCA Protein Assay Kit . Proteins were boiled and loaded (40 µg/lane) alongside a molecular weight marker  on a 10% SDS-polyacrylamide gel (SDS-PAGE). After the gel was run, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. After the membrane was blocked with 3% BSA (Bovine Serum Albumin) in TBS  for 1 h at 4 °C, the membrane was incubated with primary antibodies against FAS , SREBP , PPARα , or β-actin . After washing three times with TBST buffer , the membrane was further incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody  for 90 min at 4 °C. After washing the membrane three times with TBST buffer, immunoreactive bands were enhanced using chemiluminescence reagents. Membrane photos were then captured. Expression levels of target proteins were compared to β-actin protein levels using ImageJ software  to obtain quantitative protein expression. All experiments were performed in triplicate.® 96 system. Primers used in this study are listed in −ΔΔCT method [Frozen samples of the right lateral lobe of the liver were ground with liquid nitrogen to powder. Total RNA was isolated with a Takara MiniBEST Universal RNA extraction kit  according to the manufacturer’s instructions. After confirming the integrity of the RNA sample by agarose gel electrophoresis, RNA concentration and purity were determined with an ND-1000 spectrophotometer. For cDNA synthesis, gDNA was removed from the RNA with a gDNA eraser . After cDNA was synthetized using a Takara PrimeScript RT reagent kit following the manufacturer’s instruction, 1 µg of cDNA was mixed with 10 µL of SYBR Premix Ex Taq  and 0.5 µM of each primer of a primer pair in 20 µL Roche LightCyclerT method .TM Stool DNA mini kit  according to the manufacturer’s protocol. The V3-V4 region of the bacterial 16S rRNA gene was amplified using barcoded universal primers 341F and 805R [The intestine was immediately dissected. Feces were collected into tubes and stored at −80 °C before use. For metagenome analysis, DNA was extracted with an Exgeneand 805R . Microbit-test to determine statistical significance. Differences between groups were considered statistically significant when the p-value was equal to or less than 0.05.All data are shown as mean ± SD. The difference between the control and experimental groups was subjected to the one-way analysis of variance (ANOVA) or two-sample Limosilactobacillus reuteri species, sharing 99% similarity. This indicates that MJM60668 is closely related to L. reuteri  had no cytotoxicity to HepG2 cells. On the other hand, it slightly improved the survival rate of HepG2 cells compared to OA-C treatment. This result suggests that MJM60668 is safe for hepatocytes (MTT assay was used to assess the cytotoxicity of MJM60668 to hepatocytes. As shown in atocytes a.8 and 1 × 109 CFU/mL) in a medium containing fatty solution (Oleic acid and Cholesterol) for 6 h to measure lipid accumulation level. At 1 × 108 and 1 × 109 CFU/mL, MJM60668 reduced lipid accumulation by 50.5% and 81.29%, respectively, compared to the negative control  (HepG2 cells were treated with MJM60668 at two doses (1 × 10173.76%) b. Moreov173.76%) b.MJM60668 showed non-hemolytic activity on blood agar . MoreoveEscherichia coli K99, Salmonella gallirarum KCTC 2931, Escherichia coli O1 KCTC 2441, Escherichia coli ATCC25922, Salmonella chloreraesuis KCTC 2932, Salmonella typhi KCTC 2514, and Pseudomonas aeruginosa KCCM 11802). MJM60668 and LGG also showed moderate inhibitory activities against Esherichia coli O138 . However, log CFU units of both MJM60668 and LGG were significantly decreased after gastric stress by 0.23 and 0.38, respectively, compared with the initial CFU. MJM60668 and LGG showed significant decreases of 0.52 and 1.33 log CFU units (both p < 0.01), respectively, at the end of the OGT assay compared to the initial CFU  transit assay. MJM60668 and LGG were not affected by oral stress , and MJM60668 (109) after 12 weeks of treatment were 34.23 ± 1.92, 35.96 ± 3.88, and 35.88 ± 3.67 g, respectively.As shown in Additionally, our results showed an increase in food consumption of the control group compared to HFD feed groups c. This cAfter sacrifice, morphologies of mice livers were compared between groups. Results showed that liver sizes of HFD-treated mice were increased. Additionally, the livers of mice in the HFD group showed a whitish tone compared to those in other groups e. These 8), and MJM60668 (109) groups (33.77 ± 2.44 g (p ≤ 0.0001), 33.98 ± 2.05 g (p ≤ 0.0001), and 33.84 ± 2.76 g (p ≤ 0.0001), respectively) compared to the HFD group (42.93 ± 2.36 g) (8) and MJM60668 (109) groups were significantly decreased to 1.22 ± 0.17 g (p ≤ 0.0001), and 1.17 ± 0.2 g (p ≤ 0.0001), respectively, compared to those of the HFD group (1.76 ± 0.3 g) (8) and MJM60668 (109) groups  were slightly decreased compared with those of the HFD group (0.33 ± 0.06 g)  a. Additi± 0.3 g) b. After  0.06 g) c.p = 0.0026), MJM60668(108) (3.68 ± 0.32 g) (p = 0.0009), and MJM60668(109) (3.53 ± 0.49 g) (p = 0.0001) groups, compared with that of the HFD group (4.44 ± 0.24 g). This result suggests that MJM60668 is a potential strain that can be used to decrease fatty acid accumulation in the liver, thus improving weight loss.Finally, after measuring the body weight and liver weight of each animal, their ratio was calculated. As shown in 8) and MJM60668 (109) significantly reduced the increased levels of AST  and ALT  seen in the HFD group   and MJM60668 (109)   and total cholesterol (TCHO) were measured. Our results showed significantly increased TG and TCHO levels in the HFD group  than in the control group . Such increased hepatic TG and TCHO levels were suppressed by the administration of MJM60668 (10ctively) c,d.8) and HFD groups showed no significant difference . Nevertheless, the MJM60668 (109) group showed a slight decrease in HDL-C level (109.5 ± 1.08 µmol/L) compared with the HFD group, suggesting that MJM60668 could be potentially used as a probiotic to decrease HDL levels in the body in a dose-dependent mannerAs shown in 8) group, 2 ± 0.8; and MJM60668 (109) group, 1.5 ± 0.57; HFD group, 3 ± 0). Thus, MJM60668 could dose-dependently influence liver regeneration and reverse steatosis induced by high-fat diet.NAFLD is characterized by an increase of intracytoplasmic lipids and ballooned hepatocytes  as the principal histological findings in steatohepatitis ,46. In oExpression levels of ACC, Fas, PPARα, CPT1A, and IL-6 mRNA levels in the liver are shown in As shown in PPARα has a key role in lipid metabolism and glucose homeostasis under the catabolism of fatty acids  by regul8) and MJM60668 (109) significantly increased serum levels of adiponectin expression  compared with the HFD group (Adiponectin is a novel therapeutic target associated with diabetes and metabolic syndrome due to its anti-inflammatory and insulin-sensitizer effect . Leptin FD group f.8) and MJM60668 (109) significantly decreased serum leptin levels  increased by HFD.As shown in 8) and MJM60668 (109) significantly reduced serum levels of IL-1β to 8.14 ± 0.57 pg/mL and 8.43 ± 0.63 pg/mL, respectively, increased by HFD (Interleukin 1β (IL-1β) is a pro-inflammatory cytokine secreted by Kupffer cells. It has a key function in liver regeneration under inflammatory conditions . Our resd by HFD h. Taken Bacteroidetes, Deferribacteres, Firmicutes, Tenericutes, Verrucomicrobia, and Proteobacteria were 0.8, 0.42, 0.02, 83.82, 0.1, 14.32, 0.1, and 0.4% in the control group, 1.1, 0.62, 0.17, 97.6, 0.3, 0.02, 0.07, and 0.1% in the HFD group, and 0.47, 0.7, 0.4, 95.7, 0, 2.65, and 0.05% in the MJM60668 (109) group, respectively.The association between LAB intake and changes in gut microbiota was evaluated at the phylum and family levels. At the phylum level, as shown in Atopobiaceae, Bacteroidaceae, Deferribacteraceae, Chirstensenellaceae, Clostridiaceae 1, Clostridiales vandinBB60 group, Family XIII, Lachnospiraceae, Peptostreptococcaceae, Ruminococcaceae, Akkermansiaceaea, and others  group. MJM60668 treated mice showed an increase in the Akkermansiaceae family known to be associated with a healthy gut barrier with an anti-inflammation effect on the gut and decrease weight gain in animals [At the family level, mice microbiota was mainly composed of d others b. Relati animals   or MJM60668 (109) inhibited lipid accumulation in a dose-dependent manner in HepG2 cells -alpha (PPARα) is the most abundant isotype in the liver and hepatocyte that regulate genes involved in fatty acid beta-oxidation and homeostasis in the body regulating the expression of genes and metabolic function expressing its receptor into adipose tissue  Its delePrevious report has shown that NALFD develop pathway is responsible for activation of IL-1 β, which contributes to liver disease by activating pro-inflammatory cytokines . Our resL. reuteri MJM60668 can positively improve gut microbiota dysbiosis, markedly inhibit the inflammation characteristic of non-alcoholic fatty liver disease, and significantly inhibit adipogenesis by regulating serum adiponectin levels in treated mice.In conclusion,


