Background: A posterior tooth's occlusal surfaces and the proximal surface can be restored by using an inlay, which is an intra-crown cast reconstruction without affecting the cusps of the tooth. When an inlay is prepared using an indirect approach, issues with traditional filling approaches, including poor morphology of the occlusal aspect or proximal aspect, inadequate resistance to wear, or subpar mechanical qualities of the directly inserted filler substance, are overcome.Aim: The current study was conducted in order to compare and assess the resistance to fracture of dental materials used in the preparation of inlay restorations indirectly, like composite restorations prepared by laboratories indirectly, inlays formed indirectly of monolithic translucent ceramic derived from zirconia, and inlays formed indirectly of traditional monolithic ceramic derived from zirconia.Methods and materials: For the investigation, 100 human premolars of the maxilla that were extracted recently were chosen. A self-polymerizing acrylic resin was used to incorporate the tooth roots in a band made up of polyvinyl chloride up to 2 mm below the cement-enamel junction. The dimension of the band was 1.3 cm by 1.9 cm. Five categories of 20 specimens of such teeth were formed. Category one, featuring teeth in good condition, acted as the positive control category. The remaining four categories of teeth received inlay tooth preparation. The research samples underwent thermocycling after having been preserved for a full week following the cementation of inlay replacements. Then, in a universal testing apparatus, every sample endured axial compressive force with a metal globe delivered vertically at a crosshead rate of 1 mm/minute. The amount of force necessary to cause a fracture was measured in Newtons (N).Results:The mean values of resistance against fracture in specimens in categories 1-5 were 1208.87 N, 614.89 N, 733.05 N, 1179.14 N, and 1148.49 N, respectively. The values of fracture resistance in specimens where an inlay cavity preparation was done but not filled were lower than those in traditional monolithic ceramic derived from zirconia and tooth specimens with inlays formed of monolithic translucent ceramic derived from zirconia, and the difference was significant statistically (p=0.001). The values of fracture resistance in composite inlay restorations prepared by laboratories were indirectly lower than those of monolithic ceramic derived from zirconia and tooth specimens with inlays formed of monolithic translucent ceramic derived from zirconia, and the difference was significant statistically (p=0.004).Conclusion: Within the constraints of the current investigation, we can state that indirect zirconia-based ceramic products offer adequate fracture resistance, but additional research is needed to determine how well these materials hold up under different types of pressures before employing them in clinical tooth restoration. A posterior tooth's occlusal surfaces and the proximal surface can be restored by using an inlay, which is an intra-crown cast reconstruction without affecting the cusps of the tooth. When an inlay is prepared using an indirect approach, issues with traditional filling approaches, including poor morphology of the occlusal aspect or proximal aspect, inadequate resistance to wear, or subpar mechanical qualities of the directly inserted filler substance, are overcome ,2. The mThe requirement for restoration of teeth in the posterior region is linked to the remaining dental structure's biomechanical properties, biocompatibility properties, as well as aesthetic considerations. Zirconia is one of the materials that is frequently used as tooth-colored indirect inlay restorations in teeth in the posterior region. Particularly in terms of utility, zirconia possesses certain qualities that include better aesthetics, good physical strength, and increased mechanical strength ,6. The vA lack of knowledge was found in the literature concerning the impact of composite restorations prepared in laboratories and inlays formed indirectly from ceramic derived from zirconia on teeth's ability to withstand fracturing ,10. The For the investigation, 100 human premolars of the maxilla that were extracted recently were chosen. The study was conducted at Awadh Dental College and Hospital, Jamshedpur, India. The Ethics Committee of Awadh Dental College approved the study . A self-polymerizing acrylic resin was used to incorporate the tooth roots in a band made up of polyvinyl chloride up to 2 mm below the cement-enamel junction. The dimension of the band was 1.3 cm by 1.9 cm. Five categories of 20 specimens of such teeth were formed. Category one, featuring teeth in good condition, acted as the positive control category. The remaining four categories of teeth received inlay tooth preparation.Each sample had a standardized inlay cavity constructed for it. After four consecutive preparations, the No. 271 bur was changed to ensure cutting effectiveness. Procedures for the preparation of cavities in each category followed standards for size as given in Table In order to prepare an indirect inlay restoration with composite, there was compression of the composite in layers. Each layer was cured under light for 40 seconds. These procedures were carried out on a prototype die. After forming the occlusal framework, the inlay was heated and cured for 15 minutes at 100°C in the oven. With delicate diamond tips and carbide burs operating at low speeds and gentle pressure, each inlay was meticulously finalized.To achieve an optical picture, an antireflection varnish was sprayed onto every single die prototype. A completely computerized optical strip-light scanning device was used to scan the model, which was linked to a computer-aided design/computer-aided manufacturing (CAD/CAM) workstation. A specialized version of software designed for inlays was used to generate the framework after accurately marking all the margins of the cavity on the digital photograph. The physical prototypes, after being designed, were machined using VITA In-Ceram® YZ Cubes . After that, a carbide bur was used to finalize the milled prototype. Following the drying step, all of the structures were put on a firing rack and then sintered for roughly 7.5 hours at 1530°C, comprising the phase of cooling at 200°C, in a sintering kiln. In order to finish the glaze firing period, all restorations were subsequently heated in a furnace with a vacuum at 700°C and subsequently glazed at 500°C.The preliminary frameworks were machined from Cercon XT cubes  using machining burs within a CAD/CAM machining center (Dentsply Sirona) after similarly preparing inlay patterns, as described in category four. All of the preliminary frameworks were subsequently put on a firing rack, and then sintering was carried out at 1500°C for 4.5 hours, also incorporating a cooling period at 200°C following drying. All ceramic restorations were then finished using a sandblasting procedure with Al2O3 after being heated in a vacuum kiln at 920°C and glossed at 45°C  are shown in Table The data obtained were tabulated and put to statistical analysis. The category heterogeneity was examined using a two-way analysis of variance (ANOVA) statistical analysis. The significance threshold was established at p<0.05. To determine the degree of significance of the differences among each category, post hoc Bonferroni test was used.The mean values of resistance against fracture in specimens of the five categories are given in Table The fracture resistance was maximum in Category 1 as it consisted of tooth specimens that were completely intact. The values of resistance against fracture in Category 1 were greater than that of Category 2 and Category 3 and the difference in findings of Category 1 compared to Category 2 and Category 3 was statistically significant. (p=0.002). The values of resistance against fracture in Category 1 were greater than that of Category 4 and Category 5, however, the difference in findings of Category 1 compared to Category 4 and Category 5 was statistically non-significant (p=0.671). On comparing Category 2 and Category 3, the values of fracture resistance were greater in Category 3 as compared to Category 2; however, the difference was non-significant statistically (p = 0.891). On comparing Category 2 with Category 4 and Category 5, the values of fracture resistance in Category 2 were lower than in Category 4 and Category 5 and the difference was significant statistically (p=0.001). On comparing Category 3 with Category 3 and Category 5, the values of fracture resistance in Category 3 were lower than in Category 4 and Category 5 and the difference was significant statistically (p=0.004). On comparing mean values of resistance against fracture in Category 4 and category 5, the the difference in values was non-significant statistically (p= 0.897) .The values of fracture resistance in composite inlay restoration prepared by the laboratory indirectly were lower than monolithic ceramic derived from zirconia and tooth specimens with inlay formed of monolithic translucent ceramic derived from zirconia and the difference was significant statistically (p=0.004).There is a dearth of information in the literature about how indirect zirconia-derived ceramic inlays and indirect laboratory-prepared composite restorations affect a tooth's resistance to fracture. The goal of the current study was to compare and evaluate the resistance to fracture of dental materials used in the indirect preparation of inlay restorations, such as composite restorations, monolithic translucent ceramic derived from zirconia, and traditional monolithic ceramic derived from zirconia.The mean values of resistance against fracture in specimens in categories 1-5 were 1208.87 N, 614.89 N, 733.05 N, 1179.14 N, and 1148.49 N. The values of fracture resistance in composite inlay restorations prepared by laboratories indirectly were lower than those of monolithic ceramic derived from zirconia and tooth specimens with inlays formed of monolithic translucent ceramic derived from zirconia, and the