The production of active and biodegradable packaging materials is an emerging and efficient alternative to plastic packaging materials. By combining poly (PVA), pectin, and itaconic acid (IA), biodegradable and water-soluble packaging materials can be obtained that can also increase the shelf-life and quality of foodstuff. In the present study, the generated film-forming solutions were enriched with organic or phenolic extracts from apple by-products (apple pomace). These extracts possess an efficient antioxidant activity of 9.70 ± 0.08, and 78.61 ± 0.24 μM Trolox/100 g fresh weight, respectively. Furthermore, the lyophilization of these by-products increased the extract’s organic and phenolic content and the antioxidant activity to 67.45 ± 0.28 and 166.69 ± 0.47 μM Trolox/100 g fresh weight, respectively. These extracts influence the physical-chemical properties of the biofilm solutions by facilitating the polymerization process and thus positively influencing their viscosity. The resulting biofilms presented low water vapor permeability and reduced solubility in water. Adding IA and organic/phenolic compounds facilitates the resistance against intrinsic and extrinsic factors; therefore, they might be applicable in the food industry. With regards to food packaging, these bio-based and biodegradable polymers have an active role in conserving and shielding the food product from the external environmental stressors. Given the numerous advantages of these polymers, the production of active packaging with antioxidant effects impacts positively the shelf-life of foodstuff, and improve their quality [Plastic and plastic-based packaging rise serious pollution concerns worldwide regardless of their consistent contribution to the global economy . Nowaday quality ,11,12.Aspergillus terreus. Still, several microorganisms are genetically engineered to synthesize this organic acid in significant quantities and on various substrates [Itaconic acid (IA) is an essential chemical for producing polymers that originates from renewable resources. IA has been manufactured industrially since the mid-twentieth century using the filamentous fungus bstrates ,13. It ibstrates . IA’s chbstrates ,13,15. Sbstrates ,17.Poly (PVA) is a water-soluble synthetic polymer with high structural capacity, strong compound characteristics, good biodegradability, and ease of usage. The efficient physical properties of PVA are attributed to its hydroxyl groups, which support the creation of hydrogen bonds . Due to Pectin is a branched heteropolysaccharide composed of long-chain galacturonan segments toghether with other neutral sugars, including galactose, arabinose, and xylose . Pectin Malus domestica Borkh) is one of the world’s most popular fruits due to its great flavor and nutritional content, ranking fourth in fresh fruit consumption globally [Apple  for food packaging applications while determining its morphologic and mechanical properties. Moreover, the extract’s antioxidant and antimicrobial activity and their effect on film-forming solutions, and rheological characteristics have also been determined.Throughout the experiments, the used reagents were of analytical grade, such as ethanol, acetic acid, DPPH , Trolox , and pectin from apple were acquired from Sigma-Aldrich . For biofilm preparation, the used PVA was of high molecular weight (98–99%) hydrolyzed; it was obtained from ThermoFisher . For the antimicrobial and antioxidant assays, the used chemicals, Mueller–Hinton broth and agar, peptone, tryptic soy broth, and resazurin were acquired from BioMerieux .2PO4 was provided by Merck , and water was purified with a Direct-Q UV system by Millipore . The pure standard of malic, ascorbic, citric, succinic, and fumaric acid (purity 99% HPLC) was purchased from Merck, Germany. Acetonitrile, HPLC-gradient, was provided by Merck  and water was purified with a Direct-Q UV system by Millipore . The pure standard of cyanidin, catechin, rutin, gallic and chlorogenic acid (99% HPLC) were purchased from Sigma . Apple pectin was procured from Sigma® . Calcium chloride (CaCl2) and glycerol were bought from VWR Chemicals. CaCl2 anhydrous was procured from Cristal R Chim SRL, Romania.Chemical reagents and materials for HPLC. NaHThe apple variety selected for this study was Red Delicious, bought from a local market . AP was prepared in the University of Agricultural Science and Veterinary Medicine Cluj-Napoca biotechnological laboratory. Phenolic compounds can act as a substrate in enzymatic browning, primarily caused by polyphenol oxidase (PPO). As a result, AP is a very vulnerable substrate to oxidation processes . For theThe apparatus used for the freeze-drying process was a Telstar LyoQuest -55 Plus with a condenser at −55 °C . Apple by-products were added in a ratio of 1:3 into a 1000 mL capacity flask and were immediately frozen at target temperatures (−80 °C) for 24 h. After freezing, the samples were placed in the lyophilization apparatus and kept for nearly 72 h. The parameters for the drying process were set to 0.001 mBar vacuum and −55 °C temperature. The samples were dried until they reached a constant weight (±0.005 g). The water content evaporated by the freeze-drying process is expressed as a percentage (%), considering the mass before and after drying .Sample preparation. A 1 g sample with 5 mL distilled water was vortexed for 1 min, followed by 15 min ultrasonic treatment at room temperature, then centrifuged at 10,000 rpm for 10 min. The supernatant, containing extracted organic acids, was filtered through a nylon filter (pore size 0.45 μm), and 20 μL was injected into the high-performance liquid chromatography (HPLC) system.Chromatographic condition. The HPLC analysis was performed utilizing an HP-1200 liquid chromatograph equipped with a quaternary pump, manual injector and VWD detector . The column was an Acclaim OA  from Thermo Fisher Scientific, Waltham, MA, USA. The mobile phase was NaH2PO4 50mM solution at pH = 2.8, elution was done for 10 min at room temperature with a flow rate of 0.5 mL/min. Chromatograms were recorded at wavelength λ = 210 nm, and data acquisition was made with the Agilent ChemStation software.Quantitative determination. The organic acid content was determined using a five-point calibration curve for each organic acid. The chromatograms of standards, frozen AP, and lyophilized AP are presented in Sample preparation. A 1 g sample with 5 mL methanol was vortexed for 1 min, followed by 15 min ultrasonic treatment at room temperature. The supernatant, containing extracted polyphenols, was filtered through a nylon filter (pore size 0.45 μm) and 20 μL was injected into the HPLC system [C system .The 1 g frozen sample, respectively, 0.25 g of dry lyophilised samples, were mixed with 5 mL ethanol 70%, vortexed for 1 min followed by 15 min ultrasonic treatment at room temperature, then centrifuged at 10,000 rpm for 10 min at 40 °C. The supernatant was collected and kept in a dark place. Over the pellet, the solvent was re-added, and the sonication-centrifugation procedures were repeated 3 times.The extract collected after each centrifugation was concentrated by evaporation under low pressure (150 mbar), at low temperature at 35 °C, and 180 rpm with a rotary evaporator   to a volChromatographic condition. Analysis was carried out using an HP-1200 liquid chromatograph equipped with a quaternary pump, autosampler, DAD detector, and MS-6110 single quadrupole API-electrospray detector . The positive ionization mode was applied to detect the phenolic compounds; a different fragmentor was applied in the range of 50–100 V. The column was a Kinetex XB-C18  from Phenomenex, Torrance, CA, USA.The mobile phase was (A) water acidified by acetic acid 0.1% and (B) acetonitrile acidified by acetic acid 0.1%. The following multistep linear gradient was applied: start with 5% B for 2 min; from 5% to 90% of B in 20 min, hold for 4 min at 90% B, then 6 min to arrive at 5% B. The total time of analysis was 30 min, flow rate 0.5 mL/min, and oven temperature 25 ± 0.5 °Cm/z 120–1500. Chromatograms were recorded at wavelength λ = 280, 340, and 520 nm and data acquisition was done with the Agilent ChemStation software.Mass spectrometric detection of positively charged ions was performed using the Scan mode. The applied experimental conditions were: gas temperature 3500 °C, nitrogen flow 7 L/min, nebulizer pressure 35 psi, capillary voltage 3000 V, fragmentor 100 V, and 2 = 0.9978) in the linearity range of 10–100 μg/mL. The hydroxycinnamic acid content was determined using a five-point calibration curve of chlorogenic acid at 340 nm, (R2 = 0.9937) in the linearity range 10–50 μg/mL. The flavonol content was determined using a five-point calibration curve of rutin at 340 nm (R2 = 0.9981) in the linearity range of 10–100 μg/mL. The anthocyanin content was determined using a five-point calibration curve of cyanidin at 520 nm (R2 = 0.9951) in the linearity range of 10–100 μg/mL. The flavanol content was determined using a five-point calibration curve of catechin at 280 nm (R2 = 0.9994) in the linearity range 10–100 μg/mL.Quantitative determination. The hydroxybenzoic acid and dihydrochalcone content were determined using a five-point calibration curve of gallic acid at 280 nm  free radical scavenging capacity technique developed by Brand-Williams et al. . To detew/w dry mass PVA) dissolved in hot distilled water (90 °C) under constant stirring for two hours. Further, the solvent casting technique was used to prepare the polymer films, as indicated by Szabo et al. in their study [v/wt. IA was added under stirring conditions and at a temperature of 65 °C for 30 min, to obtain the formulation PVA+Gly+IA. This solution was enriched with two different AP extracts (9% phenolic extract (PE) and organic extract (OE)), resulting in two other formulations (PVA+Gly+IA+PE and PVA+Gly+Ia+OE). The extracts were incorporated into the PVA+Gly+IA solution under continuous stirring for 30 min at room temperature (21 °C). Further, all the biofilm solutions were analyzed regarding their antimicrobial activity and rheological measurements. Then, the process continued with the casting of the biofilm solutions into Petri dishes, and they were left for solidification at room temperature (21 °C) for 48 h. The solidified biofilms were peeled off and subjected to several investigations .The biofilm solutions were prepared by developing a base solution (PVA+Gly), the control sample. This solution was made with 3% wt. PVA and Gly (30% ir study . To the w/w) was dissolved under a gentle stirrer in pre-heated distilled water (75 ± 5 °C) for 1 h. The solution temperature was kept to 75 ± 5 °C. Then, Gly (30% w/w dry mass pectin) and CaCl2 (1% w/w dry mass pectin) were added. The whole mixed solution was homogenized to 9500 rpm for 5 min  while pectin (2% w/w) was gradually added. The homogenization was continued for another 5 min to 15,000 rpm using Ultraturax. When necessary, apple extract (PE and OE) was incorporated into the obtained mixed solution at 15,000 rpm for 5 min. Then, the mixed solution was kept at room temperature (25 °C) until the next day for better pectin hydration. The foam was removed with a spoon, and the mixed solution was immersed into the ultrasonic water bath  at 55 ± 5 °C for a minimum of 30 min to remove air bubbles. Films were obtained using the casting method. A total of 15 g of a mixed solution were poured into Petri dishes (9 cm in diameter) and stored at room temperature for a minimum of 4 days before further analysis. Control