Alanine aminotransferase (ALT) levels are frequently determined in serum and plasma samples and are a primary measure to quantitate hepatocellular injury in rodents, humans, and other organisms. An accurate, reliable, and scalable assay is hence of central importance. Here, we describe a methodology that fulfills those requirements, and demonstrates an excellent performance similar to a commercial ALT kit, with a long stable performance over several subsequent runs. Further, anticoagulation of blood samples with ethylenediaminetetraacetic acid (EDTA) or heparin results in similar ALT concentrations with this assay, whereas no anticoagulation significantly increases ALT levels. Mild hemolysis does not significantly increase ALT levels; however, moderate to severe hemolysis does lead to higher ALT levels. The assay provides stable results over a wide range of associated triglyceride concentrations that can be expected in serum and plasma samples from rodents and humans with dyslipidemia. It also performs well in diluted samples with a reduction of ALT levels corresponding to the factor used to dilute the samples. The described ALT reagent is also very affordable, costing less than 1/80 of comparable commercial kits. Based on the characteristics above, this methodology is suitable for a broad spectrum of applications in mice and possibly humans, where ALT concentrations need to be determined. The activity of ALT is quantitated by determining the oxidation rate of NADH. The assay has an excellent performance similar to commercial ALT kits, but is also easily scalable, inexpensive, and very stable.Alanine aminotransferase (ALT) levels are a primary measure to quantitate liver cell injury in a wide spectrum of liver pathologies, including alcohol-associated liver disease ,2,3,4, nTris Base ;Alpha-ketoglutarate ;L-alanine ;Ethylenediaminetetraacetic acid (EDTA) ;LDH ;NADH ;Phosphate-buffered saline (PBS) ;Validate GC3 ALT Linearity Test Kit .96-well flat-bottom plates ;15 mL or 50 mL Falcon tube ;Whatman Puradisc 25 mm 0.2 µM polyethersulfone (PES) membrane filter ;Disposable reagent reservoirs ;TM Absorbance Microplate Reader ;VersaMaxSoftMax Pro GxP Software version 5.4 .Multiple ALT reagents were prepared following the instructions per protocol described under point “3. Procedure” below, with the exception of different NADH concentrations, which were chosen to demonstrate the importance of the NADH concentration for the success of the assay. A “low NADH” reagent was prepared with 8 mg/100 mL = 0.12 mM NADH concentration; an “intermediate NADH” reagent was prepared with 50 mg/100 mL = 0.75 mM NADH concentration; and a “high NADH” reagent was prepared with 100 mg/100 mL = 1.5 mM NADH concentration, which corresponds to the NADH concentration per protocol under “3. Procedure” below.A commercial ALT kit  was used to evaluate the performance of the developed ALT reagent. The commercial ALT kit included the buffer reagent “R1” and co-enzyme reagent “R2”, which were mixed 5:1 right before the start of the assay per manufacturer’s instructions, to prepare a reagent containing 500 mM L-alanine, >1200 units/L LDH, 100 mM Tris buffer pH 7.5, 15 mM 2-oxoglutarate , 0.18 mM NADH, and stabilizers and preservatives.Blood was obtained from the inferior vena cava from 14 to 15 6- to 10-week-old wild-type C57BL/6 mice, bred at the University of California San Diego. To elicit hepatocellular injury and hence higher ALT levels, a single dose of ethanol (5 g/kg body weight) was gavaged to the mice 9 h prior to sacrifice and blood draw. Immediately prior to sacrifice, mice were anesthetized with 400 µL sodium chloride with 6% ketamine and 1% xylazine per 20 g bodyweight per intraperitoneal injection. Complete anesthesia was confirmed by cessation of physical movements after stimulation. Mice expired by exsanguination; however, additional cervical dislocation was performed after blood was obtained. For the anticoagulation studies, 100–150 µL of blood was added to a sterile Eppendorf tube without any anticoagulant (“no anticoagulant group” = serum), another 100–150 µL of blood from the same mouse was added to a sterile Eppendorf tube with 10 units of heparin (“heparin group” = heparin-treated plasma), and another ~300 µL of blood also from the same mouse was added to a sterile Eppendorf tube with 5 µL EDTA (“EDTA group” = EDTA-treated plasma).For the hemolysis experiments, ~300 µL of blood was added to a sterile Eppendorf tube with 5 µL EDTA (“no hemolysis group”). Another 100–150 µL of blood from the same mouse was added to a sterile Eppendorf tube with 5 µL EDTA and the red blood cells were then lysed by using the Mini-BeadBeater-96  for 30 s (“severe hemolysis group”). Two additional groups, the “mild hemolysis group” and “moderate hemolysis group” were prepared by mixing volumes of the “no hemolysis group” and the “severe hemolysis group” in 2:1 and 1:2 mixtures for each mouse sample, respectively.For the dilution experiments, “EDTA group” plasma samples were diluted with sterile water in a 1:2, 1:5, and 1:10 dilution, respectively. Animals were maintained in a temperature-controlled room (22 degrees C) on a 12:12-h light-dark cycle. Animal breeding and harvesting were approved by the Institutional Animal Care and Use Committee of the University of California San Diego.Varying concentrations with purified triglyceride mixes including 20% glyceryl tridecanoate, 20% glyceryl tridodecanoate, 20% glyceryl trimyristate, 20% glyceryl trioctanoate, and 20% tripalmitin  were prepared. Purified triglycerides mixed with sterile water were added to respective ALT standards to achieve final ALT levels.n = 14–15, unless denoted differently in the figure legends. Two technical replicates were performed for each group. Pearson correlation was employed to calculate correlation coefficients between maximum reaction velocity maxV values and ALT values. Significance was evaluated using one-way paired analysis of variance (ANOVA) or–if there are missing values–mixed-effects analysis with Holm-Šídák’s post hoc test. A p value < 0.05 was considered to be statistically significant. Statistical analysis was performed using GraphPad Prism 8.4.0 for Mac.Results are expressed as mean ± s.e.m. Numbers for biological replicates are Obtain blood from rodents, humans or other organisms. We recommend adding 5 µL of EDTA per 500 µL blood to prevent coagulation.g for 15 min at 4 degrees Celsius.Spin blood at 10,000× Transfer clear supernatant (=plasma) to a new sterile Eppendorf tube and store at −80 degrees Celsius or use immediately for the ALT assay.Preparation of 100 mL ALT reagent–with 97 millimolar (mM) Tris Base (stored at room temperature), 13 mM alpha-ketoglutarate (stored at 4 degrees Celsius), 440 mM L-alanine (stored at room temperature), 5 mM EDTA (stored at room temperature), 3200 units/L LDH (stored at −20 degrees Celsius), and 1.5 mM NADH (stored at −20 degrees Celsius):Add 1.175 g Tris Base to a glass beaker.Fill up to ~50 mL with sterile water.Start stirring with a magnet stir bar in the beaker.Add 190.0 mg alpha-ketoglutarate.Add 3.92 g L-alanine.Add 1 mL of 0.5 M solution of the preservative EDTA.Dissolve 1000 units of LDH in a vial with 1 mL PBS, and add 320 µL of LDH solution (=320 units) to ALT reagent .Continue stirring with the magnet stir bar, until all particles are dissolved in the ALT reagent solution.Adjust pH to 7.80 at room temperature.PAUSE STEP store at 4 degrees Celsius protected from a light source  until use.Fill up to final volume of 100 mL with sterile water, transfer this prepared ALT reagent to a sterile bottle, and CRITICAL STEPPrior to use, obtain the required volume and add the same amount of NADH in mg as the required volume in mL  and vortex for 15–30 s . If no ALT values above 250 U/L are expected, one