difference was significant statistically (p=0.004). On comparing Category 3 with categories 4 and 5, the values of fracture resistance in Category 3 were found to be lower and the difference was significant statistically. (p=0.004).This outcome was consistent with earlier research, which showed that restorations involving zirconia-based ceramic inlays may restore tooth structural rigidity more effectively than composite inlays -16. The Numerous investigations have shown that samples repaired using indirect composite resin display a greater degree of fracture than samples repaired using ceramic material . This beThe values of resistance against fracture in intact teeth were greater than those of tooth specimens with inlays formed of traditional monolithic ceramic derived from zirconia and tooth specimens with inlays formed of monolithic translucent ceramic derived from zirconia; however, the difference in findings was statistically non-significant. The values of resistance against fracture in Category 1 were greater than those in categories 4 and 5; however, the difference was statistically non-significant (p=0.671).The values of resistance against fracture in intact teeth were greater than those of specimens where an inlay cavity preparation was done but not filled and specimens where composite inlay restorations were prepared by a laboratory indirectly, and the difference in findings was statistically significant. The fracture resistance was maximum in Category 1, as it consisted of tooth specimens that were completely intact, and it was more than that in categories 2 and 3, and the difference in the findings in Category 1 compared to categories 2 and 3 was statistically significant (p=0.002).The biomechanical and biocompatibility characteristics of the remaining dental structure, as well as aesthetic concerns, are related to the need for the restoration of teeth in the posterior region. One of the materials typically used for tooth-colored indirect inlay restorations on posterior teeth is zirconia ,12. ZircIn this study, after being kept for a full week following the cementation of inlay replacements, the research samples underwent thermocycling. The samples were then subjected to axial compressive force by a metal globe applied vertically at a crosshead rate of 1 mm/minute in a universal testing device. The values of fracture resistance in specimens where an inlay cavity preparation was done but not filled were lower than those in traditional monolithic ceramic derived from zirconia and tooth specimens with inlays formed of monolithic translucent ceramic derived from zirconia, and the difference was significant statistically (p=0.001). On comparing Category 2 with categories 4 and 5, the values of fracture resistance in Category 2 were lower than those in categories 4 and 5, and the difference was significant statistically (p=0.001).The nanocomposite used in this study uses cutting-edge, contemporary light dispersion technology. It has uniformly dispersed single-spread nanofillers that provide exceptional flexible durability and durability against wear, and it is globally sculptable ,19. An iAny pre-existing repair flaws that may appear throughout the production procedure and cyclic loading assessment could further encourage the formation of a fracture. This is why CAD/CAM technology was employed in the research. This prevents the formation of cracks by fabricating an indirect replacement in just one appointment. Zirconia's higher flexibility or fracture load resilience, which is around 2.5 times higher than pressable glass ceramic-based substances, was similarly observed in earlier investigations. Therefore, materials based on zirconia might be better suited for stress-bearing restorative areas ,23.The study has some limitations. In vitro procedures were used to conduct this investigation. As a result, values of resistance may change under clinical circumstances. Only vertically axial loads were used in this laboratory experiment; however, clinically, lateral pressure and fatigue load also have a significant effect. It would have been possible to evaluate the resistance to fracture of direct restoration components compared to that of indirect restorations.Within the constraints of the current investigation, we can state that indirect zirconia-based ceramic products offer adequate fracture resistance. This implies that these materials have the potential to withstand external forces without breaking, but additional research is needed to determine how well these materials hold up under different types of pressures before employing them in clinical tooth restoration. This could include analyzing their response to biting forces, chewing, or other forms of mechanical stress that teeth may experience during everyday use.


Ocean acidification is progressing rapidly in the California Current System (CCS), a region already susceptible to reduced aragonite saturation state due to seasonal coastal upwelling. Results from a high-resolution (~ 3 km), coupled physical-biogeochemical model highlight that the intensity, duration, and severity of undersaturation events exhibit high interannual variability along the central CCS shelfbreak. Variability in dissolved inorganic carbon (DIC) along the bottom of the 100-m isobath explains 70–90% of event severity variance over the range of latitudes where most severe conditions occur. An empirical orthogonal function (EOF) analysis further reveals that interannual event variability is explained by a combination coastal upwelling intensity and DIC content in upwelled source waters. Simulated regional DIC exhibits low frequency temporal variability resembling that of the Pacific Decadal Oscillation, and is explained by changes to water mass composition in the CCS. While regional DIC concentrations and upwelling intensity individually explain 9 and 43% of year-to-year variability in undersaturation event severity, their combined influence accounts for 66% of the variance. The mechanistic description of exposure to undersaturated conditions presented here provides important context for monitoring the progression of ocean acidification in the CCS and identifies conditions leading to increased vulnerability for ecologically and commercially important species. Rapid reductions in pH and carbonate ion 2. Of particular concern is decreasing calcium carbonate mineral The global ocean has absorbed approximately 25% of anthropogenic At 4. In spring and summer, equatorward winds intensify along the west coast of North America. Upwelling favorable winds drive offshore transport of the surface Ekman layer, which is then replaced near the coast by waters originating from depths between 50 and 100 m7. These waters are rich in nutrients and both natural and anthropogenic CO2, making them simultaneously low in pH and 9. During upwelling events, enhanced nutrient content in upwelled water supports elevated primary production, which increases 2 levels, but also reduces 2 in a naturally low pH and low 11. The depth at which 12.The effects of ocean acidification are exacerbated in the California Current System (CCS) due to coastal upwelling13. Such events have been captured in synoptic surveys as well as long-term time series, which documented exposure to corrosive conditions during periods of enhanced upwelling-favorable wind stress14. Alongshore variability in coastline topography also modulates atmospheric wind patterns and nearshore ocean circulation, thereby impacting the local magnitude of upwelling intensity, primary production, and pH on the shelf19. Furthermore, upwelling and primary production exhibit strong interannual variability due to changes in atmospheric and oceanic forcing associated with modes of climate variability, such as the El Niño Southern Oscillation (ENSO)21, Pacific Decadal Oscillation (PDO)23 and North Pacific Gyre Oscillation (NPGO)25. Due to their highly variable nature, the physical and biogeochemical drivers of low 30. Exposure to low pH conditions  at 1/30° (~ 3 km) resolution forced by an outer domain for the broader CCS  at 1/10° (~ 10 km) resolution with physical data assimilation. The physical circulation is forced at the surface by the 0.25° resolution Cross-Calibrated Multi-Platform (CCMP) winds33, and physical initial and boundary conditions for the outer domain are derived from the Simple Ocean Data Assimilation (SODA) reanalysis34. A complete description of the physical model configuration can be found in Fiechter et al.18.The physical circulation is resolved by the Regional Ocean Modeling System (ROMS)35, with the addition of carbon and oxygen cycling based on formulations of Hauri et al.28, and Fennel et al.36, respectively. Boundary and initial conditions for the outer domain are derived from the World Ocean Atlas monthly climatology for nutrients and oxygen37, from the Global Ocean Data Analysis project for total alkalinity (TAlk)38, and from the empirical relationship of Alin et al.39 for dissolved organic carbon (DIC) based on monthly temperature and oxygen. Due to their climatological nature, the boundary conditions do not reflect global and basin-scale trends in biogeochemical properties. Atmospheric 19.Biogeochemical processes are simulated using NEMUCSC, an adaptation of the North Pacific Ecosystem Model for Understanding Regional Oceanography (NEMURO)20. While the full simulation spans 1980–2010, the analysis is limited to 1988–2010 to eliminate spin-up effects.The analysis presented here is based on the higher-resolution inner domain at ~ 3 km resolution and focuses on the region between Pt Conception and Cape Blanco (35–43° N), which is known to exhibit substantial alongshore variability in physical and biogeochemical processes influencing pH (and presumably 40. Because the observations (2017–2019) and simulation (1988–2010) do not overlap in time, the model is evaluated for its ability to reproduce the mean, range, and monthly climatological properties of measured pH, temperature, and dissolved oxygen (DO). An event analysis (as described below) is applied to simulated and observed variables to further