films were obtained from pectin, glycerol, and CaCl2 following the same procedure.Films were prepared according to  and adapEscherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Streptococcus pyogenes ATCC 12344, and Salmonella enterica NCTC 6017. The strains were grown in a test tube containing 10 mL sterile Mueller–Hinton broth , Tryptone Soya broth  and Brain Heart Infusion broth , respectively, at 37 °C for 24 h. A loopful of grown inoculum was streaked on growth agar medium: Tryptic Soy agar  for E. coli and P. aeruginosa, Mueller–Hinton agar  for S. epidermidis, S. aureus, S. enterica, and Brain Heart Infusion agar for E. faecalis and S. pyogenes. Plates were incubated for 24 h at 37 °C. Bacterial morphology was confirmed by optical microscopy.The following seven standard strains were tested: 8 CFU/mL). Then, a 1.5 × 105 CFU/mL bacterial suspension was prepared to be added to each microplate.Several colonies of each strain cultivated on the media were transferred in 9 mL of sterile NaCl solution (8.5 g/L) and adjusted to match the turbidity of McFarland 0.5 standard (1.5 × 105 CFU/mL) was added to all the wells. The positive control was gentamicin . The biofilm solutions without the extracts were added as a negative control.The MIC was determined using the resazurin microtiter plate-based antibacterial assay ,40,41. OThe microplates were incubated for 20–22 h at 37 °C, and 20 µL of 0.2 mg/mL resazurin aqueous solution was added to all the wells. The microplates were subjected to a subsequent 2 h incubation at 37 °C. After this period, resazurin (a blue non-fluorescent dye) was oxidized to resorufin (fluorescent pink) wherever the wells contained viable bacterial cells. Thus, the concentration in the last well on each raw that remained blue was considered to entirely inhibit bacterial growth, the MIC. The results are presented in The flow behavior of the biofilm solutions was executed with a modular compact rheometer Anton Paar MCR 72 , equipped with a Peltier plate-plate system (P-PTD 200/Air) with temperature control (temperature range 5 °C to 150 °C). Each sample (~3 mL) was positioned between the two plates, the upper plate with a smooth parallel plate geometry, and 50 mm diameter, and the lower one with a preset temperature at 25 °C and at a gap of 1 mm ,43,44. Sn = 10).Thickness (µm) was measured with a digital caliper in mm  at five random points for each film. The measurement was run in duplicate. The final thickness was calculated as the mean value of all measurements (n = 6).Diameter (mm) of each sample was measured with above-mentioned digital caliper at three random points. The measurement was run in duplicate and density values were established as the mean values of all measurements (Mass (g) of solid films were weighed with an analytical scale  with a precision of 0.0001 g. The final weight was the mean value of two measurements.3) of solid films was calculated according to [w is the weight of sample (g), a is the area of sample (cm2), t is the thickness of the sample (cm). The final thickness was calculated as the mean value of two measurements.Density (g/cmrding to . The wei2 anhydrous (0% RH). An air gap of 6 mm was kept between CaCl2 and film. A total of 10 cm2 of film was exposed to water vapor. The cups were then placed into pre-equilibrate desiccator containing a saturated NaCl aqueous solution to 25 ± 1 °C (75% RH) [WVTR was calculated by following equation:S is the slope of the straight line (g/h), A is the cup mouth area (m2), and WVTR is the rate of water vapor transmission (g/h·m2). WVTR was conducted in duplicate.Water vapor transmission rate (WVTR) was determined according to the ASTM E96/E96M-10 standard. Films were sealed onto permeability cups  containing approximately 7.5 g CaCl(75% RH) . The cup0) and Whatman paper no. 1 (W0) having a diameter of 14.5 cm were weighed to the nearest 0.0001 g. The samples were put in beakers containing 50 mL of double-distilled water and stirred at 300 rpm for 30 min at room temperature (25 °C) and at 60 °C. The biofilms solutions were then filtered through the Whatman papers and dried at 50 °C for 24 h. The filter papers were cooled in the desiccator and weighed (W1) again. Samples were measured in duplicates, and the percentage of total soluble matter expressed as solubility (%) was calculated by using the following equation:0—weight of biofilm pieces (in grams), W1—weight of Whatman filter papers before drying (in grams), W1—weight of Whatman filter papers after drying (in grams).The obtained biofilms were also subjected to a water solubility test at different temperatures, a protocol adapted after Maizura et al., 2007  and Sapun = 2). Results are expressed as mean value ± standard deviation (SD). One-way ANOVA with post hoc Tukey test was used to determine if there were significant differences between Control films (PVA+Gly and PEC+Gly) and films with addition of active compounds  regarding their diameter, mass, thickness, density, water solubility, and WVTR, respectively. Tests were applied for each batch of film (PVA and PEC) separately. For diameter and thickness (n = 6 and n = 10), tests were applied after descriptive statistical analysis. Skewness values were considered for normal distribution and variance values for homogeneity.Statistical analysis was performed in Microsoft Excel 2016 and all experiments were run in duplicate , addition of active compounds , and interaction between them influence water solubility to 25 °C and 60 °C, respectively.For WVTR, a t-test assuming unequal variances was used to determine the significant differences between the content of each organic acid and phenolic acid extracted from frozen apples and lyophilized apples. A t-test assuming unequal variances was also used to determine significant differences for antioxidant activity of OE and PE from frozen apples and lyophilized apples, as well as of OE and PE from frozen apples, and OE and PE from lyophilized apples. All tests were performed at the confidence level of 95%. The symbols used were as follows: ** p < 0.01, * p < 0.05, NS p > 0.05.For OE and PE, a The AP was used as an organic substrate for the isolation of the organic acids and phenolic compounds. The Red Delicious apple type was analyzed frozen and in lyophilized form, both for organic acid content and for the individual phenolic composition, as presented in In the case of organic acids profile, malic, citric, succinic, and fumaric acids were identified, and the lyophilization process proved significantly higher efficiency in preserving the content of the total organic acids towards freezing. That is to say, the elevated content of malic, citric, and succinic acids found in the lyophilized fraction might influence the polymerization process and the formation of strong hydrogen bonds in connection with the base matrices, both in PVA and pectin-based films . As show2−, H2O2) in freshly harvested fruits. Thus, their integration in active packaging might alter the modifications in pH or even the action of oxygen-related enzymes and maintain the antioxidant activity in fruits or vegetables during storage [Besides being efficient cross-linkers in biopolymer packaging, organic acids have high antioxidant activity  after the lyophilization process of 17.3% (723.07 ± 0.06 μg/g), and 25.86% (736.21 ± 0.44 μg/g). Based on Ezati and Rhim, they possess powerful antimicrobial and antioxidant agents and can effectively protect against UV by diminishing the transparency of biofilms [Among the flavonols group, the highest concentration of the seven identified phenols was quercetin-glucoside (Isoquercitrin) at 217.84 ± 0.08 μg/g in frozen AP, and epicatechin 736.21 ± 0.44 μg/g in the lyophilized AP powder. Quercetin-glucoside, besides quercetin-rutinoside (Rutin), quercetin-arabinoside (Avicularin), quercetin--glucoside, and quercetin-rhamnoside (Quercitrin), are derivatives of quercetin, and are polyhydroxy compound bioflavonoids. In addition to the quercetin derivatives, other phenols were also identified, such as epicatechin and procyanidin dimmer, both with a significant increase (biofilms .Three hydroxycinnamic acids were identified 5-Caffeoylquinic acid (Chlorogenic acid) with the highest content in the lyophilized form of 1173.13 ± 0.19 μg/g, and with a substantial increase of 20.77% and 20.42% of caffeic acid-glucoside, and p-coumaroylquinic acid, respectively. Although they are easily oxidized and unstable at increased temperature, through lyophilization, there was only an increase in their concentration. These compounds can be found in apples and also in berries, prunes, pear and, especially in coffee, and they also possess high antioxidant activity, and anti-viral, inflammatory and microbial activity . Among oConsequently, several organic and phenolic compounds can be found in the by-products of AP, which possess multiple advantages to health, such as enhanced oxidative stress-related diseases, diabetes, obesity, and others ,62,63.p < 0.01) and the organic extracts of 47.16% (p < 0.01).The modifications in antioxidant activity of the organic and phenolic extracts from AP were evaluated by the DPPH free radical scavenging capacity, and the obtained results are displayed in Regarding the bioactive potential of AP, several similar studies presented increased results after lyophilization ,65 or evThe high antioxidant activity observed in the organic extracts can be attributed to the fact that they have acidic properties and are identified as weak acids. Citric acid  is an acknowledged antioxidant, especially in oils and fats, due to the large number of metallic salts present in its structure comprising iron, magnesium, copper, calcium, and manganese . All fouThe antibacterial activity of AP phenolic and organic extracts is presented in The effect of IA and the integration of phenolic and organic acid extracts in the biofilm solutions have also been evaluated through the film-forming solution’s viscosity measurements. These rheological studies display the deformation of the sample due to the influence of mechanical stresses and allow the examination of the final characteristics that help in the optimization of the manufacturing process.2 were added, as can be observed in These measurements have been analyzed at three different temperatures between 20 and 40°. The viscosity and shear stress of the solutions were measured for a shear rate ranging from 5 to 300 1/s. Both control biofilms based on PVA or pectin presented a shear-thickening (dilatant) behavior when only glycerol or, in the case of pectin glycerol and CaCl2+ ions was selected to achieve an acidic state that promotes gelification [Pectin is a polysaccharide delineated primarily of an α-1,4-D-galacturonic acid residue and constructed from at least 17 distinct monosaccharides . In thisfication . With thfication .PVA also encompasses unique and favorable characteristics that promote its use in biodegradable active packaging. Carbon atoms delineate its structure, and the presence of a hydroxyl group is advantageous for forming firm hydrogen bonds among hydroxyl groups . The intThe highest value for viscosity was observed with the integration of IA in the biopolymer solution, reaching values of 22.6 ± 0.41 mPa·s at 20 °C. In the case of pectin, the integration of IA produced an outset of 4627 ± 33.40 mPa·s, at 20 °C and a final viscosity of 280.8 ± 0.49 mPa·s at 20 °C. Nevertheless, with the temperature growth, the viscosity decreased in both PVA and pectin biofilms and in the case of IA integration. The addition of