can consider adding a lower amount of NADH, i.e., 5 mg NADH to 10 mL prepared ALT reagent.CRITICAL STEP Filter the required volume of ALT reagent using Whatman Puradisc 25 mm 0.2 µM PES membrane filter to remove possible remaining small particles from the solution.CRITICAL STEP Warm up the required filtered volume in a 15 mL or 50 mL Falcon tube for 5 min in a water bath maintained at 37 degrees Celsius immediately prior to pipetting to induce the kinetic reaction required for the assay to work appropriately.15.Add 10 µL of ALT standards ranging from 0 (=water) to 1000 units/L  and plasma samples in duplicates or triplicates into 96-well flat-bottom plates.16.Add 90 µL of filtered, warmed up ALT reagent to each sample using a multi-pipet and a disposable reagent reservoir. It is good practice to prepare 1 mL in excess of the calculated required volume to ensure rapid pipetting with the multi-pipet. Shake plate slightly after completion of pipetting. Remove (large) bubbles by bursting them with a small needle.17.CRITICAL STEP Use the kinetic enzyme assay function with 24 reads at 340 nm every 13 s for a total of ~5 min. The wavelength of 340 nm is chosen to quantitate NADH, as the reduced form (NADH) can absorb light at 340 nm, whereas the oxidized from (NAD+) does not [maxV is displayed in milliunits/min.Read plate without lid in VersaMax Microplate Reader with SoftMax Pro Software, or similar microplate reader and software. does not . The mordoes not . The maxThe dependance of the standard curve on the NADH content was examined. The stability and accuracy depend on the NADH concentration with a higher NADH content  and CV of 4.92 for another sample , and high total precision over three runs with a CV 3.65 for sample 1  and CV of 4.83 for sample 2 , consistent with recommended performance per Clinical and Laboratory Standards Institute (CLSI) [To validate the prior results, we performed a head-to-head comparison of the high NADH ALT reagent , CV of 4.35 for ALT standard 62.5, CV of 4.62 for ALT standard 125, and CV of 2.04 for ALT standard 250; and across a wide biological more relevant triglyceride range 0–500 mg/dL with a CV of 0.92 for ALT standard 31.25, CV of 1.70 for ALT standard 62.5, CV of 1.24 for ALT standard 125, and CV of 1.51 for ALT standard 250. Our results with a high stability over a wide range of triglyceride concentrations are in line with results from commercial ALT kits [Since hyperlipidemia and hypertriglyceridemia often co-exist with elevated transaminase levels in particular in non-alcoholic fatty liver disease , we inveNext, we wanted to investigate whether ALT levels are still reliable in diluted plasma samples employing our ALT assay. We found that the ALT values diminish according to their dilution factor, i.e., one would expect a similar decrease in ALT value in the same magnitude of the factor that the samples were diluted with. No significant differences were noted between actual values and theoretical values A–C. AlthAs described above, in order to prepare 100 mL ALT reagent, one requires 1.175 g Tris Base , 190.0 mg alpha-ketoglutarate , 3.92 g L-alanine , 1 mL of 0.5 M EDTA , 100 mg NADH , and 320 units of LDH . This corresponds to $17.28 (=$0.30 + $0.27 + $2.38 + $0.13 + $5.20 + ~$9.00). Using 90 µL ALT reagent per sample, 100 mL would allow 1111 reactions. The final cost for the described assay would be approximately 1.56 cents per sample. On the contrary, a representative commercial assay used in this manuscript for comparison  costs $200 and allows for 150 reactions if used per manufacturer’s instructions, corresponding to $1.33 per reaction. This demonstrates how cost-effective the described ALT assay is compared with commercial assays.In conclusion, here we present an inexpensive, accurate, reliable, and scalable methodology to quantify liver cell injury. This can be applied to a broad spectrum of liver diseases in mice and possibly humans.


Raw milk (a highly perishable food)was preserved at variable roomtemperature (RT) under hyperbaric storage (HS) (50–100 MPa)for 60 days and compared with refrigeration (RF) under atmosphericpressure (AP) on quality, nutritional, and endogenous enzyme activityparameters. Overall, a comparable raw milk preservation outcome wasobserved between storage under AP/RF and 50/RT after 14 days, withsimilar variations in the parameters studied indicating milk degradation.Differently, even after 60 days (the maximum period studied) under75–100/RT, a slower milk degradation was achieved, keepingmost of the parameters similar to those of milk prior to storage,including pH, titratable acidity, total solid content, density, color,viscosity, and volatile organic and fatty acid profiles, but withhigher free amino acid content, signs of an overall better preservation.These results indicate an improved preservation and enhanced shelflife of raw milk by HS/RT versus RF, showing HS potential for milkand highly perishable food preservation in general. One of the reasonswhy HS is rising substantial research interest for food preservationis the fact that it results in significant energy reduction comparedto refrigeration (RF) since it can be applied at variable room temperature(RT) with no energy spent in maintaining constant low temperaturesas in RF, with energy being only required to pressurize and depressurizethe vessel, where food would be stored.1 estimatedthat compared to RF, storage of 800 kg of strawberry juice for 15days at HS/RT would allow a 26-fold lower energy cost but would requireso far higher investment in HS equipment. However, the equipment forecastin this study is based on the ones currently available for HPP, whichare highly more complex and demanding, as they need to achieve fastand elevated pressures (up to 600 MPa), thereby requiring a more robustvessel that can endure higher pressures than the ones for HS.1 Also, Bermejo-Prada et al.1 reported an estimated reduction of almost 25.8-fold forHS in carbon footprint (per kg strawberry juice), resulting in a moresustainable preservation methodology that in return would accountfor negligible emission tax when compared to RF.In fact, Bermejo-Pradaet al.2 Later, non-acidjuices (melon and watermelon juice) at and above RT but for shorterstorage periods, up to 60 h, have shown great stability in most ofthe parameters studied, requiring pressures of up to 50 MPa to achievea similar preservation to RF, while pressures above 75 MPa alloweda greater microbial stability, even at 25–37 °C, causingmicrobial inactivation.3 Further, longerstorage periods (10 days) were assessed in watermelon juice (50–100MPa) and whey cheese (100 MPa) at RT, reporting an initial microbialgrowth inhibition under 50 MPa, while 75–100 MPa allowed microbialinactivation in both food products, resulting in an increased shelflife compared to RF.4 More recently, thefeasibility to store fish/meat products under HS was studied for longerstorage periods (up to 60 days), with promising results. Both foodproducts revealed great microbial stability by HS above 50 MPa, withmicrobial reductions being verified in both endogenous and inoculatedmicroorganisms.5 Thus, although HS at RThas proven its capability to control microbial growth, it is importantto perform more insightful analysis in other important foods, suchas milk, not only regarding the microbial quality but also the nutritional,physicochemical, and biochemical quality parameters, to gain furtherknowledge about the potential of HS to possibly substitute RF withprolonged shelf life.Another mainreason for HS research interest is the potential considerableincrement in food shelf life, with very interesting microbial resultsbeing reported for several types of food products. Initially, HS wasstudied for juice preservation as case a study, while recently otherfoods, including solid food matrices, are being evaluated. The HSstudies on juices were carried out on strawberry juice (an acidicjuice), having been found that most of the physicochemical parametersremained stable even under low pressures such as 25 MPa at 20 °Cfor 15 days, as the low pH acts synergistically with HS to restrainthe microbial growth.6 with raw milk stored under HS conditions, 75–100MPa at RT showing considerable increased microbial stability at RTfor 60 days, compared to RF, with microbial inactivation observedto undetectable counts for endogenous microbial load (up to more than5 log units reduction) and inoculated