assess the capacity of the model to capture low pH events  Cruise between 35 and 42° N by comparing simulated daily mean values for May–June 2007 to synoptic measurements from 0 to 300 m depth at the 4 stations closest to shore along each transect. The ability of the model to reproduce temporal and alongshore properties of undersaturation events is also evaluated using daily-averaged model output compared to daily-averaged observations from nearshore moorings deployed at approximately 12-m depth within kelp forests at ~ 35.2, 36.0, 38.9, and 39.3° N27, events are characterized by their intensity, duration, and severity. Duration (D) represents the number of consecutive days I = T−S = D × I). Although biological thresholds are presumably species- or even population-specific, a heuristic threshold of The event analysis focuses on determining the properties of undersaturated events with respect to aragonite, where “events” are defined as periods of time when 21 and, therefore, of the conditions that eventually modulate coastal exposure to undersaturated events. Physical and biogeochemical water mass properties associated with events are determined by averaging annually over days during which conditions along the bottom of the 100-m isobath were undersaturated. These properties are then individually regressed against event severity to identify variables most closely associated with interannual variability in event properties at each latitude. While averaged meridional wind stress within 50 km from the coast is included in the regression as a proxy for coastal upwelling intensity18, the influence of regional upwelling dynamics and water mass properties is assessed more extensively by examining the depth and physical and biogeochemical properties associated with the 26.0 kg/m3 isopycnal , except for the multiple linear regression and associated statistical tests done using R 45.Intensity, duration, and severity are calculated using daily averaged model fields extracted along the 100-m isobath as a location characteristic of alongshore, seasonal, and interannual event variability affecting coastal waters in the central CCS. The bottom of the 100-m isobath is also indicative of upwelled source water properties2 values of 0.75 for the observations, and 0.85 for the model. R2 values for a regression between DIC and pH are higher, with 0.91 for observed and 0.99 for simulated values. Regressions of TAlk with DIC and pH reveal weaker relationships, with respective R2 values of 0.5, 0.22 for the observations, and 0.85 and 0.75 for the model. These comparisons indicate that in both model and observations, DIC explains a substantially greater fraction of pH (and presumably saturation state) variability than TAlk over the coastal region and depths considered here. Overall, the model evaluation demonstrates reasonable agreement between simulated and observed variables, especially for DIC and pH, across the shelf at latitudes (35–42° N) spanning the central CCS. The assessment is consistent with that of Cheresh and Fiechter19 based on the same model output and in situ observations, which identified that biogeochemical discrepancies were generally associated with physical conditions not exactly matching the synoptic timing of upwelling along transects.The ability of the simulation to reproduce observed variability across the shelf at several latitudes spanning the model domain is evaluated in terms of the agreement between observed and simulated properties via Taylor diagrams   . For physical variables, temperature is generally not a good predictor of event severity (R2 < 0.3) and the explanatory power of meridional (upwelling favorable) wind stress is limited to latitudes ranging between 38 and 41° N and explains 30–50% of the interannual variance in event severity  alongshore variations over the region where undersaturated conditions occur most frequently i.e., 38–43° N). Average water mass properties during undersaturated conditions along the bottom of the 100-m isobath indicate alongshore similarities between event properties and DIC and TAlk  and biogeochemical  processes controlling changes in these properties along the bottom of the 100-m isobath. Advection emerges as the dominant mechanism influencing interannual variability in DIC and TAlk during the upwelling season, with average contributions at most latitudes greater than any other process by at least two orders of magnitude  changes in upwelling intensity, whereby weaker or stronger upwelling would lead to lower or higher DIC concentrations, and (2) changes in DIC concentrations near the source depth of upwelled water independent of upwelling intensity.To explore the relative importance of changes in upwelling intensity vs. changes in upwelled source water DIC content , regional variations of the two following properties are considered: (1) depth of While the first EOF mode for the depth of ion Fig. a. The di}m3 Fig. d. The la2 value of 0.66, and a p-value of 1.967e−05 . Using upwelling season alongshore mean values for the depth of Previous work has identified a region-wide susceptibility to ocean acidification in the CCS, and the present study describes recent spatiotemporal patterns and drivers of exposure to undersaturated conditions. The results demonstrate that event intensity, duration, and severity in the central CCS exhibit substantial interannual variability governed by changes in the strength of coastal upwelling and DIC concentrations in upwelled source waters.19. The tendency of the model to simulate slightly higher than observed pH values suggests that the present assessment of exposure to undersaturated conditions is likely conservative and provides lower bounds for event properties. While noticeable differences exist in the magnitude of pH values, the alongshore pattern of event occurrence and overall magnitude of event properties are generally consistent between the model and observations. The focus of the present study is to identify dominant drivers of interannual event variability rather than provide exact estimates of event properties in terms of their magnitude. As such, the interpretation relies on regional spatial and temporal patterns to describe event properties along the 100-m isobath, while in reality there are additional physical and biogeochemical mechanisms, such as tidal mixing and benthic processes, that will further influence event properties at the local scale of nearshore environments. The lack of a global anthropogenic signal in the model boundary conditions and the relatively short simulation period also preclude any conclusions about trends in corrosive events from the present analyses.Comparison of simulated pH, dissolved oxygen, and temperature against observed data from nearshore moorings indicates the model reproduces, in a climatological sense, natural variability experienced in coastal waters on monthly time scales. Episodic excursions of observed pH below the minimum simulated values are attributed to a model bias towards higher pH levels near the source depth of upwelled water identified in previous evaluations19 and evidenced by higher POC remineralization rates in Fig. 46, who also found DIC to be the dominant variable driving changes in pH within 100 km from the coast in the central CCS. The results presented here do not contradict previous work identifying the role of remineralization in reducing 9, but rather establish that it has limited explanatory power for year-to-year variability in event intensity. Based on the interannual variability of simulated remineralization rates during events at the bottom of the 100-m isobath , it cannot by itself explain the simulated interannual variability in event severity. For example, moderate to strong La Niña events occurred in 2000, 2006 and 2009, but resulted in below average simulated event severity, suggesting that a moderate to strong La Niña event  may not solely dictate undersaturation events at 100-m depth. It is more likely that interannual modes of variability modulating physical and biogeochemical properties in the central CCS, such as ENSO, superimpose on lower frequency modes of basin-scale variability, such as the PDO, and potentially exacerbate or mediate exposure to undersaturated conditions.Turi et al.27, in which changes to simulated event properties from the preindustrial era through 2050 were quantified under a high-emissions scenario. Event properties calculated here are higher than those reported in Hauri et al.27 for the 5-year time period approximating 2010. The discrepancy could be attributed to wind forcing differences between the two simulations. Hauri et al.27 used climatological wind forcing, which likely underestimates synoptic variability and, therefore, the intensity of coastal upwelling events. Aditionally, Hauri et al.27 reported a model bias towards higher 27 included shallower shelf regions where events may be shorter and less intense, reducing spatially-averaged event property statistics.The present study builds on the work of Hauri et al.11. Most observational studies examining the ecological effects of ocean acidification have necessarily related snapshots of organisms’ conditions to concurrent conditions. However, the duration, intensity and severity of exposure is likely important in governing species’ responses. The effects of exposure to low 47). Thus, the results detailed here add to a growing body of evidence that the impacts of ocean acidification along the U.S. west coast will not be experienced uniformly with latitude and vary significantly from year to year. The mechanistic exploration of the drivers of events in high-resolution model simulations provides a valuable framework for identifying geographic hotspots or oceanographic conditions  associated with increased vulnerability to ocean acidification at local and regional scales.The current findings have important implications for marine species and ecosystems in upwelling regions. In the CCS, exposure to undersaturated conditions has been linked to shell or carapace dissolution of ecologically important speciesSupplementary Information.