phenolic and organic extracts diminished biofilm solution viscosities, which was also observed in similar studies ,43.The physical aspect of the solid biofilms with pectin or PVA, based on IA and enriched with phenolic and organic extracts, is presented in Biofilms with PVA were transparent, and the integration of IA did not affect the biofilm’s transparency or color. In the case of PVA, only the phenolic extract gave a slightly yellowish color. Pectin films were orange, but without IA, they were also transparent. The integration of IA gave an opacity to biofilms. In addition, the integration of the phenolic extract gave a more intense color. Although transparent films are more appreciated, the positive effect of opaque packaging on light’s degradative effect is constantly increasing .Eight types of biofilms were attained in the current study, represented as PVA/pectin (PEC)+Gly, enriched with IA and afterwards also enriched with organic (OE) or phenolic (PE) extract, as presented in p < 0.05) from 92 ± 23 µm to 74 ± 10 µm with extract integration, while in case of pectin biofilms, the thickness increased (48 ± 17 µm) significantly (p < 0.01), especially with organic extract enrichment (75 ± 24 µm). The mass of biofilms increased with the incorporation of IA and extracts. At the same time, the density was the highest in PEC+Gly+IA+PE biofilm with 1.63 ± 0.14 g·cm−3, and the lowest in PEC+Gly+IA+OE with a final density of 1.05 ± 0.21 g·cm−3.The thickness of PVA biofilms decreased significantly  and pectin+Gly (20.60 ± 1.27 g/h·m2), respectively (p < 0.05) and pectin (p < 0.01) films, WVTR significantly decreased to 12 ± 3.82 g/h·m2 and 11.25 ± 1.20 g/h·m2. IA contains two carboxylic (-COOH) groups [2O) and the formation of one less hydrophilic carbonyl group (-C=O) for each –COOH. Therefore, the cross-linking of IA to both PVA and pectin matrices leads to more compact films (due to eliminated H2O molecules) and less hydrophilic films (due to the formed –C=O groups), which may explain the lower WVTR values. Furthermore, when OE and PE were added, WVTR significantly decreased in PVA and pectin films (p < 0.01). OE reduced WVTR by 94% (1.30 ± 0.14 g/h·m2) for PVA films and by 63% (7.55 ± 0.07 g/h·m2) for pectin films. OE was the most effective active compound for reducing WVTR of PVA and pectin films in this study.PVA and pectin contain hydroxylic groups (−OH) that are strongly hydrophilic. Due to their high hydrophilicity, the moisture barrier of PVA and pectin films is very low. Therefore, in this study, WVTR had the highest values for PVA+Gly (22.30 ± 1.70 g/h·mectively . When IA) groups . The COOp < 0.05) between WVTR values obtained for addition of IA with or without PE in both PVA and pectin films. In conclusion, addition of IA with or without apple extracts can significantly reduce WVTR in both PVA and pectin matrices. Incorporation of OE had the highest effect to reduce WVTR of PVA (by 94%) and pectin (by 63%) films.Organic acids also contain -COOH that acts as strong cross-linkers and reduce the availability of free -OH groups in the PVA and PEC structures. For example, WVTR was reduced when citric acid was added to chitosan films . The sam2). LDPE films were recently developed by melt extrusion and hot pressing [Furthermore, WVTR of PVA and PEC films incorporating apple extracts was lower than WVTR of pure low-density polyethylene (LDPE) films  affected by the film matrix base (PVA and pectin) and by the addition of active compounds at both 25 °C and 60 °C. Moreover, the water solubility is significantly affected by the interaction between the film matrix and active compounds  in the case of dissolution at 60 °C (p < 0.05).The solubility test represents the physical-chemical exam for samples to resist water activity. It also brings valuable information about the polymeric structures or the coating matrices in close connection with their possible applications . For thip < 0.05) diminished the water solubility of the PVA biofilms, both at 25 °C and 60 °C. This aspect can be attributed to forming strong hydrogen bonds between PVA hydroxyl units and the analogous groups [p > 0.05) influence on water solubility at 60 °C by comparison with PVA+Gly was observed. By comparing our results for the solubility percentage of PVA-based samples, we found that these are similar to those reported by Oyeoka et al. [The increase in temperature positively influenced the samples’ dissolution and the filtration time in the case of PVA films. In contrast, the solubility percentage for pectin-based films was higher at room temperature, as seen in s groups . Moreovea et al. . They aca et al. . In anota et al. . As the a et al. , who obsa et al. .p < 0.05) influences the water solubility at 60 °C in both PVA and pectin-based films in comparison to the addition of IA without extracts. At 60 °C, even though the filtration rate was improved towards that achieved at 25 °C, the total soluble matter was lower at elevated temperature, as it was obtained: 85.58 ± 1.05% (pectin+Gly), 62.14 ± 2.71% (pectin+Gly+IA), 33.54 ± 3.49% (pectin+Gly+IA+OE), 36.84 ± 6.61% (pectin+Gly+IA+PE). Surprisingly, the addition of organic and phenolic extracts to pectin+Gly+IA biofilms gave firmness and resistance against water activity to the initial polymeric matrix. This effect can be attributed to the presence of other organic acids besides IA, such as fumaric, succinic, malic, and citric acids, and phenols such as flavonols, hydroxybenzoic and hydroxycinnamic acids that have the capacity to strengthen the cross-linking among the bioactive molecules and to generate more hydrogen bonds between polysaccharides by giving improved mechanical properties to the entire structure. The results reported for trials based on pectin are comparable with those shown in the scientific literature. For example, Guadarrama-Lezama et al. [2) at low pH (around 4). At a pH value of 4, the water solubility percentage decreases to 83  ±  5.0% with the increase of density to 5.8 mg/cm2. When the pH values reach 8, the solubility percentage is around 78% (at a density of 2.5 mg/cm2) and decreases to 53  ±  5.0% with the increase of density (5.8 mg/cm2) [In the case of pectin-based biofilms, these were slightly more rigid compared with the PVA-based ones and with facile dissolution in water. It was observed that the highest water solubility percentage (97.07 ± 0.33%) was recorded at 25 °C for pectin-based samples plasticized with glycerol, but with no addition of other compounds. The addition of IA, organic extract, and phenols brought higher resistance and lower water solubility to the biofilm samples, as it was obtained a water solubility percentage of 72.98 ± 7.91% (pectin+Gly+IA), 61.37 ± 4.33% (pectin+Gly+IA+OE), 62.43 ± 0.68% (pectin+Gly+IA+PE). That is to say, the addition of organic and phenolic extracts significantly . Knowledge acquired from this experiment will be an essential precursor for the practical application of AP that can be suitable for bio-composite packaging materials.


According to the THQ, arsenic contents in infant formulas showed a THQ > 1, indicating potential health risk concerns for newborns or children. Minimal carcinogenic risks were observed for the elements considered carcinogenic. Metabolic and nutritional interactions are also discussed. This study indicates the need to improve infant formula surveillance concerning contamination by potentially toxic and carcinogenic elements.Children are highly vulnerable to chemical exposure. Thus, metal and metalloid in infant formulas are a concern, although studies in this regard are still relatively scarce. Thus, the presence of aluminum, arsenic, cadmium, tin, mercury, lead, and uranium was investigated in infant formulas marketed in Brazil by inductively coupled plasma mass spectrometry, and the Target Hazard Quotients (THQ) and Target Cancer Risk (TCR) were calculated in to assess the potential risk of toxicity for children who consume these products continuously. Aluminum ranging from 0.432 ± 0.049 to 1.241 ± 0.113 mg·kg Adequate and safe nutrition is essential to ensure optimal health, growth, and development during early childhood. Breast milk combines ideal nutritional characteristics with optimal nutrient balance available at all times, at the ideal temperature, and free of microbial contaminants, also favoring numerous immunological and psychological advantages, paramount in reducing infant morbidity and mortality ,2. NumerWhen breastfeeding is impossible or not recommended, infant formulas comprise the most suitable source of nutrients for children under 1 year old, replacing or partially complementing breastfeeding . Infant Environmental contaminants present in infant formulas are a concern, as both the raw materials and/or their manipulation can be subject to inadequate agriculture practices and/or processing methods. In this regard, milk and its derivatives may be contaminated during agricultural management by harmful chemical compounds, such as toxic metals and metalloids, which may be taken up by both plants and animals. Furthermore, industrial processing product packaging also comprise toxic metals and metalloids sources in infant formulas .status, as well as synergistic or antagonistic effects due to the presence of other chemical compounds [Metal toxicity in humans depends on several factors, such as their physicochemical properties, ingested frequency and amounts, and individual health ompounds . These compounds . In thisompounds .The high degree of toxicity of some elements, such as mercury (Hg), cadmium (Cd), arsenic (As), lead (Pb), uranium (U), tin (Sn), and aluminum (Al), makes them of great importance for public health, mainly due to its ability to bioaccumulate in the body ,12. Due In this context, the purpose of the present work was to determine the level of toxic elements in infant formula brands recommended for children from 0 to 6 months of age  and for children aged 6 to 12 months  marketed in Brazil and identify the potential health risk from detected levels to both age classes who consume these products continuously.Three batches of each infant formula widely marketed in Brazil were purchased from different commercial establishments located in the metropolitan region of the city of Rio de Janeiro. All these infant formulas are marketed in a great number of commercial establishments, located at the most populated region of Rio de Janeiro municipality.All products are registered at the Brazilian regulatory agency (ANVISA) and were packaged in labeled cans in powdered form. Regarding composition, the following inclusion criteria were used for infant formula selection: all formulas should (1) be composed essentially of cow milk; (2) contain unhydrolyzed cow milk proteins; (3) contain lactose as the main carbohydrate; and (4) be supplemented with essential long chain fatty acids as docosahexaenoic acid (DHA) and arachidonic acid (ARA) for children aged from 0 to 6 months (phase 1 starting formulas) and from 6 to 12 months (phase 2 follow-up formulas).Infant formulas containing protein sources other than cow milk, such as soy or wheat proteins, or developed for specific needs, such as lactose-free or hydrolyzed, were not included in this study. Considering products sold in the Brazilian market that meet the inclusion criteria, ten brand formulas from three different manufacturers were selected. Therefore, five phase 1 and five phase 2 formulas comprising three distinct batches of each brand were included, totaling thirty samples (N = 30). Due to ethical reasons, manufacturers and brands are not disclosed, and samples were coded for the analyses. Brands are represented by two capital letters, followed by 1 or 2 representing phase 1 or phase 2 formulas and the three batches of each brand were identified using capital letters  .