surrogate and pathogenic microorganisms, as well as bacterial spores (Bacillus subtilis endospores) throughout storage. These results clearly indicate thegreat potential of HS for raw milk preservation, compared to RF, withpotentially increased shelf life and microbial safety.Raw milk is a highly perishable food productwith short shelf lifedue to its high nutritional profile, near-neutral pH, and high-wateractivity, resulting in a good environment for the development of severalmicroorganisms, that jeopardize the overall quality and safety ofmilk as well as dairy products produced from it, thus requiring refrigeratedstorage to slow down the microbial growth, prior processing. So far,there are no results for the effect of HS for milk preservation, withthe exception of a study carried out recently by our group,Therefore,in the present study, several raw milk quality/nutritionalparameters and activity of endogenous enzymes were studied, with rawstored under HS (50–100 MPa) at variable RT (18–22 °C)and stored at atmospheric pressure (AP) at RF and RT for 60 days.An overall assessment in milk pH, titratable acidity, total solidcontent, density, color, viscosity, volatile organic and fatty acidprofiles, lipid oxidation, total protein, soluble protein (SP), freeamino acids (FAAs), and alkaline phosphatase and lactoperoxidase activitieswas performed and compared to the milk prior to storage and milk storedin the different storage conditions.22.1Cow raw milk was collectedfrom a local dairy farm associationcompany, kept under RF during transportation, and then packaged underaseptic conditions inside a laminar flow cabinet . The samples were double-packedin UV-light-sterilized, low-permeability polyamide–polyethylenecommercial food packaging bags  and heat-sealed individually,avoiding as much as possible, leaving air inside.6 the studyof that storage condition was stopped.Then, thepackaged raw milk was stored under three different vessels at 50,75, and 100 MPa at RT (18–22 °C) using an SFP FPG13900Model  system, equipped with pressurevessels of 30 mm inner diameter and 500 mm height, with a mixtureof (40:60) propylene glycol and water used as the pressuring fluid.For comparison, the raw milk samples were also stored at RT and RF(4 °C) at AP (0.1 MPa) for 7, 14, 28, 39, and 60 days and keptin the dark to simulate the same conditions of HS samples. Once theraw milk stored under the different storage conditions was consideredmicrobiologically unsuitable,2.2pH wasmeasured directly in the sample at constant RT with a proper calibratedpH meter . The total solidcontent and density were determined using a portable density and brixmeter  at 20 and15 °C, respectively. The titratable acidity of the raw milk sampleswas determined by titrating 5 mL of diluted raw milk (2 mL of milkin 3 mL of distilled water) to pH 8.4 with a previously standardizedsodium hydroxide 0.01 M solution using an automatic titrator . The results wereexpressed as grams of lactic acid per liter of milk based on eq 2.3a*), yellow/blue color (b*), and luminosity (L*) parameters. Thecolor parameters L*, a*, and b* were measured, and the total color change (ΔE*) was calculated by eq E* representsthe total color difference between a respective sampleand the initial one prior to storage, with L0*, a0*, and b0* representing the respective parameter at day 0.The color parameters were measuredusing a Minolta Konica CM 2300d equipment , calibrated before each sample measurement. The colorparameters were recorded in the CIELab system and directly computedthrough the original SpectraMagic NX software  according to the International Commission on Illumination regulations:red/green color (2.4p-nitrophenylphosphate (p-NPP) as a substrate,as described by Negrão et al.,7 withsome modifications . Initially,raw milk was mixed with 4 mM p-NPP in buffer solutionand incubated for 30 min at 37 °C. The reaction was stopped bythe addition of 2 M NaOH, and the p-nitrophenol releasedwas measured at 405 nm using a microplate spectrophotometer . The activity was expressedin ΔAbs405nm/min .Thealkaline phosphatase (ALP) activity was assayed with 8 Briefly, raw milk was mixed witha solution of 0.325 mM ABTS  and left for 30 min at 20 °C, and then 0.1 mM hydrogen peroxidewas added and mixed quickly to initiate the reaction, with the absorbance(Abs412nm) measured for 1 min. The enzymatic activity wascalculated as the slope of the curve relating Abs increment versustime and expressed as ΔAbs412nm AU/min.The lactoperoxidase (LPO)activity assay was performed based on the method described by Marínet al.A is the enzymaticactivity in raw milk samples after storage and A0 is the enzymatic activity of the sample at day 0.Allenzymatic assays were performed in triplicates for each storagecondition, with the residual activity calculated by eq 2.5–1, within the Newtonian region, was usedto compare among samples.Milk viscosity was assessedthrough a controlled stress rheometer  equipped with a cone-and-plate geometry . Prior to the analysis, the sampleswere allowed to achieve a constant temperature (20 ± 0.5 °C)on the rheometer equipped with a fixed flat plate at the bottom for300 s. A circulatory thermostatic bath  was connected to this plate, ensuringthat the target temperature was achieved and maintained. Before placingthe samples on the rheometer plate, each sample was mixed gently andcarefully transferred onto the rheometer measuring system, avoidingthe trapping of air bubbles between the cone and the plate. Flow curveswere obtained by applying a continuous stress ramp from 0 to 2 Pafor 3 min. Rheological results were monitored using a TA Instrumentssoftware package. The apparent viscosity measured at a shear rateof 300 s2.69 It was performedby headspace solid-phase microextraction, followed by gas chromatography–massspectrometry (GC–MS). Raw milk (2 mL) was pipetted into 35mL vials containing the internal standard (50 μL of cyclohexanoneaqueous solution at 25 μg/mL) and immediately sealed with ametallic cap with silicon septum. After equilibration at 50 °Cfor 30 min, with agitation (500 rpm), the SPME fiber  was exposed to the sample’s headspacefor 30 min at 50 °C for adsorption of volatile compounds. Thefiber was inserted in the injection port of the GC equipment, an AgilentGC-7890 gas chromatographer, equipped with a an Agilent 5977B massspectrometer, and a DB-5 MS Capillary GC column . The injectorport was heated to 260 °C, and injections were performed in thesplitless mode with helium at a linear velocity of 1 mL/min. The oventemperature was set at 35 °C for 5 min, increasing to 100 °Cat a rate of 4 °C/min, followed by an increase of 10 °C/minuntil 225 °C, and held for 0.25 min . Theion source and interface temperatures were maintained at 230 and 280°C, respectively, and the electron impact ionization mass spectrawere recorded with an ionization energy of 70 eV. Mass spectra werescanned from 20 to 350 m/z in thefull scan mode. Identification of the volatile compounds was basedon computer-matching with the reference mass spectra of the MS libraryof the National Institute of Standards and Technology 2011 (NIST 11),retention times, retention index, and with individual standards whenavailable. Using cyclohexanone as the internal standard equivalentbasis, the volatile profile was semi-quantitatively determined fromthe full scan areas, and the results were expressed in μg ofthe internal standard equivalents per mL of milk.Volatileorganic compound (VOC) profile determination was based on the methoddescribed by Yue et al.2.710 was performed. Briefly, 100 μLof the internal standard solution  was evaporated to dryness under a gentle stream of nitrogen, and 1 mL of milk was added, followedby the addition of isopropanol (2 mL) for protein precipitation, cyclohexane(2 mL), and NaCl aqueous solution (1%) (1.5 mL). After agitation andcentrifugation , the supernatant was collected, evaporatedunder a nitrogen stream at RT, and redissolved with heptane (2 mL).For the preparation of fatty acid methyl esters (FAME), 2 M potassiumhydroxide (200 μL) was added, and the samples were carefullyvortexed for 1 min. Finally, 50 μL of injection standard [20mg/mL of methyl tridecanoate (C13:0 methyl ester)] was added. Thehexane layer containing FAME was transferred into 1 mL GC vials.For the determinationof fatty acid profile, a method similar to that described by Sobralet al.The FAME profile was analyzed using a gas chromatograph  with