Posidonia oceanica. Through a space-for-time substitution study, Fatty acid (FA) profiles on the second and fifth leaf of the shoots were quantified at eight sites in Sardinia along a natural sea surface temperature (SST) summer gradient (about 4 °C). Higher mean SST were related to a decrease in the leaf total fatty acid content (LTFA), a reduction in polyunsaturated fatty acids (PUFA), omega-3/omega-6 PUFA and PUFA/saturated fatty acids (SFA) ratios and an increase in SFA, monounsaturated fatty acids and carbon elongation index  ratio. Results also revealed that FA profiles were strongly influenced by leaf age, independently of SST and spatial variability within sites. Overall, this study evidenced that the sensitive response of P. oceanica FA profiles to intra-shoot and spatial variability must not be overlooked when considering their response to temperature changes.Global warming is expected to have inexorable and profound effects on marine ecosystems, particularly in foundation species such as seagrasses. Identifying responses to warming and comparing populations across natural temperature gradients can inform how future warming will impact the structure and function of ecosystems. Here, we investigated how thermal environment, intra-shoot and spatial variability modulate biochemical responses of the Mediterranean seagrass  Evaluating early warning stressor-specific responses of foundation species is pivotal to understand the effects of future warming and to predict their status, and consequently their structure and distribution. In this context, for marine macrophytes temperature is one of the most important limiting factors7.Predicting the consequences of global warming and identifying mitigation and restoration actions in the future is critical, particularly in the sea12 and coastal protection from erosion14. Several local anthropogenic stressors, such as sediments and nutrient inputs, physical disturbance such as trawling, anchoring, invasions of exotic species have put seagrasses under high pressure over the past decades19. Moreover, concern is growing over the impacts of warming on seagrasses, increasing seagrass shoot mortality22 and limiting meadow recovery and structure23. Therefore, understanding their response to climate change is pivotal to determine their future ecosystem functioning and services. Thermal environment is likely to shape the structure and density of a meadow, driving morphological adaptations and acclimations26, physiology6, and reproductive phenology28. However, defining the effects of temperature changes on seagrass species remains a controversial topic that has not found unequivocal descriptions likely because results can be affected by local species adaptations making the acclimation responses difficult to measure and interpret31. However, depending on the intensity and duration of the heat, sublethal effects can be observed at different plant organization levels: changes in morphology may include leaf fall and leaf necrosis33, remodelling leaf biochemistry as carbohydrates and lipids35, and adjusting the transcriptomic regulation at molecular level37.Seagrasses, one of the most important and productive marine coastal ecosystems, have a key role in ecosystem functioning and services, providing blue carbon sequestration and nutrient cycling, forming habitats, nursery grounds, sediment stabilization, trophic transfer to other habitats6, and among these the composition of Fatty acids (FA) can represent a valuable physiological descriptor. FA are vital structural compounds of primary producers, constitute the structure of the membranes of the thylakoids40 and play a fundamental role in the photosynthetic activity41. The remodelling of leaf FA due to temperature increase has been found in a wide range of plant species living in several biomes42 and in particular in seagrass species40. Results consistently indicate that heat reduces polyunsaturated fatty acids (PUFA), counterbalanced by the increase in saturated fatty acids (SFA) and monounsaturated fatty acids . However, although the use of FA analysis for understanding marine ecosystem functioning is promising, the variability in FA composition, either as single or grouped fatty acids, needs to be estimated to ensure relevant and appropriate interpretations45.Several seagrass traits are widely used to assess coastal ecosystem status in the past years47, while the majority have been conducted in mesocosms with artificial induced changes. Remodelling in FA profiles related to a heat stress, such as a reduction in PUFA, in total Fatty acids, and an increase in SFA47, is connected to the healthy functioning of photosynthetic activity, and consequently, to the plant resilience to the environmental changes.With the aim of evaluating the potential sources of variability in seagrass leaf FA composition at different environmental conditions, we realized that only relatively few studies have investigated variations in FA composition along natural environmental gradientsPosidonia oceanica and Zostera noltii (e.g.48. and50 respectively), while recently only young leaves were considered . Very little attention is paid to the specific leaf analysed: only Park et al.51 measured differences between fresh and senescent leaves of Z. marina, and Viso et al.52 compared chlorophyllous to non-chlorophyllous part of the same leaf in P. oceanica. However, because in each bundle there are differences across leaves, younger leaves internally enclosed by the external older leaves9, each leaf corresponds to a different time of production, exposition, morphology , and epiphytic cover54. Analysing the entire shoot material or focusing only on a specific leaf of the shoot could hinder some leaf-type functioning, metabolism, and phenology features. Previous evidence highlighted differences in biochemical composition and physiological processes along within-shoot leaf variability as in isotopic composition55, nutrient mass balance57, phenolic compounds59 and investigating intra-plant variation in FA composition will be crucial in determining the interaction outcomes between seagrasses and their surrounding environment. Thus, here we hypothesize that the FA composition would depend on the leaf age within the same shoot.Furthermore, the material collected for analyses is often inconsistent for seagrasses, even within samples belonging to the same species: most authors referred to generic leaf biomass of the same shoot both in Posidonia oceanica using a space-for-time substitution approach, where the relationships between ecological variables are studied at sites assumed to be at different stages of development60. The study also estimated the variability in FA composition due to leaf material and spatial variability at a local scale, across the gradient. The first hypothesis is that FA profile varies along a temperature gradient resembling the responses to changes due to future warming, mainly with an expected decrease of PUFAs relative to SFAs at higher temperatures. A further approach was used to test the second hypothesis that FA profiles can depend also on intra-shoot variability by comparing the second vs fifth leaf of the same shoot, with an expected higher level of PUFAs in the former due to higher productivity. Lastly, we tested the third hypothesis that spatial variability among meadows reflects differences in local conditions rather than temperature. With these aims, we considered meadows along a natural gradient of water temperature in Sardinia (western Mediterranean Sea) that allowed examination of the association of P. oceanica FA composition for a wide range of Sea Surface Temperature  comparable to the increase in SST according to future warming scenarios61, with minimum interference of factors associated to a narrow latitudinal range . Overall, results provide knowledge about the substantial source of variability of seagrass leaf FA composition contributing to produce accurate sampling practices and monitoring procedures and rely on valuable warming descriptors.In this context, this study investigated the dependence of FA composition on a thermal gradient in the seagrass The thermal environment at the eight sites in the 10 days before sampling was widely different, mean SST ranging from 20.72 °C (SE ±0.14) in AHO to 24.84 °C (SE ±0.26) in ARB Table .Table 1FAs expected, FA groups were all related to the SST Table . HoweverP < 0.0001) and LTFA  were associated to L2, while higher values of SFA and n-3/n-6 were associated to L5  and n-3/n-6 (P = 0.00018) which differed only among sites and areas, respectively , but also on the area within each site; the exceptions were LTFA  with warmer temperatures can be ascribable to different homeoviscous conditions as an effort to preserve membrane fluidity properties and electron transport65. Higher proportions of SFA would be produced to provide optimal cell membrane structural rigidity, counteracting the increasing fluidity produced in warmer conditions66. Consequently, in natural environment warm-acclimated populations exhibit higher levels of SFA than their cool-acclimated counterparts. Similarly, the decrease in polyunsaturated n-3 group (C16:3 n-3 and C18:3 n-3) relative to n-6 group (C16:2 n-6 and C18:2 n-6), and thus the n-3/n-6 (seagrass photosynthetic productivity index), could reflect the increase in SFA and MUFA (which are substrates of polyunsaturated FA) to maintain membrane fluidity, adjusting their metabolism in