® INTEGRAL 10 system . Nitric acid  was purified by sub-boiling followed by distillation in a quartz still . A multi-element standard solution Merck IV , comprising 29 elements diluted in nitric acid, was employed to construct the calibration curves.All reagents were of analytical grade. Ultra-pure water (resistivity > 18.2 MΩ cm) was obtained from a Milli-Q® BD150 and Non-Fat Milk Powder 1549) were also analyzed, in triplicate, to ensure method accuracy.Metals and metalloids were determined according to the US EPA method 6020B  by induc−1. The limit of detection (LOD) and limit of quantification (LOQ) were determined according to the Brazilian National Institute of Metrology, Quality, and Technology—INMETRO [The total metal and metalloid contents in the infant formula samples were expressed as mg·kg—INMETRO , where Lelement is the concentration of each toxic metal or metalloid in the infant formula (mg·kg−1), RfDo comprises an estimate of the safe daily oral exposure, expressed as units of mg·kg−1∙day−1 .The target hazard quotient (THQ), which measures the toxicological risk of certain contaminants, non-carcinogenic ones, over a specific population , was calngestion , Celemen−1∙day−1 , WAB is If the THQ value for non-carcinogenic metals or metalloids is lower than 1, an unlikely health risk is suggested, while values over 1 indicate a likely health risk, although non-carcinogenic, with an increasing probability with increasing target hazard quotients.O) stablished by each element to estimate the probability of an individual developing cancer following oral ingestion of contaminant elements considering the body mass, determined with the equation below:element is the metal concentration in the infant formula (mg·kg−1), CSFO is the oral cancer slope factor for Al (0.021 mg·kg−1), iAs (1.5 mg·kg−1), and Pb (0.0085 mg·kg−1) [−3 is the unit conversion factor. Oral cancer slope factors were established only for elements that are considered carcinogenic.The target cancer risk (TCR), which is used to assess the potential risk associated with exposure to carcinogens during a lifetime exposure period, was calculated for Al, inorganic As (iAs), and Pb, as these elements present potential carcinogenic effects ,20. Instngestion , Celemenrequency 2.5 days,or Al 0.0 mg·kg−1,F was found. The results were considered statistically significant when p < 0.05 and were expressed as the means ± standard deviations (SD). All statistical analyses were carried out using the GraphPad Prism® .Significant differences in element contents among the infant formula batches were assessed through a two-way analysis of variance (ANOVA) with repeated measures. A post hoc analysis (Bonferroni correction) was performed when a significant ® BD150 and Non-Fat Milk Powder 1549 CRMs are displayed in The observed and certified values for the reference materials Skimmed Milk Powder ERM−1, As from 0.012 ± 0.009 to 0.034 ± 0.006 mg·kg−1, Sn from 0.007 ± 0.003 to 0.095 ± 0.024 mg·kg−1, and U from 0.002 ± 0.001 to 0.016 ± 0.007 mg·kg−1, displaying significant heterogeneity between the investigated formulations (p < 0.01)  .Metals and metalloids are present in all environmental compartments and, thus, present in virtually all foodstuff . Metal u−1; DM1, 0.016 ± 0.021 mg·kg−1, ME2, 0.023 ± 0.033 mg·kg−1, and NC2, 0.011 ± 0.001 mg·kg−1). The toxic elements reported herein are similar to those reported in twenty-six infant formulas marketed in Nigeria, where Al ranged from 0.41 to 2.47 mg·kg−1 and As ranged from 0.02 to 1.56 mg·kg−1. Hg, Sn, U, and Pb contents, however, were not assessed [−1, Cd from 0.01 to 0.002 mg·kg−1, and Pb from 0.05 to 0.09 mg·kg−1 [−1 and Pb, ranging from 0.82 to 1.50 µg·L−1, with Sn, Hg, and Al not assessed [The lowest concentrations were observed for Cd and U, whereas Hg was not detected, and Pb was detected in only three sample . Furthermore, in the majority of phase 1 and phase 2 infant formulas, the Hg was not detected, and Pb was detected in only three sample, NC1C, DM1B, and NC2A , from agricultural practices, irrigation with As-contaminated water, improper application of arsenical fertilizers, insecticides, or herbicides, mining activities, and petroleum refineries ,38.As in organic forms and bound to macromolecules are known to be less toxic than iAs species. There is no evidence that As is essential to human cellular processes, but on the other hand, exposure to As has been linked to severe health conditions such as hyperkeratosis, gangrene, hypertension, peripheral vascular disease, melanosis, and keratosis ,38. As h−1 and 4.5 mg·kg·day−1 for adults and children, respectively, approaching a 95% confidence level. Furthermore, the CSF estimated for Al and Pb were 0.021 mg·kg·day−1 and 0.0085 mg·kg·day−1, respectively [−4 to 4.0 × 10−4), As ranges from 0.0002 to 0.0009 (2.0 × 10−4 to 9.0 × 10−4), and for Pb ranges from 0.000001 to 0.000006 (1.0 × 10−6 to 6.0 × 10−6)  is determined by multiplying the exposure to iAs, Al, and Pb by the appropriate cancer slope factor (CSF) . For As,ectively ,21. The  × 10−6) .−5 mg·kg·day−1 and by U.S. EPA, ranging from 10−4 to 10−6 mg·kg·day−1 [Younger populations may be particularly at risk for long-term health effects of chronic exposure to iAs from milk or rice-derived based products for infants and young children because of the massive exposure during earlier years of life and while they are still developing different systems and the presumed long-term exposure. As exposure seems to provoke internal cancers in organs such as skin, lung, liver, kidney, and bladder. Therefore, iAs has been classified by the International Agency for Research on Cancer as a non-threshold since even small doses may be associated with cancer risk, being considered a class 1 human carcinogen ,38. Howekg·day−1 .Studies have shown that breastfed babies have less exposure to As than formula-fed babies and receive better nutrition. Attention should be taken if infant formulas offered to infants have rice-derived based products in their formulation, which are commonly contaminated by abundant species of As, including the most toxic to humans . AdditioThe Brazilian phase 1 and 2 infant formulas contained undetectable Hg values, but low contents of Cd, Pb, and U. However, Al, As, and Sn were found in high contents. The THQ values estimated for Hg, Cd, Pb, Al, Sn, and U were inferior to 1 (THQ < 1), indicating that Brazilian infant formulas do not represent a health risk for early childhood for the aforementioned toxic metals. On the other hand, the THQ calculated for iAs was over 1 (THQ > 1), indicating potential risks regarding As ingestion in most phase 1 and phase 2 infant formulas. However, negligible TCR for infants were noted regarding As, Al, and Pb, as these toxic metals/metalloids were within the safe limits established by international regulatory organizations.Based on the data reported herein, caution is recommended during the inspection of the investigated formulas by public health authorities regarding contamination by toxic metals and metalloids, especially the excess As consistently identified in most infant formulas. Further attention given to the inspection of infant formulas should guarantee the reliability of these products in relation to international public health organizations recommendations on toxic metals and metalloids, beyond nutritional quality, the protein source used to supply the needs of early childhood, and labeling information.The present study encourages a complete risk assessment of the entire production chain in order to guide regulatory and sector risk managers on maximum ingestion limits for newborns and infants that should be established for metal or metalloids concerning infant formulas marketed in Brazil.


Lactobacillus casei TH14 improves silage quality, which might be utilized as an alternative feed source for ruminants.Ruminant feed is one potential application for cassava pulp (CSP), an industrial waste product generated during the manufacturing of cassava flour. However, because of its high fiber content and low protein content, the quality should be enhanced. We observed that fermenting CSP with urea, molasses, and Lactobacillus casei TH14, urea, and molasses on its chemical composition, the fermentation end-product of silage, and aerobic stability. A 2 × 2 × 2 factorial arrangement with a randomized complete block design was employed. The first factor: level of L. casei TH14 , the second factor: level of molasses , the third factor: level of urea , and the number of days of fermentation  were evaluated using a statistical block. There were interactions among CSP fermented with different additives on DM content (p < 0.05). The control group (CON) and CSP fermented with L, L×M, and L×U had lower DM contents than U, U×M, and L×U×M. The crude protein of CSP was increased by interaction of L×U and U×M additives . Interaction effects between L and U and NDF content were detected (p < 0.05). The L×U combination resulted in a significantly lower NDF than the other groups. The interaction between L×U×M had no effect on the change in the CSP fermentation process (p > 0.05). The combination of U×M caused a poorer pH than other groups (p < 0.01). The ammonia-N content was higher than others, when CSP was fermented with L×U (p < 0.01) or U×M (p < 0.05), respectively. The lactic acid levels in fermented CSP were higher (p < 0.01) than in other groups through the L. casei. The interaction between L×U×M had an influence on lactic acid bacteria (LAB) (p < 0.01) and aerobic bacteria (p < 0.01). The highest LAB population (p < 0.01) at 106 cfu/g FM was found in CSP fermented with L. casei and molasses. In conclusion, the current study shows that CSP treated with L×U×M resulted in good preservation by recovering DM, a low number of aerobic bacteria, and greater LAB than other treatments, with the exception of the L×U×M addition. A 21-day fermentation period is advised because it produces products with greater levels of crude protein, lactic acid, acetic acid, and propionic acid.This study evaluated the effects of cassava pulp fermented with  Manihot esculenta Crantz), which ranks third in economic significance in Thailand behind rice and rubber, is a major crop. Global production of cassava was 277 million tons in 2018, and 302 million tons are expected to be produced in the crop year of 2021/22, an increase of 9.0% [Cassava ( of 9.0% . The was of 9.0%  and 7.3  of 9.0%  million  of 9.0%  illustraSaccharomyces cerevisiae [Lactobacillus casei TH14 (LcTH14), molasses, and cellulase [S. cerevisiae affects animal diets and how it improves the quality of byproducts, such as CSP, and they recommend feeding animals [S. cerevisiae has a high protein content and is rich in the essential amino acids animals require. Phesatcha et al. [S. cerevisiae to fattening cattle enhanced nutrient digestibility and growth performance. Additionally, the capability of yeast cells to scavenge oxygen may be one of the primary factors that justify the beneficial effect that live yeasts have on the quality of silage as well as fiber-degrading bacteria in the rumen [Lactobacillus casei strain TH14, Lactobacillus plantarum for three strains, and Lactobacillus rhamnosus. They discovered that strain TH14 has a high lactic-acid-production