flame ionization detection (FID).1 μL of the extracted FAME was injected into the GC system,with the injector and detector temperatures set to 250 and 270 °C,respectively. Separation of the fatty acids was achieved on a SelectFAME (50 m × 0.25 mm × 0.25 μm) column using helium as the carrier gas (pressure of 140 kPa), heated from120 °C (3 min hold) to 220 °C (5 min hold) at a rate of3 °C/min. Fatty acid identification and FID calibration wereaccomplished with a certified reference standard mixture , and the results were expressed in relativepercentages of their FAMEs.2.811 Briefly, 1 mL of rawmilk was mixed with 2 mL of 7.5% trichloroacetic acid and then vortexedfor approximately 60 s, followed by centrifugation at 4000g at 4 °C for 20 min . After filtration (Whatman no. 1), 1 mL of theresulting extract was added to 1 mL of 46 mM 2-thiobarbituric acid,vortexed and immersed in boiling water for 40 min, and then cooleddown in cold water. Triplicates were measured using a microplate spectrophotometer with a Brand plateof 96 wells, at 532 nm. Standard solutions of MDA in 7.5% trichloroaceticacid were prepared from 1,1,3,3-tetramethoxypropane, and a calibrationcurve was prepared at a concentration ranging from 0.2 to 10 μg/L.TBARS results were expressed as μg of MDA per mL of milk.Lipid oxidation was determined by malondialdehyde(MDA) quantificationusing the 2-thiobarbituric acid reactive substances (TBARS) methodwith adaptations.2.912 with few modifications. Initially,milk was diluted in distilled water (1:100 v/v), followed by centrifugationat 4000g at 4 °C for 15 min . Then, 50 μL of the supernatantwas added to 250 μL of dye Coomassie Blue G25 in a microplate,shaken for 30 s, and incubated for 20 min at RT. The absorbance wasmeasured at 595 nm , and SP was expressed in mg per 100mL of milk. A calibration curve was prepared using BSA as the standardat concentrations ranging from 0 to 0.5 mg/mL. For FAA determinationand quantification, the milk was centrifuged , and the supernatant was collected and centrifugedagain. The secondary supernatant (100 μL) was used for the analysisof FAAs using the EZ:Faast Amino Acid Analysis Kit (GC-FID),13 and the results were expressed in nmol per mLof milk.The overall proteinprofile was assessed by determining the total nitrogen (TN) usingthe Kjeldahl method, SP by the Bradford method, and FAAs using theEZ:Faast Amino Acid Analysis Kit available for GC-FID. The micro-Kjeldahlprocedure was performed with a Kjeltec system 1002 Distilling unit, and the crude protein content was determined bymultiplying the total nitrogen content by 6.38 . The total SP was determined based on the Bradfordmethod2.10All experimentsand analyses were carried out in triplicate. Analysis of variancewas performed under all the different storage conditions, followedby a multiple comparison post hoc test, Tukey’s HSD test, ata 5% level of significance. Additionally, principal component analysis(PCA) was performed in order to identify the statistical patternsin VOC data.33.114 As expected, storageconditions that allowed fast and considerable microbial growth6 resulted in an increasing acidity (p < 0.05), just after 7 days, in the case of AP/RT with a pH of4.14 ± 0.02 (p < 0.05)decrease to 6.53 ± 0.01 for storage under AP/RF after 14 daysand to 6.43 ± 0.01 at 50/RT after 28 days was also observed.The raw milk stored under 75 and 100 MPa also presented a slight decreasein pH throughout storage; despite being statistically significant(p < 0.05), the decrements were much smaller,with the pH decreasing slightly to around 6.51 ± 0.2 for bothstorage conditions after 60 days .The pH,titratable acidity, total solid content, and density were assessedin all samples under the different storage conditions 1. The in4 ± 0.02 1. A less15 A substantial increase was observed for samplesstored under AP/RT, with titratable acidity increasing to 11.66 ±0.18 g/L after 7 days  comparedto samples prior to storage. The milk acidity continued to increasefor all storage conditions, however at different rates, with samplesstored under AP/RF and 50/RT reaching similar values, just after 14and 28 days, 2.22 ± 0.16 and 2.41 ± 0.05 g/L, respectively,while when stored under 75 and 100/RT, the milk reached a maximumof 2.24 ± 0.13 and 2.26 ± 0.03 g/L after 60 days, respectively.Regarding titratable acidity,the initial value observed was similarto the values reported in the literature, 1.73 ± 0.02 g/L.r 7 days 1. As men16 Under AP/RT and AP/RF after 7 and 14 days, respectively,a significant decrease (p < 0.05) was observed,reaching a minimum of 9.17 ± 0.26% and 10.67 ± 0.38%, respectively.Storage under pressure maintained a similar TS value throughout thestorage period with slight variations observed (p > 0.05), with final values of 11.25 ± 0.42 and 11.58 ±0.14 after 60 days at 75 and 100/RT, respectively.For total solids, the samples prior storage had a value of 11.83± 0.63%, similar to the values reported in the literature, 9.7–12.5%.15 The only variation(p < 0.05) observed within all the storage conditionswas for AP/RT that presented a significant decrease from 1.036 ±0.003 to 1.029 ± 0.001 g/mL after 7 days, with storage underRF and HS resulting in no modifications in milk density (p > 0.05).As for density,the values obtained were within the ones observedin the literature for refrigerated raw milk, which should be between1.023 and 1.040 g/mL, as values outside this range may indicate adulteration,such as water addition.3.2L*, a*, and b* were monitoredin milk stored under the different conditions, and the total colorchange (ΔE*) was calculated, followed by comparisonto the initial values of milk prior to storage  ranged from 55.79 ± 0.03 (AP/RTat day 7) to 52.68 ± 0.14 (100/RT at day 60), with no significantchanges (p > 0.05) observed compared to the initialvalue.The colorparameters  storage 1. Raw mip > 0.05) in the raw milk L* parameter,but with no statistical significance (p > 0.05).Regarding a* (greenness), the only significant variationwas observed under 75/RT at days 28 and 60 (p <0.05), to values of −0.89 ± 0.02, for both periods. Asfor b* (yellowness), no changes were observed underthe different storage conditions (p > 0.05), rangingfrom 2.51 ± 0.04 to 3.09 ± 0.04. In milk HPP studies, the L* parameter is usually the most affected (p < 0.05), decreasing after processing,17 which could be due to disintegration of casein micelles into smallerfragments that increase the translucency of milk, thus affecting thiscolor parameter. However, the HPP studies apply significant higherpressures compared to HS, which in the present work presented onlya slight decrease (p > 0.05) throughout the storage.Overall, HS presented a slight decreasing tendency  to values of1.70 ± 0.03, compared to storage under AP/RF, 0.56 ± 0.16.On the 14th day, a slight increase (p > 0.05)wasobserved for most storage conditions, compared to the respective storageat day 7, which tended to increase as the time passed. On the 28thday, samples at 50/RT presented a significantly increased ΔE* value of 1.37 ± 0.30, while storage at 75 and 100/RTmaintained a ΔE* value similar to that of day7. However, at the end of the storage, at day 60, the values increasedto 1.89 ± 0.17 and 1.48 ± 0.15 under 75 and 100/RT, respectively,possibly related to the observed decrease in the L* parameter in these samples. According to Drlange,18 all of the ΔE* values for samplesin this study are considered to have a “small difference”(0.5 < ΔE* < 1.5) perceptible by theconsumer’s eyes compared to the initial raw milk color, withsamples under AP/RT at day 7 and 75/RT at day 60 having “distinctdifferences” (1.5 < ΔE* < 3).Relatively to the overall color changes, Δ3.319 The residual activityof ALP under all storage conditions throughout storage is describedin p < 0.05) of35%, at AP/RT with the rest of the storage conditions presenting residualactivities similar to AP/RF (around 81%). Overall, after 7 days ofstorage, the ALP activity tended to decrease slightly over time, butglobally without statistical significance (p >0.05),compared to the value at day 7. Similarly, Fidalgo et al.20 also observed a decrease in acid phosphatasein Atlantic salmon under 75 MPa at 25 °C after 25 days to 23%opposingly to storage under 60 MPa at 10 °C after 30 days, whichmaintained the acid phosphatase activity stable (around 111%).Alkaline phosphatase (ALP) is an enzyme naturally present in milk,mainly bounded to the fat globule