direct response to the warming stress, as mentioned above.Differences in FA profiles in plant cells membrane lipids could be interpreted as a required metabolic pathway to retain the photosynthetic thermostability and acclimation to temperature conditions64, this result could suggest that PUFAs are proxy of productivity of seagrass plants, especially in younger leaves where photosynthetic activity is higher57. Therefore, depending on the influence of temperature, leaf-type effect on specific functioning and metabolism would be missed if the seagrass material collected is not accurately decided by a procedural protocol and each leaf within a shoot was treated as a possible replicate55. Indeed, the amount of variability in FA groups associated to the range of temperature considered is very similar to that occurring between the leaves of the shoot, supporting the need of carefully evaluating the plant material to collect for the analysis not to add undesired variability to the results. Thermo-tolerance of plants is known to be strongly influenced by leaf age and life span with a general higher resistance of the more mature leaves to the heat69. In monocotyledonous plants a strong vertical age gradient is also present within the same leaf, as the undifferentiated cells are located at the base of the blade and the most mature cells occurring at the tips70. The gradient of leaf developmental stages reflects the differences in biochemical and physiological processes along the leaf length and across leaves71. The higher levels of FAs associated to photosynthesis in the second leaf (younger or intermediate57) suggest this plant material is more suitable to detect temperature influence on seagrass species. The variation in photosynthetic activity is considered one of the most sensitive seagrass physiological responses to heat69, and this study supports the hypothesis that the effect of temperature on the FA profiles is consistent between leaves. These findings are in accordance with previous studies that investigated the nutritional role of different leaves in the same shoot57. Overall, this evidence fosters the need of accurately selecting the seagrass material to produce comparative evaluations and possibly rely on solid descriptors of heat stress.Furthermore, we evidenced a significant source of intra-shoot variability in FA composition between the second and fifth leaf of the same shoot. Specifically, the contribution of different FA groups (except for the CEI) changed and leaf 5 contained higher SFA and n-3/n-6 and lower PUFA, MUFA and LTFA content than leaf 2. As higher values of PUFAs are associated to higher photosynthetic activity43). Some manipulative experiments have instead tested the effect of a 4 °C temperature increase (treatment similar to the range considered in this study and comparable to climate change scenarios) in a relatively short time43 to simulate a warming event. Although different in the time of warming, the response of FA in plants subjected to the temperature treatment in mesocosms resembled the FA composition associated to SST found in this study in untouched plants.The much less obvious result is the fact that we have identified associations of FA groups with SST in a very field range of mean temperature (about 4 °C) and a relatively restricted spatial range. Descriptive studies that investigated the remodelling of FA composition in seagrasses have generally dealt with wider temperature ranges, often larger than 10 °C, both because of either wide temporal or latitudinal ranges  were found among areas at the same temperature. This result confirms how environment-induced variation can affect FA profiles, as Schmid et al.72 described for seaweeds. If disregarded, the role of temperature may be confounded by effects due to local conditions; therefore, the choice of dealing with a hierarchical sampling design  should be encouraged.Overall, obtaining biochemical changes in P. oceanica FA groups as possible descriptors in a thermal summer gradient resembling future warming scenario. Although we are aware that the use of satellite-derived SST data as proxy of thermal environment at 10 m depth is less accurate than in-situ measures, we highlight the reliability of this approach when testing wide range gradients as in this study, where the effect of variations in summer Mediterranean temperature, resembling the future climate changes, on P. oceanica biochemical response in the field was evaluated. Nevertheless, further research is needed to predict global warming effects on plant species and define if FA profiles are good candidate to explain species response to the heat stress, whose adjustment to temperature shifts needs to be deeply investigated. However, the significant role of spatial variability and plant material on the highly sensitive response of FA profiles of P. oceanica leaves here evidenced, suggest the necessity that methodology and sampling design in seagrass studies and in monitoring plans will be carefully decided to obtain reliable indicators.This study proposed the response of several 73, while the eastern coast is affected by the warm Algerian Current74.This study was done in Sardinia  and four of the east coast , from 40°34’ to 39°15’N. The seagrass meadows sampled were all dense (on average 581 shoots/m225), and far from anthropogenic disturbances. Collection of samples followed a hierarchical sampling design, as at each site three areas 100 s of m apart were randomly selected . Posidonia oceanica (L.) Delile shoots were identified by Arianna Pansini and deposited as voucher specimens at the University of Sassari Herbarium . In the laboratory, the intermediate second leaf, more than 5 cm long without basal sheath (L2 from 72 shoots) and the adult fifth leaf, more than 5 cm long with basal sheath  of each shoot, were identified washed in filtered sea water to remove extraneous materials, cleaned from epiphytes, and kept frozen at − 20 °C. For each leaf, the photosynthetically active part  and finely ground into powder using a homogenizer (Beadmill 4 machine at 5 m s−1 for 3 min).Samplings were done in meadows at 10 m of depth, from the 202SO4. To quantify total and individual FA composition and contents, the standard Nonadecanoic acid, C19:0  was added as an internal standard before the transmethylation process. To prevent oxidation, vials were sealed with nitrogen gas before being heated at 80 °C for 2 h under stirring conditions. After transmethylation, 1 mL of Milli-Q water was added, and FAMEs were extracted using 0.25 mL of n-hexane. Identification of FAMEs was conducted using a Clarus 500 Gas Chromatograph  equipped with a flame ionization detector and a fused silica capillary column . Identification of FAME was achieved by co-chromatography with commercially available FAME certified standard material .Fatty acids methyl esters (FAMEs) were determined by direct transmethylation of ~ 10 mg of powdered biomass with dry methanol containing 2% (v/v) H39, n-3/n-6 (the sum n-3 PUFA/sum of n-6 PUFA as an index of seagrass photosynthetic productivity39), and an additional biomarker proposed as carbon elongation index . Finally, leaf total FA content (LTFA) was calculated and expressed in % mg DW.The FA composition was determined and the proportion was expressed as relative contribution (%) of the grouped fatty acid SFA , MUFA , and PUFA  groups. Moreover, the following FA ratios were considered: PUFA/SFA as an index of thermal stressP. oceanica shoot collection day , was obtained by the Group for High Resolution Sea Surface Temperature (GHRSST) daily . SST was used as proxy of the site 10 m subtidal temperature77 and due to the resolution, it allowed assuming the same thermal conditions for the three areas within each site. For each site, daily SST data were averaged through the ten days before sampling. Despite satellite-derived SST data are less appropriate than in-situ measures, they allow investigating the interaction between biotic and abiotic variables on ecological dynamics78.For each site the SST for the ten days preceding the specific 79 using the nlme package80. Because FA data presented variance heterogeneity among the factor, the variance leaf structure with different spread per level of the factor (“VarIdent” Variance Structure) was included into the GLS analyses81. The best model was selected according to minimum Akaike information criterion  and SST (continuous variable), leaf (factor with two levels: L2 and L5), and their interaction were tested using Generalized Least Square (GLS) models. GLSs are weighted linear regressions which consider the inequality of variance in the observations, allowing for heterogeneity. Before running the models, the collinearity between the response variables was inspected with pair-plots, and variance inflation factors (VIFs) were calculated. Analyses were only run on SFA, MUFA, n-3/n-6, CEI and LTFA; in fact, PUFA and PUFA/SFA were negatively correlated with SFA, as well as n-3 and n-6 with n-3/n-6 . Analyses were performed in RTo estimate relevant scales of spatial variability in FA composition, for each response variable  obtained only from L2, a two-way ANOVA was run to test the effect of site  and area (three levels) random and nested in site. Homogeneity of variances was checked by Cochran’s test.The authors declare that the seagrass samplings have been carried out in accordance with relevant guidelines and regulations.Supplementary Information.