capacity and can produce more lactic acid, which verified its usefulness as a possible silage inoculant and showed that this strain was more successful in enhancing silage quality than inoculants or other strains.Many studies have been interested in improving the quality of CSP in animal diets through physical, chemical, and physicochemical means . CSP is revisiae , replacirevisiae , Lactoba animals ,14,15. The rumen ,13. In ahe rumen  found thhe rumen  comparedL. casei TH14, urea, and molasses. Cherdthong et al. [L. casei TH14 (105 cfu/kg FM of rice straw) provides better results, by preventing CP loss during fermentation while also increasing beef cattle digestibility, rumen bacterial population, and propionic acid. Similarly, sugarcane bagasse fermented with L. casei at 105 cfu/kg and 50 g/kg molasses can increase bagasse quality while also improving feed utilization, rumen ecology, and milk production in Holstein Friesian cows [L. casei TH14 and cellulase increased CP and decreased neutral detergent fiber in sweet corn stover silage, according to Kaewpila et al. [L. casei TH14 (LcTH14), urea, and molasses on chemical composition, the fermentation end-product of silage, microbial population, and aerobic stability. This is due to the fact that adding urea and molasses as cheap and accessible silage additives uses them as sources of NPN and carbohydrates for silage additives, respectively. Lactic acid bacteria inoculation aids in successful fermentation processes. Furthermore, urea was typically added to low nitrogen feedstuffs, and the current cassava pulp contained a low quantity of nitrogen. A source of nitrogen can also be used in the silage as a substrate for the growth of microorganisms . A previg et al.  discoverian cows . The add5 colony forming unit; cfu/kg FM) [(1)No LcTH14 + Urea 0% DM + Molasses 0% DM; CON(2)No LcTH14 + Urea 0% DM + Molasses 4% DM; M(3)No LcTH14 + Urea 4% DM + Molasses 0% DM; U(4)No LcTH14 + Urea 4% DM + Molasses 4% DM; UM(5)5 cfu/g FM + Urea 0% DM + Molasses 0% DM; LLcTH14 1 × 10(6)5 cfu/g FM + Urea 0% DM + Molasses 4% DM; LMLcTH14 1 × 10(7)5 cfu/g FM + Urea 4% DM + Molasses 0% DM; LULcTH14 1 × 10(8)5 cfu/g FM + Urea 4% DM + Molasses 4% DM; LUMLcTH14 1 × 10In this experiment, a randomized complete block design with a 2 × 2 × 2 factorial arrangement was used, wherein the first factor: the level of LcTH14 (0 and 1 × 10u/kg FM) , the secu/kg FM) , the thiu/kg FM) , and blo11 cfu/g LcTH14  [The CSP (wet form) material was obtained in Khon Kaen, northern Thailand. The CSP had fermented while being kept and covered by a tarp for a few days for the experiment. The recommended LcTH14 was purchased, containing 10hailand) . On the hailand)  were botThe samples were dried in an oven at 60 °C for each replication of each treatment, then mashed into smaller particles using a 1-mm colander screen to measure the chemical compositions, including dry matter (DM-viz. 34.01), organic matter (OM-viz. 942.05), crude protein (CP-viz. 976.05), and ether extract (EE-viz. 920.39), following the standard method of the Association of Official Analytical Chemists . The neu3-N) concentration was evaluated by a spectrophotometer  based on the method of Fawcett and Scott [At the end of fermentation, a blend of 10 g of sample and 90 mL of sterilized distilled water was placed in an incubator for 12 h at 4 °C . The pH nd Scott .The silage samples for microbial analysis were obtained in a vacuum bag and stored at room temperature (28–30 degrees Celsius). They were collected at four different points of the silo; point 1: top left side; point 2: top right sides; point 3: intermediate regions between the center and left side at the bottom of the silo; point 4: intermediate regions between the center and right side at the bottom of the silo; and these subsamples were manually collected and homogenized, forming one composite sample per silo, following the methods of Carvslho et al. . The samOn each of the fermentation days for all silages, the aerobic stability was assessed. A total of 350 g of each treatment’s three duplicates were kept loosely packed in silos and allowed to degrade aerobically at the ambient temperature (27–34 °C). Each container was covered with two layers of cheesecloth to prevent drying and contamination while allowing air to flow through . AerobicijklY = observation values; µ = overall mean; i = effect of block at I when i = 1, …, r; jα = effect of main effect A at j when j = 1, …, a; kβ = effect of main effect B at k when k = 1, …, b; lγ = effect of main effect C at l when l = 1, …, c; jkαβ = interaction of A and B at jk; jlαγ = interaction of A and C at jl; klβγ = interaction of B and C at kl; jklαβγ = interaction of A, B, and C at jkl; ijklε = error term. The block was three days of fermentation. The chemical composition, pH values, and fermentation characteristics of CSP after fermentation were measured in a 2 × 2 × 2 factorial in a randomized complete block design by the effect of two levels of LcTH14, two levels of urea, and two levels of molasses, and the block was three days of fermentation. We used SAS version 9.00  with the following model:ijkY = observation values; µ = overall mean; iα = effect of main effect A at i when i = 1, …, a; jβ = effect of main effect B at j when j = 1, …, b; kγ = effect of main effect C at k when k = 1, …, c; ijαβ = interaction of A and B at ij; ikαγ = interaction of A and C at ik; jkβγ = interaction of B and C at jk; ijkαβγ = interaction of A, B, and C at ijk; ijkε = error term. The aerobic stability and microbial population after 21 days of fermentation were analyzed with SAS version 9.00  with the following model:p < 0.05, which is accepted as a statistically significant difference.Each treatment had three replications. Using Duncan’s new multiple range tests , the meaThe microbial populations and chemical composition of CSP before ensiling are shown in p > 0.05). However, there were interactions among CSPs fermented with different additives on DM content (p < 0.05). The control group and CSP fermented with L, L×M, and L × U had lower DM contents than U, U × M, and L × U × M. CP was higher by the interaction of L × U and U × M . As a result of a single factor from U supplementation, CS’s EE decreased (p < 0.05). Interaction effects between L and U and NDF content were detected (p < 0.05). The L × U combination resulted in a significantly lower NDF than the other groups. The influence of the day of fermentation (block) on the EE and ADF contents in CSP had no effect (p > 0.05). However, the largest CP (p < 0.01) was obtained after 21 days of CSP fermentation (p < 0.05) and (p < 0.01), respectively)  C,D. Furtctively) A. p > 0.05). The combination of U × M caused a pH that was lower than in other groups (p < 0.01). The PA and ammonia-N contents were higher than others when CSP was fermented with L × U  or U × M , respectively. LA concentrations in fermented cassava pulp were higher with LcTH14 addition than without (p < 0.01). The day of fermentation of CSP had no effect on the ammonia-N level (p > 0.05). The amount of LA produced was higher when CSP fermentation had been carried out for 7 days (p < 0.01)  (p < 0.01).The characteristics of the CSP fermentation product are shown in  < 0.01) B, whereactively) A,C–E. Th2 cfu/g FM) during CSP fermentation in any additive. The interaction between L × U×M had no effect on yeast populations in fermented CSP (p > 0.05), but it has an influence on LAB (p < 0.01) and AB (p < 0.01). The highest LAB population (p < 0.01) at 9.1 × 106 cfu/g FM was found in CSP treated with LcTH14 and molasses together, while CSP treated with additives reduced the AB population (p < 0.05) compared with the CON group.The effects of LcTH14, urea, and molasses on microbial populations and aerobic stability after 21 days of fermentation are shown in In the present study, prior to improvement, the CSP contained LAB, AB, and yeasts. These findings demonstrated that LAB numbers were lower than AB and yeast, indicating that it was necessary to prevent the growth of undesirable bacteria. Additionally, a high population of yeast and AB was found in unfermented cassava pulp , which m2) was also added through processing as a bleaching and antibacterial agent, which further contributed to the low pH of CSP. Therefore, the low pH of CSP is beneficial because it assists in the long-term preservation of the pulp [In agreement with the findings of this study, Keaokliang et al.  have fouthe pulp ,37.Lactobacillus casei TH14 combined with cellulase, and molasses did not reduce DM loss. In addition, Cherdthong et al. [Lactobacillus casei TH14, molasses, and cellulase enzymes.As shown in a previous study, Yunus et al.  found thg et al.  found noThe fermentation days were lengthened in this study, which may result in lower DM and OM levels. Longer fermentation periods result in reduced OM in silage, which is consistent with Bial et al. ’s resear−). In a hydrolysis reaction, the hydroxyl ion produced by water dissociation combines with fats to produce free fatty acids, which bond to the ammonium ion to form soluble salts [The CP content of the treated CSP was increased due to urea supplementation. Urea is a low-cost protein per unit, with about 46% nitrogen, which has the main objective of increasing the CP content of the feed . The ammle salts  reportedle salts . As a rele salts . In the le salts , and appNeumann et al.  verifiedLactobacillus plantarum, Pedi coccus, and Lactococcus species. They support ensure fermentation, which produces LA and rapidly lowers the pH to 4, inhibiting the degradation of the crop’s protein and sugar [3 released from urea buffered the silage [LcTH14 was the homofermentative type of LAB; it has a lactic capacity, and could produce more LA, which is more effective in improving silage quality . Homofernd sugar ,56. Durind sugar . Yunus end sugar  found the silage . Accordie silage , this ise silage . Moreovee silage . This mae silage , TMR silPropionibacterium spp., which may ferment lactate to propionate, have been identified sometimes in low pH silages, according to Woolford [Propionibacterium that convert glucose and LA to AA and PA have been found in silages, but it is doubtful that natural populations can flourish in most silages [Propionibacterium spp. in habitats with high LA concentrations  [Woolford , who als silages . Lactatesilages) . Studiessilages) . 3-N/kg total nitrogen [Ammonia-N production is affected by the CP breakdown of silage, where the fraction of proteolysis is shown by the generation of ammonia-N, and well-preserved silages should contain no more than 100 g NHnitrogen . In the nitrogen  reportednitrogen  discovernitrogen . Garcia nitrogen  reportednitrogen . Howevernitrogen .L. casie TH14, cellulase, and molasses in combination has resulted in the promotion of the best qualities of rice straw and sugarcane bagasse, respectively [Ensilage, also known as ensiling, is a method of conserving forage for use as animal feed under anaerobic conditions. The purpose is to prevent oxygen contact with forage for a period of time due to yeast and mold that will develop as a result of aerobic microbial activity, causing the material to degrade into an unusable, unpalatable, and always poisonous product . Silage ectively ,69. In aectively . L. plantarum with sorbate resulted in an overall increase in mean aerobic stability of 77 h for untreated silage and 153 h for treated silage [Lactobacillus casei TH14, urea, and molasses could enhance silage quality.While silage is exposed to the air, the ambient temperature and the silage temperature are observed. Aerobic stability is the period of time until the silage effectively exhibits signs of heating, or when the silage’s condition is 2 °C or higher above ambient. As a result, the microorganisms oxidize acids and water-soluble carbohydrates to produce water and carbon dioxide, which increases the temperature of the silage above the ambient air . Pahlow d silage ,72. Mored silage . In thisLactobacillus casei TH14, urea, and molasses. The combination of treatments causes CSP to regain DM, have low levels of aerobic bacteria, and have greater amounts of LAB. We recommended fermenting CSPs for more than 21 days in this experiment since it produced higher CP and LA content than other procedures. However, further research on in vitro methods for ruminant feed should be assessed.According to the current findings, CSP is satisfactorily preserved when treated with a combination of