membrane, that can catalyze thehydrolysis of phosphate monoesters, yielding phosphate and the correspondingalcohol. ALP is also commonly used as a standard assay for rapid validationof the milk pasteurization process as it is slightly more resistantto thermal treatment than the non-sporogenic pathogenic microorganismspresent in milk.21 At AP/RT,the LPO activity was significantly reduced (p <0.05) to 24% of its initial value after 7 days (p < 0.05) whencompared to its initial activity to around 79 and to 47% after 7 and14 days, respectively. Storage under 50/RT presented a behavior alikeAP/RT, with a more pronounced reduction (p < 0.05)in the LPO activity up to 33 and 5% of the initial value after 7 and14 days, respectively. This enzyme might be more susceptible to thechanges observed under these storage conditions, AP/RT, AP/RF, and50/RT, namely, high microbial activity, decrease in pH, and increasein acidity, which all together can promote LPO denaturation and/orreduce its activity.22LPO is also an enzyme commonly present in milk, being one of itsindigenous antimicrobial agents. This enzyme catalyzes the oxidationreactions in the presence of hydrogen peroxide and helps the productionof products with a wide antimicrobial activity, such as pseudohalogens,thiocyanates, or halogens.r 7 days 1. Under p < 0.05) and 123% (p < 0.05) for 75 and 100 MPa, respectively. After 14 and 28 days,despite the decrease in LPO activity observed for both storage conditions,no significant variations were observed compared to the initial LPOactivity (p < 0.05). Overtime, the LPO activitywas slightly more affected during storage under 75/RT, ending at day60 with an activity of 56% of the initial value, while storage under100/RT maintained an LPO residual activity similar to the one observedat days 28 and 39, around 80% of the initial value, at the end ofthe storage period (p < 0.05).Storage under75 and 100/RT presented overall a much better maintenancein LPO residual activity throughout storage, compared to the otherstorage conditions performed. At day 7, both storage conditions presentedan increased LPO activity, compared to the initial one, to valuesaround 113%  activity during HS/RTin watermelon and strawberry juice, for 10 and 15 days, respectively.25 Bermejo-Prada and Otero25 reported aconstant residual POD activity throughout storage, decreasing onlyto 85% under 200 MPa at 20 °C. As in Pinto et al.’s4a work, a significant reduction overtime in PODresidual activity was reported, to values of 54.6 and 16.8% after10 days under 75 and 100 MPa, respectively. In the present study,milk stored at 100/RT resulted in the decrease of LPO residual activityto 80% on the 28th day of storage, remaining stable until the endof the storage period, possibly retaining the LPO antimicrobial activity.Knowledgeabout the HPP effect, especially for low and mild pressures,in enzyme activity is scarce, and the HPP effect is not always straightforward,being dependent in several variables, such as HPP pressure level,duration, temperature, pH, and the matrix environment, and can bespecific for a determined enzyme.3.426 Storage at AP/RF presented an almost 10-fold increase (p < 0.05) in milk viscosity to 31.12 ± 5.98 mPa·safter 7 days, while under HS conditions , it remainedunchanged (p > 0.05) for this storage period.After14 days, samples under 50/RT presented a viscosity of 10.63 ±1.39 mPa·s (p < 0.05), while samples under75 and 100/RT showed no changes in viscosity (p >0.05) throughout the entire storage period, with values of 3.00 ±0.02 and 2.96 ± 0.07 mPa·s, respectively, at the end ofthe storage (60 days). The considerable microbial growth observedin samples at AP/RF and 50/RT6 may inducechanges in milk composition, such as a decrease in pH and an increasein extracellular proteases and polymeric substances released by lacticacid bacteria, that have shown to increase the viscosity of milk.27 Several studies have shown that HPP causes anincrease in milk viscosity, directly dependent on treatment intensityfrom pressures above 200 MPa for 30 min, with a slight increase inmilk viscosity also observed in treatments below that pressure.28 These changes are mostly related to the HPPeffect in casein micelles, promoting changes in caseins shape, fromspherical to non-spherical, micelle disruption, or even reductionin particle size.28 Apparently, under HSat 75 and 100 MPa, such changes seem to not to occur, or at leastnot at a level enough to cause observable changes in viscosity.Apparent viscosity wasdetermined for all studied storage conditions 1, with t3.5n = 5), namely,acetic, butanoic, hexanoic, octanoic, and decanoic acids, followedby 3-hydroxybutan-2-one, similar to what is reported for milk in theliterature.9 In lower concentrations, someethyl esters (n = 3), alcohols (n = 2), and aldehydes (n = 2) were also found, with3-methylbutanal being detected only in the initial raw milk, priorto storage.A totalof 19 VOCs were identified in almost all samples, mostly free fattyacids (FFAs) and their ethyl esters, alcohols, and aldehydes 2. FFAs wp < 0.05) in the major VOC classes, except for aldehydes,despite the slight increase in hexanal concentration. Overall, a significantincrement (p < 0.05) in all FFAs was observed,almost up to 10-fold for acetic, butanoic, hexanoic, and decanoicacids, which can result mainly from the microbial action and lipaseactivity on fatty acids, and in a smaller degree, degradation of lactoseand amino acids, that all together can be responsible for perceptiblerancid flavor in milk.14 Esters were alsomore abundant (p < 0.05) in samples stored underAP/RT at day 7, when compared to the initial milk, which was particularlymore pronounced for ethyl acetate, butanoate, and hexanoate. Additionally,ethyl octanoate and decanoate that were absent in the initial milkwere now detected abundantly. The content in alcohols also increasedconsiderably (p < 0.05) compared with milk priorto storage, especially for 3-methylbutan-1-ol (64-fold higher), with2-methyl-1-butanol being now present. Both alcohols and esters caninfluence the flavor of dairy products when present in high concentrations,with alcohols being mainly derived from amino acid metabolism or fermentationof lactose and esters from esterification of short-chain alcoholsand FFAs, both potentially indicating high microbial and enzymaticactivities,29 which is coherent to whatwas reported for this storage condition  for AP/RT samples at day7).6 In what concerns aldehydes, for thesesamples only hexanal was found, showing a significant 5-fold increaseunder AP/RT (p < 0.05) that may derive from unsaturatedfatty acids oxidation.30Storage under AP/RT at day 7 resulted in significant changes , when compared to milk prior to storage,with ethyl octanoate and decanoate now present .6At the 7thday of storage under AP/RF, the evolution of milk VOCprofile was similar to AP/RT samples; however, the increments of themain VOC occurred at a slower rate, namely, for most FFA and esters,as under low temperature, the microbial growth and enzymatic activityare slowed down, as it was observed in a previous study regardingthe microbial evolution between these two storage conditions, AP/RFand RT (around 5 log units for AP/RF after 7 days).p > 0.05) with AP/RF samples forthe same storage period, with the exception for total FFAs, that werestatistically found at a lower concentration (p <0.05), while aldehydes were considerably higher (p < 0.05) when compared to the corresponding AP/RF samples. Althoughthese samples presented a slower degradation rate pattern overallwhen compared to AP/RF, they presented significant increases (p < 0.05) in all identified VOCs when compared to theinitial milk, with the presence of ethyl octanoate and decanoate beingdetected at the 28th day of storage, possibly resulting from the increasedmicrobial load observed for this storage condition at the end of thestorage period (above the acceptable limit).6 Toluene is a common compound reported in milk and dairy products,resulting from β-carotene degradation, detected in HS samples.Under HS at the lowest pressure (50 MPa), the VOC concentrationwas comparable (p < 0.05) under 50 and100/RT. Between storage under 75 and 100/RT, the latter one presenteda VOC profile more similar to that of the initial milk, with lowerchanges in all FFAs, with no nonanoic acid formation being detected(similar to milk prior storage) and also relatively for esters , without the