At the parameter, the smallest particle size and size distribution range of MP were 190 nm (9.51%). Furthermore, the interplay between CA–DI and MPs contributed to a reduction in the protein particle size and intrinsic fluorescence. In summary, the combination of CA–DI and MPs played a crucial role in inducing protein unfolding and disintegration.Spice and its extracts have gained widespread utilization as natural and eco-friendly additives, imparting enhancements in flavor, color, and antioxidative attributes to meat-based products. This work aims to study the effect mechanism of capsaicin (CA) and dihydrocapsaicin (DI) in capsicum (chili pepper) on the structure and function of myofibrillar proteins (MPs) in duck meat during thermal treatment. The results showed that at a CA–DI to MP ratio of 1:500 (g/g) following a 12 min heat treatment, the carbonyl content of MPs in duck meat decreased by 48.30%, and the sulfhydryl content increased by 53.42%. When the concentration was 1:500  after 24 min of heat treatment, the •OH and DPPH radical scavenging rates were highest at 59.5% and 94.0%, respectively. And the initial denaturation temperature of MPs was the highest at 96.62 °C, and the thermal absorption was lowest at 200.24 J g Duck meat, which is well known for its distinct flavor, aromatic nuances, elevated protein content, large polyunsaturated fatty acid composition, and economic affordability, comprises a substantial proportion of consumption patterns, particularly across Asia ,2. Howevhardness, adhesiveness, gumminess, and chewiness, alongside the regulation of elasticity loss during refrigeration [Spices, botanical entities imbued with multifarious aromatic and gustatory attributes encompassing fragrant, pungent, numbing, bitter, and sweet nuances, have garnered increased attention as natural antioxidant compounds that may impact meat processing . This pageration . The reageration . An overgeration .However, scientific scrutiny of the influence of capsicum (chili pepper) on myofibrillar proteins within duck meat remains relatively scant. Capsicum, imbued with connotations encompassing culinary and medicinal dimensions, serves as an effective approach within the antioxidant and antibacterial domains in animal feeding studies and pharmacological investigations ,24. In mFrozen duck leg meat samples  were sourced from Hubei Chia Tai Egg Industry Co. Ltd. . The CA (purity: HPLC ≥ 98%) and DI (purity: HPLC ≥ 98%) compounds were procured from Shanghai Tongwei Biotechnology Co. Ltd. . It is imperative to note that all chemicals and reagents employed in the experimental procedures were of analytical grade, unless specified otherwise.2, and 1 mmol/L PMSF, maintaining a specific proportion of 1:10 . The mixture was homogenized at 10,000 rpm for 30 s, , followed by centrifugation at 2205 g and 4 °C for a duration of 30 min . The supernatant obtained was discarded, and the precipitate was subjected to a wash sequence, repeated three times, employing a buffer solution composed of 50 mmol/L potassium chloride alongside 5 mmol/L β-mercaptoethanol. Finally, the resultant pellet underwent extraction via buffer solution, thereby culminating in the generation of a homogenized suspension constituting the MPs extract. The quantitative assessment of protein concentration was undertaken through the utilization of the Coomassie brilliant blue method [Duck MPs were prepared in accordance with the method outlined by Park et al. incorporating discerning modifications . The duce method , with thIn accordance with established laboratory protocols, the extracted MPs were initially subjected to incubation within a water bath (40 °C) for a duration of 7 min. Subsequently, a composite solution comprising capsaicin (CA) and dihydrocapsaicin (DI) in a ratio of 2:1 (g/g) was introduced to the MPs matrix at ratios of 1:1000, 1:500, 1:250, and 3:500 . This amalgamation was then subjected to thermal treatment at 90 °C over a period of 36 min. At defined intervals of 12 min, corresponding to time points encompassing 12 min, 24 min, and 36 min, aliquots of the resultant mixed solution were used for subsequent analysis and detection. Notably, the binding modality between CA–DI and MPs is elucidated in the −1 × cm−1 was invoked, resulting in the expression of protein carbonyl content in units of nmol/mg protein. Analysis for each individual sample was repeated three times.The quantification of carbonyl content was conducted utilizing a method described in . A total−1 × cm−1, which finally expressed the outcomes in units of nmol/mg. Analysis of each individual sample was performed three times to ensure robustness and reproducibility of results.The quantification of free hydroxyl content was conducted following a method that was modestly adapted . For thiThe evaluation of MP surface hydrophobicity was carried out by employing 1-benzenedioic acid-8-sulfonic acid (ANS) as the pivotal fluorescent probe, following an established methodology with minor refinements . To initThe assessment of hydroxyl radical scavenging activity was conducted employing the methodology outlined by Zhu et al. . A total0: absorbance value of sample solution replaced by distilled water;A1: absorbance measured at 510 nm;A2: absorbance value of salicylic acid–ethanol solution replaced by anhydrous ethanol.AThe evaluation of DPPH radical scavenging potential was undertaken following the protocol of Nunes et al. . A combi0: absorbance value of a mixture of water and DPPH solution at 517 nm;Ai: absorbance value of a mixture of sample solution and DPPH solution at 517 nm;Aj: absorbance values of a mixture of sample solution and methanol at 517 nm.A−1) were meticulously quantified. In order to ensure robustness and precision, each distinct sample was analyzed three times.The investigation into the thermal denaturation behavior of the protein within the MPs specimens was conducted employing a differential scanning calorimeter , as detailed in reference . The expParticle size analysis was conducted employing a protocol adapted marginally . In thisThe quantification of molecular fluorescence was conducted in accordance with the methodology outlined by Huang et al., with a slight modification . The samAll experiments were conducted independently and in triplicate using MPs from three sample preparations. Experimental data were presented in the form of the mean ± standard deviation (SD). A two-way analysis of variance (ANOVA) was performed to determine statistical significance using SPSS statistical software . The visualization of data and its systematic tabulation were facilitated by leveraging the capabilities of OriginPro (2021) 9.8 software. In order to discern and elucidate notable disparities among means, the methodology