Nonylphenol (NP) is considered to be an environmentally toxic, endocrine-disrupting chemical that affects humans and ecosystems. Adsorption is one of the most promising approaches for the removal of nonylphenol contamination from water. Herein, in order to design an adsorbent with high adsorption capacity, magnesium silicate with different Mg/Si ratios was successfully synthesized by a sol–gel method at 60 °C. Magnesium silicate with a Mg/Si ratio of 1:6 was found to possess the best adsorption performance, with maximum 4−NP sorption 30.84 mg/g under 25 °C and 0.2 g/L adsorbent dose. The adsorption was negatively affected by increasing adsorbent dose and temperature. The kinetics and isotherm of 4−NP adsorption by Mg/Si were well described by the pseudo−second−order and Sips model, respectively, and behavior was proven to be physisorption−enhanced by a chemical effect. Detailed characterization by XRD, BET, and SEM confirmed that the magnesium silicate possesses an amorphous, mesoporous structure. The study will contribute to the applicability of cheap magnesium silicate for removal of NP contamination in water. Nonylphenol (NP), a toxic xenobiotic compound, is mostly used to produce nonylphenol ethoxylate surfactants, as well as in the manufacturing of antioxidants, pesticide emulsifiers, and resin stabilizers ,2. In th2+, Zn2+, and Cu2+, with adsorption capacities of 436.68, 78.86, and 52.30 mg/g, respectively [2+ of 527.22 mg/g and 210.64 mg/g, respectively [Hence, the removal of NP from water has attracted widespread attention and various ways have been adopted to relatively eliminate NP from aquatic systems in recent decades ,13,14,15ectively . Interesectively ,33. The ectively ,35. At Mectively . Magnesiectively . A largeectively ,29. By tectively . As bothThe structure−performance relationship of magnesium silicate has not been clearly elucidated, and its adsorption performance for NP is currently unknown. To address these issues, herein, magnesium silicate was synthesized by the hydrothermal method and the sol−gel method, and the adsorption capacity of 4−NP was compared to find the most efficient magnesium silicate ratio for removal of 4−NP from water.2, 99%), guaranteed reagent (GR) grade sodium silicate nonahydrate (Na2SiO3·9H2O), analytical reagent (AR) grade hydrochloric acid (HCl), and sodium hydroxide  were purchased from Sinopharm Chemical Reagent Co., Ltd. . Additionally, 4−NP (99%) and AR grade nitric acid (HNO3) were purchased from Macklin Biochemical Technology Co. . Chromatographic−grade methanol was purchased from Merck Company . Ultrapure water was obtained from a Milli−Q reagent water system . The 4−NP stock solutions (5000 mg/g) were prepared by LC−MS grade methanol and then diluting with deionized water. The working standard solution of 4−NP was stored at −20 °C in a brown glass bottle. A small amount of methanol (<1%) was used to avoid cosolvent effects.Magnesium chloride . The concentrations of 4−NP were detected by the Waters Acquity UPLC system  and Xevo triple−quadrupole (Xevo−TQD) AB SCIEX 5500 mass spectrometer . The adsorption was carried out by a shaking incubator . The pH value was adjusted using a pH/Ion Seven Compact .2 to a 0.5 mol/L aqueous solution of Na2SiO3·9H2O (the volume was controlled according to the Mg/Si ratio of 1:1), stirring continuously in a constant-temperature water bath adjusted to 25 ± 1 and 60 ± 1 °C, respectively. Then, a white suspension was formed by vigorous stirring of both solutions. The pH value of the resulting mixture was then regulated to 9. The previous mixture was stirred successively for 2 h, then condensed and aged overnight at room temperature. The resulting products were then rinsed with deionized water to remove impurities. The dry gels were obtained by placing them in a heating oven for 12 h at 60 °C. The obtained solids were ground and stored at room temperature. Two magnesium silicate samples were individually prepared at different temperatures: sol−gel 25 °C−1Mg:1Si; sol−gel 60 °C−1Mg:1Si. Subsequently, the effect of the Mg/Si ratio was studied following the optimized synthetic way. Additionally, the five samples were prepared on the optimal synthesis route: Mg/Si 3:1, Mg/Si 1:1, Mg/Si 1:3, Mg/Si 1:6, and Mg/Si 1:7.Magnesium silicate was prepared by the dropwise addition of 0.5 mol/L aqueous solution of MgClAnother group of magnesium silicate samples was prepared using a hydrothermal method. The same procedure as described above was followed until the hydrothermal treatment step. The value of pH is regulated to 9. The above mixture was transferred to a Teflon−lined autoclave (200 mL), and then maintained in a heating oven at 110 °C for 12 h. Finally, these wet materials were dried at 110 °C for 12 h. The obtained white solids were ground and stored at room temperature. A synthetic magnesium silicate sample was named hydrothermal−1Mg:1Si.eq (mg/g) is the amount of 4−NP adsorbed per unit mass of adsorbent. C0 (mg/L) and eC (mg/L) are the initial and equilibrium concentrations of the 4−NP solution, respectively. V (mL) is the volume of the 4−NP solution and m (mg) is the mass of the adsorbent. Considering the actual conditions, subsequent experiments were carried out according to the optimal Mg/Si ratio synthesized on the optimal synthesis route.To establish a relationship between the amount of adsorbent with time, the adsorption performance of magnesium silicate for 4−NP was tested by batch techniques under pH 7 and 25 °C. Briefly, a series of 50 mL brown, glass bottles with 30 mL of 4−NP solution (10 mg/L) were put in contact with 30 mg of adsorbents placed in shaking incubator at 200 rpm to achieve adsorption equilibrium. At different intervals, the solution was promptly separated from the adsorbent by a 0.22 µm filter. The above solutions were diluted with methanol and the concentration of residual 4−NP in solution was analyzed by UPLC−MS/MS. All experiments were conducted in parallel three times, each obtained value of experiment was calculated by data from the three parallel, and the experimental values were recorded. The adsorption capacity of 4−NP at equilibrium is expressed and calculated by:The adsorption performance of several materials for 4−NP was surveyed by contacting 30 mL 4−NP solution (10 mg/L) with 30 mg of adsorbents, including magnesium silicate and commercial adsorbents  on adsorption capacity was evaluated by sufficiently contacting adsorbents with 10 mg/L 4−NP solution (30 mL). After shaking these series bottles in a thermostatic vibrator at 25 °C for 300 min, the concentration of the residual 4−NP was detected.The performance of the sample in different temperatures  was carried in 50 mL brown bottles by dispersing adsorbates (0.2 g/L) into 30 mL of 4−NP solution (10 mg/L). The equilibrium concentrations of 4−NP in solution were determined by UPLC−MS/MS. The other operation procedures were the same as above.To determine the adsorption kinetics, 30 mL of the 4−NP solution  was put in contact with 30 mg of five Mg/Si ratio samples at 25 °C and 200 rpm. Then, sampling at intervals  to detect the concentrations of the remaining 4−NP in the solution was performed. The other operation procedures were the same as above.A set of 4−NP solutions with 30 mL initial concentrations of 0.1, 1, 3, 5, 7, 9,10, 14, 20, 30, 39, and 51 mg/L (adsorbents dose of 0.2 g/L) was adopted to investigate the adsorption isotherms and thermodynamics for 4−NP. The dose of adsorbents was 0.2 g/L, and the adsorption experiments were carried out at 25, 35, and 45 °C, respectively. Magnesium silicate (dose 0.2 mg/L) was added into 50 mL brown bottles filled with 30 mL of 4−NP solution. These brown bottles were oscillated in a thermostatic shaker for 300 min at 25, 35, and 45 °C. The other operation procedures were the same as above.The recyclability was studied in 50 mL brown glass bottles with 10 mg/L 4−NP solution (30 mL) by dispersing optimal adsorbents (1.6 mg/L). These brown bottles were shaken at 25 °C (200 rpm) for 300 min. The adsorbent, Mg/Si 1:6, was then separated by centrifugation . The 4−NP−containing adsorbents were eluted several times by acetonitrile until the 4−NP could not be detected from the eluant by HPLC−MS/MS. Finally, the adsorbents, without 4−NP, were collected and dried at 60 °C for 12 h. The obtained adsorbent was reused to conduct the following adsorption experiments.The details and instruments are shown in the eq) compared to hydrothermal−1Mg:1Si. In Synthesis of magnesium silicate was performed using sol−gel and hydrolysis. In 2SiO4, well−matched with JCPDS card no. 87-2030, at 2θ~35.540°, 51.140°, and 68.206° for (220), (400), and (511), respectively. The intensity of characteristic peaks, however, is slightly less than that reported according to Temuujin et al. [2, which corresponds to the report by El-Naggar et al. [XRD: Herein, the structure, morphology, and pore characteristics of magnesium silicate were investigated using XRD, BET, and SEM. n et al. . Notably2 adsorption–desorption isotherm, obtained at 77 K, of magnesium silicate with three Mg/Si ratios at 77 K, and the pore size distribution of Mg/Si 1:6. Based on classification of IUPAC, it can be seen that Mg/Si 1:6 exhibits an obvious type H3 hysteresis loop generated by the loosely coherent aggregates of a plate−like substance [0 is related to the existence of micropores [2 is observed at high P/P0 values, the pseudo−type II isotherm caused by delayed capillary condensation can describe the isotherm of magnesium silicate [0 value reaches 0.8, a sharp rise was noticed in the adsorption amount due to multilayer adsorption of N2 on the mesopores [BET: ubstance , which aubstance . Mg/Si 1cropores . It can silicate . As the esopores ; the mea2/g) is relatively higher than that of the 1:1 (41.90 m2/g) and 3:1 (98.58 m2/g). On the other hand, the average pore diameter for the Mg/Si 1:6 (12.30 nm) is also larger than the Mg/Si 1:1 (1.706 nm) and Mg/Si 3:1 (3.827 nm). The observed increase in the pore volume can be attributed to the formation of loosely coherent aggregates with a decrease in Mg2+ content.The surface area of the Mg/Si 1:6 , the R2 (0.9770−0.9914) of pseudo−second−order kinetic model for adsorption 4−NP were slightly higher than that of the pseudo−first−order kinetic model (0.9957–0.9980). Additionally, the calculated adsorption amounts of 4−NP at equilibrium were close to that of the experimental data, and the maximum value of theoretical eq was 6.034 mg/g at Mg/Si 1:6. The pseudo−second−order kinetic could better fit the adsorption process.The adsorption rate and efficiency were determined from