formationof fatty acids and ethyl esters, thus possibly indicating a betterpreservation of raw milk under these conditions. The overall alcoholcontent remained low and constant under 100/RT (p < 0.05), despite the formation in low concentrations of 2-methyl-1-butanoland 2-ethylhexan-1-ol. As far as the authors are aware, the informationavailable regarding the effect of low pressures for extended periodson VOC of foods is very scarce and absent at variable RT. Anyway,for the sake of comparison, the results observed in the present workare in accordance with that reported by Fidalgo et al.,31 which observed a similar fresh salmon-like VOCprofile for samples stored under 60 MPa at 10 °C up to 30 days.Regarding raw milk under variable RT, a slower matrix degradationevolution for both 75 and 100/RT for 60 days was observed comparedto the sample prior to storage and a much better preservation of theVOC profile than those under AP/RF, which may also indicate a bettercontrol in microbial and enzymatic parameters.As for the upper pressures (75 and 100 MPa), a better overall preservationof the raw milk VOC profile was achieved, even after 60 days, comparedto the initial one, for all FFAs, esters, alcohols, and aldehydes,with the exception for 3-hydroxybutan-2-one, whose concentration decreasedconsiderably, especially , compared to the initial milk priorto storage. The major variations regarding 75 and 100/RT were relatedto the increase (p < 0.05) of lauric, myristic,and palmitic acids and reductions in oleic acid (±0.5%). However,when compared to storage at AP/RF after 7 days, the only significantreduction was on MUFA content after 60 days under 75/RT, presentinga significant decrease around 0.34%, which can be related to the increasinglipid oxidation values observed for that storage period (The milk fattyacid profile was characterized by a greater abundance in saturatedfatty acids (SFAs—63.15–63.30%), followed by monounsaturatedfatty acids (MUFAs—30.28–30.92%) and polyunsaturatedfatty acids (PUFAs—4.65–4.66%) 3. Regarde period 1.3.734 and the only storagecondition that presented a significant change (increase) was AP/RT,reaching values of 1.18 ± 0.07 μg MDA/mL (p < 0.05) after 7 days. All the other storage conditions showedno significant (p > 0.05) variations in lipidoxidationvalues, despite showing a tendency to increase in HS samples overtime. This resulted in a good maintenance of MDA values for all HSsamples up to 60 days of storage (particularly at 75 and 100 MPa),compared to the values of the initial milk, because after 7 and 14days of storage, these samples showed even lower MDA concentration.Noteworthy is the fact that after 60 days at RT, the samples storedunder 75 and 100 MPa presented TBARS values  below 1.3 μgMDA/mL, which was associated with the perceptible sensory changesin milk reported in Johnson et al.’s34 work.Lipid oxidation is responsible for the productionof numerous undesirablecompounds that impact negatively the sensory and nutritional qualitiesof dairy products, which can be enhanced by the presence of oxygen,light, endogenous, and exogenous metals and enzymes. The MDA contentwas monitored as an indicator of secondary lipid oxidation developmentin all milk samples, being the results presented in 3.815 novariations (p > 0.05) were observed between allthedifferent storage conditions, even after 60 days at 75 and 100/RT.Total protein wasquantified prior to and after storage at the different conditions4, with ap < 0.05)was observed, especially for longer storage periods (p < 0.05) at AP/RF, 50/RT, and AP/RT to a minimum of1.06 ± 0.10 mg/100 mL, possibly related to nitrogen uptake formicrobial metabolism. Storage under 75 and 100/RT maintained SP concentrationafter 7 days (p > 0.05), which tended to increaseas the storage period increased, with a significant variation beingobserved from day 14 to 39 (p < 0.05) of storage,under 75 and 100/RT, respectively. Especially, from day 39 until theend of the study, the SP concentration had a pronounce increase ofalmost 4- to 6-fold under 75 and 100/RT, respectively, which can beattributed to the enzymatic activity of proteases such as plasminand microbial proteases.22 The SP incrementin milk can occur in prolonged storage at RT, with Olfa et al.22 reporting an increase of 140% from day 0 to60 days at 30 °C in the SP content of UHT milk, possibly as aresult of thermoresistant microbial proteases (mainly fromPseudomonas spp.) since plasmin tends to lose mostof its activity at UHT conditions.35 TheHPP of raw milk36 seems to affect onlythe plasmin activity for pressures higher than 250 MPa (10–30min), which decreased throughout the storage at AP/RF and at 37 °Cbut in the end resulted in a significant increase in SP content, evenin treatments with reduced plasmin activity, thus pointing to theinvolvement of other proteases in this process.Regarding SP, an overall increase( periods 4. The SPr2 = 0.968). Initially, raw milkhad a total FAA of 1.3 ± 0.1 μmol/mL, being detected 20aa, with all the essential ones being present and characterized mainlyby a high abundance of glutamic acid, alanine, glycine, histidine,and proline, which is in accordance with the literature.37 Storage under AP/RT and AP/RF resulted in similartotal FAA values (p > 0.05), compared to the initialvalues, despite the tendency to increase at the end of storage forthese two conditions. On the other hand, HS presented an overall considerableincrease throughout the storage on the majority of FAAs, increasingover time and were more pronounced with the increase of pressure (p < 0.05). Milk stored under 75 and 100/RT had a similarFAAs profile, but compared to the initial FAAs, a greater variance(p < 0.05) was observed especially for tyrosine> methionine > leucine > tryptophan > serine > isoleucine> phenylalanine> threonine > cystine > valine > and histidine, with theseFAAs beingassociated with plasmin and microbial protease enzymatic activityon caseins.22 Since in this study, rawmilk without any kind of processing was employed, a variety of activemicrobial proteases can be present initially, alongside with endogenousplasmin, which can promote casein proteolysis into small peptidesor amino acids.35FAAs showeda similar behavior during the study as to what wasobserved to SP, under the different storage conditions 4, with a36 However, when Atlantic salmon was stored underHS conditions (50–75 MPa at 10–25 °C), severalproteases  have been shown to maintainpartial activity even after periods up to 50 days;20 these protease activities are more affected by the storagetemperature than by the HS pressure level during storage, with storageat lower temperature (10 °C) causing changes, to a lesser extentin the myofibrillar fragmentation index.20The informationregarding the effect of HPP in milk plasmin andother proteases usually employed higher pressures (200–400MPa) than the ones used in this study, and for shorter periods oftime, and thus, it is difficult to make a straightforward comparisonof the results obtained in these different conditions.6Overall, the quality and nutritional parameters of raw milkevaluatedin this study point to a better preservation by HS, compared to conventionalRF, particularly for pressures of 75 and 100 MPa. For instance, theonly parameter, from those studied, found to be considerably affectedby HS was the FAA content, indicating a higher proteolytic activityafter 60 days under HS. Thus, further research regarding the HS effecton the proteolytic agents of raw milk should be investigated in orderto fully understand it and its impacts on the sensorial propertiesof milk and the possible technofunctional properties in the latteron produced dairy products. All the other parameters monitored indicatea better preservation under HS (75–100/RT) of raw milk, resultingin an overall similar profile to raw milk prior to storage by instrumentalanalysis, retaining the characteristic fatty acids and VOC profiles,and corroborated by a PCA for the latter, clearly resulting in a betterpreservation methodology compared to RF for longer storage periods.In conclusion, HS at 75 and100 MPa at RT is a clear promisingfood preservation methodology for storage of raw milk, leading possiblyto considerable shelf life extension with an overall closer quality,similar to raw and refrigerated milk (but in this case for a muchshorter storage period) and should be further studied, given the highimportance of milk in the human diet.