of Duncan’s multiple range tests was harnessed, with a predetermined significance threshold of 0.05.p < 0.05). The formation of carbonyl compounds and the loss of sulfhydryl groups are some of the most common expressions of the chemical damage to oxidized proteins. This is consistent with the findings of Valquíria et al. [The quantification of carbonyl group and free sulfhydryl group content within proteins is a pivotal marker for assessing protein oxidation dynamics, as determined by previous studies ,39. As ia et al. . The dega et al. .p < 0.05). At a ratio of 1:500  within a 12 min timeframe, the carbonyl content declined by 64.77%, while free sulfhydryl content increased by 32.53%. CA–DI is fat-soluble, whereas MPs are amphiphilic macromolecules [A significantly notable mitigation of protein oxidation occurred, as evidenced by the reduced carbonyl group content, across all experimental groups incorporating CA–DI supplementation compared with the control group (olecules . The addolecules . These folecules .p < 0.05). In this configuration, the surface hydrophobicity of MPs decreased by 50.55% compared with the untreated group upon 36 min of heat treatment. High temperatures in meat processing can oxidize proteins and lead to aggregation and crosslinking between the molecules, enhancing their surface hydrophobicity [In contrast to the untreated group, the surface hydrophobicity of MPs exhibited a significant reduction in all capsaicin–dihydrocapsaicin treatment groups (hobicity . This sup < 0.05). A significant reduction for each concentration was observed in scavenging efficacy between the 24 min and 36 min intervals (p > 0.05). Notably, when the CA–DI to MPs ratio was maintained at 1:500 (g/g) over 24 min of heat treatment, MPs demonstrated their highest recorded scavenging rates for •OH and DPPH radicals, reaching 59.5% and 94.0%, respectively. This exceptional radical scavenging rate of MPs at this specific ratio can be attributed to the proper incorporation of an optimal concentration of CA–DI. This might be attributed to the excess content of CA–DI, which could not combine with the MPs, resulting in a reduced radical scavenging rate when the concentration was increased to 1:250 and 3:500 . This finding aligns with the observations that 1% clove extract exhibited elevated DPPH radical scavenging potential in comparison to the control group [Elevated thermal conditions have been observed to readily trigger the generation of hydroxyl (OH) radicals and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals within meat products, thereby causing oxidative deterioration in proteins and the consequent protein degradation, thereby impairing the quality attributes of meats . As portol group . CorrespThe investigation of meat denaturation temperature, a critical indicator of protein thermal stability during heat processing , was und−1 in instances wherein the CA–DI to MPs ratio was maintained at 1:500 (g/g). From these findings, it is inferred that under this specific ratio, the thermal denaturation of myofibrillar proteins is minimized. Protein oxidation causes the formation of protein cross-links by covalent bonding (hydrogen bonding) within a protein or between proteins [Observations from proteins ,55. Optiproteins . NonetheThe analysis of myofibrillar protein particle size serves as a pivotal indicator elucidating the extent of protein cross-linking consequent to thermal processing, thereby having direct implications on the texture properties of duck meat . The eluConversely, the addition of CA–DI caused a reduction in particle size in comparison to the control, apart from the 1:1000 CA–DI to MPs (g/g) ratio. The particle size of MPs was minimal in samples at a ratio of 1:500  in 12 min heat treatment. This empirical revelation underscores the substantive capacity of CA–DI to effectively counteract protein aggregation dynamics, thereby providing increased control over protein particle size distribution. However, when the CA–DI to MPs ratio surpassed 1:500 (g/g), it caused the enlargement of myofibrillar protein particle size compared to the control group. This tendency is ascribed to the potential accentuation of hydrophobic interactions and the corresponding augmentation of the degree of protein polypeptide chain cross-linking. Drawing a parallel, the observations of Zhang et al. reveal analogous tendencies in the context of curcumin supplementation, highlighting that the measured augmentation of curcumin concentrations (ranging from 40 to 80 μg/mL) engendered heightened stability, while an excessive curcumin dosage precipitated the formation of considerable protein aggregates .Trp and Tyr) conventionally reside within the hydrophobic core of proteins; nevertheless, underpinning the conformational changes arising from denaturation or ligand interactions may expose these residues to the ambient milieu [The evaluation of tertiary structural alterations in heat-processed MPs can be performed using fluorescence spectroscopy to analyze the fluorescence intensity of endogenous amino acids . Figure t milieu ,61. It it milieu .The addition of CA–DI into the heat treatment paradigm has a remarkable influence on protein structure via complex interactions with MPs, thereby affecting physicochemical attributes and altering the functional properties of the protein. Notably, the addition of CA–DI mitigated the formation of carbonyl groups and protected the sulfhydryl groups in MPs of duck meat. In addition, CA–DI reduced the surface hydrophobicity of protein and promoted the free radical scavenging rate during the heat treatment within MPs. DSC analyses showed that CA–DI increased the thermal denaturation temperature while concomitantly enhancing the overall thermal stability of MPs. It has also been found that the combination of CA–DI with endogenous amino acids is critical to maintaining the protein particle size of MPs. However, caution is warranted with regard to excessive CA–DI supplementation, and prolonged thermal exposure may culminate in the erosion of essential functional characteristics of MPs. Consequently, it is important to control the dose of CA–DI during the processing of meat products to inhibit the oxidation of meat products. The proper use of CA-DI could also enhance the thermal stability of duck meat and contribute to the meat quality. The objective of this study was to provide a reference for investigating the effect of capsicum on duck meat protein properties during heat treatment. To further clarify the mechanism, it is necessary to conduct more research into the changes in protein conformation, particularly amino acids, the different types of chemical bonds, and their related characteristics during the heat processing of meat.