adsorption kinetics. In Furthermore, R2, 0.9423−0.9924) and Langmuir  isotherms, and the adaptation sequence of the four isothermal models was as follows: Sips, Langmuir, Freundlich, and Temkin isotherm. Notably, R2 was the adjusted determination coefficient, which could inhibit the multiparameter isotherms [Isotherm: Here, the four widely used adsorption isotherm models, Langmuir, Freundlich, Sips, and Temkin, are used to clarify the essence of the adsorption process for 4−NP under different temperatures . The nonlinear fitting graphs of the Sips and Langmuir isotherm models are shown in sotherms . The SipR2, 0.8900−0.9635) showed a better simulation of 4−NP adsorption performance than the Temkin model . The values of 1/n at different temperatures lies in the range of 0.1−1, indicating adsorption was easy [mq of 4−NP calculated from the Langmuir model at 25, 35, and 45 °C was 46.63, 35.61, and 26.89 mg/g, respectively, which was higher than that of 4−NP on some adsorbents  [R2 value of Temkin isotherm was lower than 0.9070, which suggested that there was a weak interior molecular force between Mg/Si 1:6 and 4−NP in adsorption processes [As shown in was easy . Further49 mg/g) . As showrocesses . Therefosα) was transformed to be dimensionless [θK and 1/T was exhibited in s is the standard state Sips isotherm constant, R is the universal gas constant (8.314 J/mol·K), T is the absolute temperature (K), ΔGθ is the Gibbs free energy change (kJ/mol), ΔSθ is entropy change (kJ/K·mol), and ΔHθ is enthalpy change (kJ/mol). With the increase in temperature, the ΔGθ values decreased gradually, which indicates that the adsorption became less feasible [θ. Besides, physisorption was involved in the adsorption process when ΔHθ had a value less than 40 kJ/mol. The value of Gibbs free energy that lied in −47.83~−48.55 kJ/mol demonstrated that the adsorption was physisorption enhanced by a chemical effect. The negative value of ΔSθ presented that magnesium silicate had an affinity for 4−NP molecules in aqueous solution, which makes 4−NP molecules more regular.Thermodynamics: Adsorption mechanism can be inferred from thermodynamics. Firstly, the Sips equilibrium constant . Adsorption was negatively affected by increasing temperature and adsorbent dose. The adsorption behavior conformed to the pseudo−second−order kinetic model, while the calculated adsorption amounts of 4−NP in equilibrium were close to the experimental value. The adsorption isotherm accords with the Sips isotherm model. The thermodynamic studies confirmed that the adsorption mechanism was physisorption under the effect of chemical enhancement. Moreover, the adsorbent came from environmentally compatible elements, such as Si and Mg, and was a new adsorbent with low cost, high efficiency, nontoxicity, and environmental protection. Therefore, magnesium silicate exhibited a good adsorption performance for 4−NP, indicating that magnesium silicate is a promising material to remove NP from wastewater.In summary, it was found that optimized magnesium silicate, Mg/Si 1:6, was an efficient adsorbent to remove 4−NP from water, with its highest adsorption capacity reaching 30.84 mg/g. Through characterization, the optimized adsorbent possessed the structure of amorphous and mesoporous with a higher surface area (117.7 m


A 49‐year‐old man with stage 4 melanoma metastatic to multiple organs, including multifocal osseous lesions, was referred for evaluation of several months of persistent leukocytosis and thrombocytosis. The patient endorsed fatigue from chemotherapy and immunotherapy, but otherwise denied fevers, dyspnea, bleeding, clotting, or history of corticosteroid use. Physical examination revealed tachycardia and tenderness in the right upper quadrant of the abdomen, without lymphadenopathy or splenomegaly.9/L. White blood cell count was 18.3 × 109/L with 71% neutrophils, 19% lymphocytes, 8% monocytes, 1% eosinophils, and 1% basophils. Peripheral blood smear showed erythroblastosis, with presence of echinocytes and few dacryocytes and fragmented red blood cells, few giant platelets, and extensive platelet clumping, and large mature granular lymphocytes in the setting of malignancy (Figure Complete blood count showed a hemoglobin concentration of 10.7 g/dL, MCV 84 fL, and platelet count of 728 × 10IL‐6 is a pleiotropic immunomodulatory cytokine that promotes tumor growth by inhibition of apoptosis, and is produced in states of inflammation and malignancy by several types of cells, including melanoma cells. IL‐6 has


Legionella spp. in water samples. However, its quality could be improved in terms of recovery of Legionella spp. and selectivity properties. Modifications were introduced in GVPC manufacture: autoclaving conditions  and atmosphere during component-stirring . The use of softer autoclaving conditions  improved the growth of Legionella anisa by the spiral method and Legionella pneumophila after membrane filtration. The medium manufactured with O2 removal and autoclaving for 15 min at 115°C allowed a faster growth of L. pneumophila (colonies visible at day 2) and a notable increase of L. anisa growth . After 3 to 5 days of incubation, the improved media showed higher selectivity properties, particularly for Enterococcus faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 9027. A further improvement was achieved by the addition of N2 during ingredient stirring, leading to a statistically significant faster growth of L. pneumophila at days 2 and 3 and L. anisa at day 3. Selectivity properties were also enhanced, resulting in the complete inhibition of both E. faecalis strains and Escherichia coli and complete-partial inhibition of P. aeruginosa. Oxygen removal during GVPC manufacture using a vacuum pump system promotes the growth of L. pneumophila and L. anisa, and markedly inhibits the growth of E. coli, P. aeruginosa, and E. faecalis.Glycine-vancomycin-polymyxin-cycloheximide agar (GVPC) is a recommended medium for the detection of IMPORTANCE Currently, GVPC is a recommended medium for the detection of Legionella spp. in water samples. However, recovery of Legionella spp. and selectivity properties can be improved. GVPC medium manufactured without oxygen improved the growth of Legionella pneumophila and Legionella anisa. Oxygen removal during GVPC manufacture also improved selectivity properties. A further improvement was achieved by the addition of N2 during ingredient stirring, leading to a faster growth of L. pneumophila at days 2 and 3 and L. anisa at day 3 and enhancement of selectivity properties. The introduction of the modified GVPC medium in routine practice can allow a better detection of Legionella spp. in water samples. Legionella spp., remains an important public health threat , L. dumoffii, and L. longeachae  agar containing 0.1% α-ketoglutarate with mosphere , 19. Mos 2 weeks , thus reIn water samples, the culture plate technique is the standard method stipulated by current regulations and requires more than 10 days to obtain results. In search of a more rapid method, multiple alternatives are currently being developed, including viability qPCR  and immuLegionella in water samples, is identical to BCYE agar  pyrophosphate) except for the addition of four selective compounds: glycine, polymyxin B sulfate, vancomycin hydrochloride, and cycloheximide  pyrophosphate are added to stimulate Legionella growth.GVPC, the medium used for the isolation and enumeration of heximide . In GVPCsensible –22, whilensible –, l-cysteLegionella spp. and its selectivity properties  from the quality control requirements of this medium according to ISO 11731:2017. Difficulties in inhibiting the growth of a Enterococcus faecalis strain recommended for quality control by ISO 11133:2014  are also common. This has prompted most GVPC manufacturers to opt for the other recommended strain, E. faecalis ATCC 19433, for the medium quality control. The quality of the culture media used to isolate Legionella spp. has a great influence on the process of standardization and quality assurance. However, few studies aimed at improving GVPC medium properties have been published to date.Although it is one of the media recommended in ISO 11731:217 and one of the most frequently used selective media by laboratories, it has certain shortcomings that greatly affect recovery of operties . In partLegionella spp. in terms of growth rate, productivity (recovery of L. pneumophila and Legionella anisa) and selectivity . We found that slight modifications can significantly improve the productivity and selectivity properties of GVPC.In this context, the present study was performed to assess the factors in the manufacture of GVPC according to ISO 11731:2017 that may influence the detection of L. pneumophila and L. anisa growth. Using the spiral-pour method, similar mean numbers of CFU were observed for L. pneumophila at day 3 with conventional and lower autoclaving temperatures , and mean colony sizes were also similar  , a reduction of 6°C and 5 min in the autoclaving cycle of the thermoresistant ingredients yielded better nt data) , and 3A.L. pneumophila, a statistically significant increase in the mean number of CFU was observed after membrane filtration  at day 3, without differences being observed in the colony size (0.63 mm in both conditions) (For ditions)  and 3A.L. anisa, a notable increase in the mean number of CFU was observed by the spiral-pour method at day 5 , while no improvements in growth were detected after membrane filtration . Similar results were observed for colony sizes at day 5: 1.46 mm versus 1.06 mm by the spiral-pour method  and 0.81 mm versus 0.80 mm by membrane filtration  (In the case of nt data)  and 3A.E. faecalis, P. aeruginosa, and E. coli. Total inhibition was obtained for E. coli ATCC 25922 and E. faecalis ATCC 19433, and a high degree of P. aeruginosa ATCC 9027 and E. faecalis ATCC 29212 growth was observed in both conditions (> 1000 CFU)  ; Fig. 4.2 removal.Based on the above results, modified autoclaving (115°C for 15 min) was used in the manufacture of media with OL. pneumophila and L. anisa than modified autoclaving alone . Notably, the medium produced without O2 allowed a faster growth of L. pneumophila, with colonies being visible at day 2, particularly when using the spiral-pour method (76 CFU versus 0 CFU). Moreover, at day 3, a non-significant increase was observed in the growth of L. pneumophila assessed as a mean number of CFU by the spiral-pour method  and after membrane filtration   produced a higher growth of VA test) .L. pneumophila were obtained in the medium manufactured without O2: 1.51 ± 0.098 mm versus 0.84 ± 0.09 mm; P < 0.001; Student’s t test for independent data , and 1.01 ± 0.067 mm versus 0.51 ± 0.035 mm; P < 0.001; Student’s t test for independent data (membrane filtration)  .L. anisa is associated with a slower growth, leading to a more difficult detection, the faster growth observed in the medium produced without O2 is of particular interest. Notably, at day 3, colonies were only detected in the medium produced by autoclaving at 115°C for 15 min without O2 and using the spiral-pour method: the mean number of CFU was 59 ± 6.36 and the mean size of colonies was 0.88 ± 0.062 mm   and after membrane filtration: 1.71 ± 0.19 mm versus 1.17 ± 0.13 mm; P < 0.001   and 2. A < 0.001  and 3B.Demonstrating the reproducibility of the modified method, similar results were obtained on different days .2 and with modified autoclaving conditions exhibited higher selectivity properties than the medium with modified autoclaving