S. aureus using fluctuation analysis at 4 × minimum inhibitory concentration (MIC). Resistant mutants were characterized. S. aureus strains HG001, Mu50, N315, and USA300 had an MIC of 0.25 mg/L. The median FoM for CorA resistance was 1.5 × 10−8, 4.5-fold lower than the median FoM of 6.7 × 10−8 for rifampicin, and was reflected in a 4-fold lower mutation rate for CorA than rifampicin (6 × 10−9 for CorA vs. 2.5 × 10−8 for rifampicin). In CorA-resistant/rifampicin-sensitive strains, the majority of amino acid exchanges were S1127L in RpoB or K334N in RpoC. S. aureus Mu50, a rifampicin-resistant clinical isolate, yielded two further exchanges targeting amino acids L1131 and E1048 of the RpoB subunit. The plating of >1011 cells on agar containing a combination of 4 × MIC of rifampicin and 4 × MIC of CorA did not yield any growth. In conclusion, with proper usage, e.g., in combination therapy and good antibiotic stewardship, CorA is a potential antibiotic for treating S. aureus infections.Corallopyronin A (CorA) is active against Gram-positive bacteria and targets the switch region of RNA polymerase. Because of the high frequency of mutation (FoM) leading to rifampicin resistance, we determined the CorA FoM in  Corallococcus coralloides Cc c127 [Staphylococcus aureus and some Gram-negatives such as E. coli DH21tolC, a mutant strain with increased permeability of the outer membrane; Neisseria flava [Chlamydia trachomatis [Wolbachia spp. [Rickettsia typhi, and Orientia tsutsugamushi [Corallopyronin A (CorA) is an α-pyrone antibiotic MW 527.7 Da) produced by the myxobacterium 7.7 Da prhia spp. , Rickettugamushi . CorA isE. coli, exchanges of two amino acids in RpoC (K345 and V1351) and four amino acids in RpoB  increased the CorA MIC at least eightfold [Thermus thermophilus showed that of these sites, K345, E1279, and S1322 directly interact with myxopyronin [CorA inhibits bacterial DNA-dependent RNA polymerase (RNAP) by blocking the switch region and thus prevents the opening and closing of the RNA clamp . CorA biS. aureus SH1000 mutants with CorA, five mutated amino acids were identified: S1127, E1084, and L1131 in S. aureus RpoB and K334 and L1165 in S. aureus RpoC [E. coli  [Staphylococcus aureus L1165 is located next to K1166 (K1348 in E. coli RpoC), which also shows an interaction with myxopyronin [S. aureus HG001, a closely related strain that, however, does not harbor the UV-treatment-induced SNPs of S. aureus SH1000 [S. aureus clinical strains. After the selection of resistant Measured mutation rates are influenced by the concentration of the selective agent in the agar . In a prS. aureus strains HG001, Mu50, N315, and USA300  was determined in parallel, using rifampicin  for the preparation of selective plates. Employing directly plated 100 µL culture aliquots, the percentage of agar plates that did not show the growth of resistant colonies was significantly higher for CorA (37.5%) than for rifampicin (2.9%) . Therefore, 250 or 1000 µL of culture was pelleted, resuspended in 100 µL of buffer, and plated. Using this method, the median FoM to rifampicin resistance was 4.5-fold higher than that to CorA resistance  (CorA had an MIC of 0.25 mg/l against d USA300 . To detesistance  and the  U-test) .p < 0.0001, Mann–Whitney U-test). The plating of 1.2 × 1011 cells of S. aureus HG001 on Iso-Sensitest agar containing a combination of both agents, 4 × MIC of rifampicin and 4 × MIC of CorA, did not yield any growth after 24 h of incubation.The mutation rates  were 2.8S. aureus HG001 were sequenced and were found to harbor exchanges in two mutational hotspots, i.e., position 1127 in RpoB (favoring the exchange S1127L) and position 334 in RpoC (favoring the exchange K334N) . All sites that were affected in different strains are shown in Twenty-seven resistant mutants from nine independent cultures of e K334N) . Rifampi−8 ± 2.4 × 10−8 for resistance to CorA and of 1.4 × 10−7 ± 2.2 × 10−7 for resistance to rifampicin at 4 × MIC were determined, and the authors concluded that both FoMs were “comparable”. However, the exact methodologies of the experiment and calculations were not described [−8  were reported at 4 × MIC, which is higher than the rates of 6 × 10−9 to 9 × 10−9 obtained here using the same calculation method. Potentially unstable features have also been identified in myxopyronin. However, chemical modifications of these functional groups led to decreases in antibacterial activity [In a previous publication, FoM values of 7.2 × 10escribed . This reactivity ,16.S. aureus 8325-4 after the plating of saturated cultures on agar containing 4 × MIC, and further mutations have been found in clinical isolates [E. coli. However, using myxopyronin, nine mutations were detected, which also increased CorA MICs eightfold or more [S. aureus HG001 picked from nine different cultures yielded only four different exchanges targeting mainly two amino acids, S1127 in RpoB and K334 in RpoC, with S1127L RpoB and K334N RpoC being the most abundant exchanges . We also tested the mutational changes in a rifampicin-resistant strain, S. aureus Mu50, an MRSA with intermediate susceptibility to vancomycin [S. aureus Mu50 yielded two additional mutation sites at L1131 and E1084 in RpoB. Mariner et al. found exchanges in the same positions in eight sequenced mutants, including an additional exchange in L1165 [S. aureus Mu50 CSz1 mutant in this study. Therefore, it is possible that in these experiments relatively more mutants with a lower resistance level were able to survive the selection [The spontaneous mutation rates of growing bacterial cultures are strain-specific and constant in the absence of agents that influence the mutation rates. Therefore, the lower FoM and mutation rates measured with CorA might depend on a lower number of different mutations that convey resistance against CorA compared to rifampicin. For rifampicin, 16 different exchanges have been described in isolates . Saturat or more . In thisxchanges . Identicncomycin  that alrncomycin . Eleven S. aureus in direct selection experiments, which might explain the lower mutation rates compared to rifampicin. A stable formulation is now available [S. aureus infections. The efficacy of CorA as a chemotherapeutic agent in combination with different antibiotics for the treatment of staphylococcal disease will be investigated in the near future.In conclusion, six possible amino acid positions were identified that yield resistance against CorA in vailable ; therefoStaphylococcus aureus HG001, a derivative of the standard strain S. aureus NCTC 8325  8) that has been repaired in rsbU [S. aureus Mu50 , vancomycin-intermediate susceptibility, CC5 [S. aureus USA300 (NRS384)  and S. aureus N315  was empble, CC5 ) were alMyxococcus xanthus carrying the CorA biosynthesis gene cluster, and was purified as reported previously [CorA with a purity of >91% was produced by the Helmholtz Centre for Infection Research, Department of Microbial Drugs, using the heterologous producer strain eviously . 5 cells per ml, as specified by EUCAST [The determination of MICs was performed in Iso-Sensitest broth  with an inoculum of 1–5 × 10y EUCAST . RifampiS. aureus HG0001 from a fresh Columbia blood agar plate and incubated overnight. In the morning, 9–15 main cultures (9.9 mL Iso-Sensitest broth in a 100 mL Erlenmeyer flask) were inoculated with 0.1 mL preculture and incubated at 37 °C and 170 rpm until the cultures had reached an optical density OD600 of 1. Then, 1 mL of a culture of OD 1 (or 0.25 mL for most agar plates with rifampicin) was centrifuged  and the pellets were resuspended in 100 µL of broth. An aliquot of 100 µL of this resuspended pellet and 100 µL of undiluted culture were plated onto selective plates with CorA (1 µg/mL) and rifampicin (0.076 µg/mL). In addition, serial tenfold dilutions of the cultures were plated onto Iso-Sensitest agar without antibiotics for the determination of colony forming units (CFU). CFU were counted after 24 h of incubation at 37 °C in the dark. All culture plates that did not yield colony smears were evaluated, i.e., for CorA 26 of 40 agar plates with plated pellets were evaluated. For rifampicin, 34 main cultures were tested and it was possible to evaluate 19 of 34 plated pellets.Iso-Sensitest agar  was prepared as advised by the manufacturer, autoclaved, and equilibrated at 50 °C before the addition of antibiotics  for the preparation of selective agar plates. Agar plates (20 mL) were poured shortly before plating, wrapped in aluminum foil, and stored at 4 °C and never longer than for one week. All steps that involved the handling of selective agar plates were performed in the absence of artificial light, since both antibiotics are sensitive to light. For preculture, 5 mL broth were inoculated with one colony of the test strain https://lianglab.brocku.ca/FALCOR/, accessed on 18 March 2022) [A two-tailed Mann–Whitney-U-Test was used for the statistical comparison of the median mutation frequencies and median mutation rates. Fisher’s exact test was employed for the comparison of agar plates without growth . Mutation rates were analyzed using the FALCOR Fluctuation AnaLysis CalculatOR (ch 2022) .S. aureus HG001 in 5 mL of Iso-Sensitest broth. A total of 4.9 mL of culture was pelleted, and the cells were resuspended in 500 µL of Iso-Sensitest broth. These suspensions were plated in 150 µL aliquots on agar plates containing the combination of both antibiotics. For the determination of the total cell counts, a series of tenfold dilutions was plated on Iso-Sensitest agar without antibiotics. All agar plates were incubated at 37 °C and evaluated after 24 h.For the determination of FoM against a combination of both agents, rifampicin and CorA, fresh agar plates with 4 × MIC of both antibiotics were prepared two days before plating. Eight different overnight cultures were grown from eight different colonies of S. aureus HG001 (rifampicin-susceptible), eight cultures of S. aureus Mu50 (rifampicin-resistant), and one culture each of S. aureus USA300 and S. aureus N315 ). Resistant colonies were picked and purified by streaking on Iso-Sensitest agar (4 × MIC CorA). For the DNA preparation, strains were grown overnight on 4 × MIC CorA-containing agar, and two to three colonies were resuspended in 60 µL of sterile distilled water and heated for 5 min to 100 °C. Cells were removed by centrifugation and the supernatant served as a template for the amplification of rpoA, rpoB, and rpoC . Sanger sequencing was performed by Microsynth Seqlab  using the primers in Resistant colonies were selected on agar with CorA (4 × MIC) employing 19 independent cultures (nine cultures of