alone, particularly for Enterococcus faecalis ATCC 29212, which was completely inhibited in the absence of O2, yet grew in the other conditions . A trend toward a higher selectivity for P. aeruginosa was also observed (0.37 log reduction)  ; Fig. 4.2, together with O2 removal and modified autoclaving, yielded the best results in terms of Legionella growth. With the improved conditions, colonies were visible significantly earlier: at day 2 for L. pneumophila and day 3 for L. anisa, with higher sizes than when using the previously assessed conditions. In comparison with conditions 3, the injection of N2 produced statistically significant improvements in the number of CFU and colony size at days 2 and 3 for L. pneumophila; at day 2, a 28.9% increase was obtained in the mean number of colonies of L. pneumophila using the spiral-pour method, without statistically significant differences: 158 ± 38.88 CFU versus 99.66 ± 20.98 CFU; . Accordingly, higher colony sizes were obtained in the medium manufactured with N2 injection: 0.69 ± 0.049 mm versus 0.41 ± 0.027 mm; P < 0.001 , although the former yielded higher colony sizes: 1.75 ± 0.122 mm versus 1.37 ± 0.107 mm; P < 0.001; Student’s t test for independent data   .L. anisa, colonies were visible at day 3 in both media: after N2 injection, 36 CFU were obtained versus 50 CFU and 0.99 ± 0.072 mm versus 0.74 ± 0.05 mm (P < 0.001). Higher numbers of colonies were also determined by the spiral-pour method at day 5 after N2 injection, with statistically significant differences , whereas the results were similar after membrane filtration , with colony sizes of 2.23 ± 0.12 mm versus 2.42 ± 0.145 mm (P = 0.040) and 1.23 ± 0.09 versus 1.33 ± 0.068 mm (P = 0.075), respectively (L. pneumophila (day 3) and L. anisa (day 5)  , and 3D.Demonstrating the reproducibility of the method, similar results were obtained at different days .2 in the presence of N2 and autoclaved at 115°C for 15 min showed improved selectivity properties, particularly for P. aeruginosa and E. faecalis strain ATCC 29212. The medium prepared with N2 completely inhibited the Enterococcus faecalis strain ATCC 29212 (0 CFU) and also E. faecalis ATCC 19433 and E. coli ATCC 25922   ; Table 12 also exhibited higher selectivity properties, particularly for E. faecalis ATCC 29212 and P. aeruginosa.In comparison with other marketed GVPC media, the medium manufactured with NLegionella and inhibit the selectivity strains.Using the modified medium with improved properties, activated charcoal was removed to test if its absence would also allow the growth of L. pneumophila (at day 3) and L. anisa (at day 5) determined by the spiral-pour method and after membrane filtration. In the selectivity assays, the medium without activated charcoal allowed the growth of E. faecalis 29212  and completely inhibited E. faecalis ATCC 19433, E. coli ATCC 25922, and P. aeruginosa ATCC 9027.In the absence of activated charcoal, GVPC autoclaved at 115°C for 15 min prevented the growth of both 2 and autoclaved at 115°C for 15 min prevented the growth of L. pneumophila (direct plating and after membrane filtration). In the case of L. anisa, growth was only observed after membrane filtration (59 CFU at day 5). In the selectivity assays, complete inhibition of growth was observed for E. faecalis (both strains ATCC 29212 and ATCC 19433), E. coli (ATCC 25922), and P. aeruginosa (ATCC 9027).In the absence of activated charcoal, GVPC manufactured without O2 and autoclaved at 115°C for 15 min also exhibited improved selectivity properties, inhibiting the growth of interfering microorganisms in real water samples obtained from cooling towers, in comparison with the medium manufactured with the same conditions but with O2.GVPC manufactured without O2 versus medium with O2) was observed after filtration and filter resuspension (15.00 ± 1.41 CFU versus 30.00 ± 5.65 CFU), heat treatment (4.00 ± 2.82 CFU versus 22.00 ± 2.82 CFU) and acid treatment (7.00 ± 2.82 CFU versus 32.50 ± 6.36 CFU) (The significant reduction in growth of interfering microorganisms (medium without O.36 CFU) .L. pneumophila and L. anisa, Legionella concentrations were 300 CFU/L using the medium without O2 and 200 CFU/L with the medium manufactured in normal conditions .Analysis of results showed that the productivity and selectivity of the manufactured media were maintained until the end of shelf life (3 months). Similar CFU values were obtained for Legionella spp.  have been described   , being cumophila . A fasteL. anisa in GVPC is important, as it is the most common non-pneumophila Legionella species in the environment, can be a causative agent of legionellosis and Pontiac fever, and may be hospital-acquired (Legionella (mostly L. anisa)  , 37. In . anisa)  and osteLegionella growth by modified autoclaving conditions and O2 removal, we tested the removal of activated charcoal. However, using these conditions, growth of L. pneumophila in GVPC was not possible and only L. anisa growth was determined after membrane filtration, thus confirming the key role of activated charcoal in GVPC as a scavenger of radicals and peroxides  and E. coli was achieved, with complete or partial inhibition of P. aeruginosa.Another relevant finding of this study are the enhanced selectivity properties of the medium autoclaved at 115°C for 15 min, with OLegionella spp. due to possible contaminating microbiota  is sensitive to this antibiotic, but it is always inhibited in the absence of charcoal. We postulate that in GVPC, polymyxin B, a lipopeptide consisting of a polycationic peptide ring and a tripeptide side chain . The absence of inhibition in GVPC could also be attributed to interactions of the antibiotic with activated charcoal in the presence of O2, although the hypothesis is less plausible considering the glycopeptide vancomycin structure .As inhibition of radation , thus al2 removal with a vacuum pump system significantly enhances GVPC quality, in terms of both productivity and selectivity. The effect of N2 injection still requires further research. Oxygen removal during GVPC manufacture increased the growth of L. pneumophila and L. anisa, and markedly inhibited the growth of E. coli, P. aeruginosa, and E. faecalis. We consider that these simple modifications are easy to implement, and in the case of the autoclaving conditions, save time and energy.In summary, we have demonstrated for the first time that more moderate autoclaving conditions  and O2 removal and N2 injection during medium manufacturing, and charcoal removal. In total, six different conditions were evaluated in this study: (i) normal conditions ; (ii) presence of O2, autoclaving at 115°C, 15 min; (iii) absence of O2, autoclaving at 115°C, 15 min; (iv) absence of O2, autoclaving at 115°C, 15 min and injection of N2; (v) presence of O2, autoclaving at 115°C, 15 min in the absence of charcoal; (vi) absence of O2, autoclaving at 115°C, 15 min without charcoal  using different conditions, based on modification of autoclaving conditions, Ocharcoal .GVPC was manufactured on a pilot scale for this study  was also introduced during ingredient stirring in the reactor connected to the vacuum pump (condition 4). Finally, GVPC was also manufactured without activated charcoal, in the presence or absence of O2 in the reactor and autoclaved at 115°C for 15 min (conditions 5 and 6).To produce a more marked decrease in the concentration of OFor all the conditions, the final mixture was dispensed in 20 mL volumes into Petri dishes 90-mm diameter in a cleanroom. Plates were stored in airtight containers.E. faecalis strains, P. aeruginosa, E. coli, and BCYE for Legionella strains. Each strain was verified before use in the appropriate selective and differential media according to ISO 17025:2017 (Inocula were prepared according to ISO11133:2014/amended 1:2018 . All tes025:2017 .Legionella species), serial dilutions were prepared to obtain 50 to 120 CFU per plate. For the selectivity assays , serial dilutions were prepared to obtain ≥104 CFU per plate, according to ISO 11133:2014/amended 1:2018.Stock dilutions were prepared according to ISO 11731:2017  and ISO Enterococcus faecalis ATCC 19433 and 29212  and E. coli ATCC 25922 . P. aeruginosa 9027  was used in the selectivity assays.The strains used were those recommended by ISO 11731:2017: Legionella species, according to ISO 8199:2018 . In the productivity assays, plating was also performed after filtration with 0.45 μm pore mixed cellulose ester membrane filters of a bacterial suspension in a diluent containing < 80 CFU of 199:2018 .L. pneumophila and 5 to 10 days for L. anisa. In the selectivity assays, plates were incubated at 36 ± 2°C for 3 days. The number and size of colonies were measured at days 2, 3, and 5 in the productivity assays and at day 3 in the selectivity assays.In the productivity assays, plates were incubated at 36 ± 2°C for periods of 2 to 5 days for The colony count was performed using an automatic colony counter . The size of colonies was obtained first in pixels and then converted to millimeters using the automatic colony counter and also the mobile application Pixel Measure 1.0 .L. pneumophila  and L. anisa .Productivity assays were performed according to ISO 11731:2017 , with thLegionella generally appeared white-gray, but other colors could appear. Colonies were smooth with an entire edge and had a characteristic ground-glass appearance.Colonies of E. faecalis , and E. coli . P. aeruginosa  was also tested.Selectivity assays were performed according to ISO 11731:2017  and ISO 2 removal and autoclaved at 115°C for 15 min without N2 injection (conditions 3).Water samples from two evaporative condensers were kindly provided by Eminfor S.L. . To test the quality of the media, the water samples were analyzed using two different types of GVPC medium: GVPC manufactured in normal conditions (conditions 1) and GVPC manufactured with OL. pneumophila  and L. anisa .Water samples of 1L were collected aseptically in sterile containers containing a neutralizing agent (sodium thiosulfate) and transported to the laboratory, where they were stored at 5 ± 3°C until needed. For each sample, two aliquots (500 mL) were analyzed: direct samples and laboratory samples contaminated with Samples were prepared according to ISO11731:2017  for samp2 (conditions 3).Two aliquots of 100 μL of each portion were plated onto GVPC  and modified autoclaving in the absence of OThe plates were incubated at 36 ± 2°C for 10 days and were inspected for the first time on day 2 and manually read every 2 days until the end of the incubation period.2 and autoclaved at 115°C for 15 min (conditions 3).Productivity and selectivity controls were performed after manufacturing and at the end of shelf life (3 months) for the medium manufactured without OEach sample was plated in duplicate on all direct plating media. All data were analyzed by the general linear model form using SPSS v.21.0 . The mean and standard deviation were calculated for all measures . The numbers of CFU obtained in media manufactured with different methods were subjected to analysis of variance (ANOVA test) using the general linear model procedure to eliminate inter-day variability. The number of bacterial colonies growing on an agar plate was presumed to follow a Poisson distribution, so the square root was extracted to normalize the data and to apply the ANOVA tests.t test for independent data. In all tests, the significance level alpha was set as 0.05.The size of colonies (mm) grown in different media was compared using the Student’s