Using solid waste to replace limestone filler in asphalt concrete can not only reduce the cost of road construction, but also improve the utilization rate of solid waste. In this study, PHC pile waste concrete (PPWC) was innovatively used to replace limestone filler in asphalt mixture and its effect on the physical and rheological properties of asphalt mastics was studied. Firstly, PPWC was ground into filler particles with a diameter less than 0.075 mm. The physical properties, particle characteristics and chemical composition of PPWC filler and limestone filler were compared. Asphalt mastics were prepared with different filler-asphalt volume ratios  and the physical properties, high-temperature rheological properties and low-temperature cracking resistance of asphalt mastics were tested. The experimental results showed that the surface of PPWC filler is rougher and has lower density and smaller particle size than limestone filler. When the filler content is the same, PPWC filler asphalt mastics have lower penetration and ductility, higher softening point than limestone filler asphalt mastics, and the viscosity of PPWC filler asphalt mastics is more sensitive than limestone filler asphalt mastics. PPWC filler asphalt mastics demonstrated superior high-temperature stability, but poorer low-temperature cracking resistance compared to limestone filler asphalt mastics. In conclusion, PPWC fillers can be used to replace limestone fillers in asphalt mixtures. The finding of this study will provide a new solution for the construction of eco-friendly roads. As a kind of hollow cylindrical precast concrete component, prestressed high-strength concrete pile (PHC pile) is widely employed in the foundations of various construction, including highways and railroads ,2,3. ChiAs the primary material of pavement structure at present, asphalt concrete has the advantages of high load-bearing capacity, excellent skid resistance, and effective noise reduction ,9,10,11.Some low-utilization geotechnical materials have been utilized to prepare asphalt concrete as filler ,33,34. TIn summary, the use of solid waste materials in place of mineral fillers can increase the utilization of waste resources, lower the cost of raw materials, and improve the working properties of asphalt concrete. However, the research on using PPWC as the filler in asphalt mixtures remains limited, and it is still unclear how PPWC filler may affect the rheological performance and physical characteristics of asphalt mastics.This study used PPWC as filler to prepare asphalt mastics to evaluate the feasibility of replacing the limestone filler in asphalt mixture with PPWC filler. Firstly, by using the planetary ball mill, PPWC was crushed into powder with a particle size less than 0.075 mm. Both the PPWC filler and the limestone filler’s chemical composition and particle characteristics were investigated. Then, different volume fractions of limestone filler and PPWC filler were uniformly mixed with matrix asphalt by high-speed shearing apparatus to obtain asphalt mastics. The softening point, penetration, ductility and viscosity of asphalt mastics were tested. Furthermore, using bending beam rheometers (BBR) and dynamic shear rheometers (DSR), the rheological characteristics of asphalt mastics at low and high temperatures were investigated. The study findings will offer a theoretical foundation for using PPWC filler in asphalt concrete.The base asphalt for this study was 70# asphalt produced in Hebei Province, China. The results of the evaluation of its basic properties in line with the Chinese standard JTG E20-2011  are presThe basic properties of PPWC and limestone filler were assessed in accordance with Chinese standard JTG-E42-2005 , and theFirstly, the base asphalt was heated at 160 °C for 40 min, and the two fillers were heated at 120 °C for 60 min to remove excess water present in the fillers. Then, the two fillers were slowly added to the asphalt at 20%, 30% and 40% of the asphalt volume, respectively, and pre-stirred with a glass rod for 10 min. After the filler is initially dispersed, the high-speed shear apparatus was used to shear the asphalt mastics at 150 °C for 40 min at 3000 rpm to guarantee that the filler in the asphalt mastics was evenly and completely dispersed. The particle characteristics of limestone filler and PPWC filler were analyzed by scanning electron microscopy  and laser particle size analyzer . The chemical composition and phase distribution of the two fillers were analyzed by X-ray fluorescence (XRF) test and X-ray diffraction (XRD) test. According to the study of Gautam et al. , a layerThe penetration, softening point, and ductility of asphalt mastics were evaluated in accordance with Chinese standard JTG E20-2011 to evaluate the impact of two fillers on the physical characteristics of asphalt mastics, at 25 °C for the penetration test and 5 °C for the ductility test, respectively. The Brookfield viscosity of asphalt mastics was also tested at five temperatures  to evaluate the machinability of asphalt mastics.Asphalt mastics were subjected to a temperature sweep test by DSR. The high-temperature rheological properties of asphalt mastics were analyzed by complex modulus (G*), phase angle (δ) and rutting factor (G*/sinδ). The temperature range was set to 30 °C–80 °C, the heating rate was set to 2 °C/min, and the frequency was set to 10 rad/s.The frequency range of 0.01–10 Hz was used for the frequency sweep test of asphalt mastics, and the viscoelastic behavior of asphalt mastics was analyzed by G*. All tests were carried out at five temperatures of 46 °C, 52 °C, 58 °C, 64 °C, and 70 °C.The low-temperature crack resistance of asphalt mastics was studied by BBR test. The test temperature was set to −12 °C, −18 °C, and −24 °C. Continuous load was applied to the beam specimen prepared by asphalt mastics at low temperature, and the creep stiffness (S) and m-value (m) were obtained by the creep response of the beam specimen.The SEM images of PPWC filler and limestone filler are shown in 2/g) is higher than that of limestone filler (1.44 m2/g). This may be because PPWC filler not only has smaller average particle size and density than limestone filler, but also has rougher surface than limestone filler.The particle size distribution curves of the two fillers are shown in 2 are the primary oxide in both PPWC filler and limestone filler. Limestone filler is primarily composed of calcium carbonate, resulting in a higher CaO content (56.92%) than that of PPWC (35.10%). In contrast, PPWC filler exhibits a higher SiO2 content (32.4%) compared to limestone filler (18.35%), which may be because PPWC contains aggregates rich in SiO2.2 and CaCO3. However, the XRD curve of PPWC also shows the diffraction peak of Ca(OH)2. According to the study of Han et al. [The XRD curve of PPWC filler and limestone filler are presented in n et al. , this maThe softening point, penetration, and ductility test results of asphalt mastics are shown in η denotes viscosity, A denotes regression coefficient, R is the universal gas constant (R = 8.314 J/mol k), T denotes the absolute temperature, and E denotes flow activation energy. The larger ηE represents the more energy required for the flow of asphalt mastics, indicating that asphalt mastics are more difficult to flow and less sensitive to temperature changes.The Brookfield viscosity test, as one of the characterization methods for asphalt performance, can assess the processability of asphalt mastics and reflect the high-temperature stability of asphalt mastics. According to the research of Gong et al. , the con2) greater than 0.98, indicating that Equation (1) can accurately represent the connection between asphalt mastics’ viscosity and temperature. The results suggest that the incorporation of filler will increase the flow activation energy of the asphalt mastics. When the filler content is the same, asphalt mastics with PPWC filler have a lower ηE than asphalt mastics with limestone filler, which suggests that they are more sensitive to temperature, that is, the viscosity changes more rapidly with temperature.The viscosity–temperature curve can be built using Equation (2) using the viscosity of asphalt mastics at various temperatures, as shown in The ambient temperature has a considerable impact on the rheological characteristics of asphalt. This study used DSR test to examine the rheological characteristics of asphalt mastics at various temperatures. The complex modulus (G*) and phase angle (δ) of the asphalt mastics at high temperatures were measured using the temperature sweep test, as shown in It can be observed from The curves in The rutting coefficient can be used to objectively examine the rutting resistance of asphalt mastics, which is defined as the ratio of G* and sinδ. Asphalt mastics exhibit greater rutting resistance as rutting coefficient increases. According to the standard AASHTO , the G*/It has been reported that the frequency sweep test can be used to investigate the viscoelastic response of asphalt mastics under dynamic load . The resTo assess the low-temperature crack resistance of asphalt mastics, BBR test was conducted. A smaller S and a larger m value under low-temperature conditions show that the asphalt mastics have better adaptation to deformation and stronger crack resistance. The S and m values of asphalt mastics with various filler contents at −12 °C, −18 °C, and −24 °C are shown in (1)By grinding treatment, PPWC can be reused, reducing waste emissions and complying with environmental requirements. This not only reduces the cost of waste disposal but also maximizes resource utilization, thereby lowering overall costs.(2)PPWC, as a solid waste, does not require a mining process, whereas limestone extraction adds additional costs. Therefore, the processing and utilization costs of PPWC are lower, thus reducing the raw material costs.(3)PPWC is typically generated near construction sites. Therefore, using PPWC filler can reduce the transportation distance of raw materials, leading to decreased transportation costs.(4)In some regions, the government has policies and regulations related to waste management and resource utilization. Grinding PPWC for reuse complies with these policies and regulations. This approach might garner government support and incentives, ultimately lowering production costs.Based on the following factors, grinding PPWC into particles smaller than 0.075 μm to replace limestone filler can reduce the production cost of asphalt concrete:(1)3), smaller particle size (12.54 μm) and rougher surface, and its hydrophilicity coefficient (1.77) is higher than limestone filler (1.44).Compared to limestone filler, PPWC filler has a lower density (2.67 g/cm(2)As the filler-to-asphalt volume ratio increased from 0% to 40%, the softening point of the asphalt mastics containing PPWC filler improved by 15.1%, but the penetration and ductility decreased by 38.9% and 47.4%, respectively.(3)In comparison to the asphalt mastics with limestone filler, the performance of the asphalt mastics’ rutting resistance and high-temperature stability can both be greatly improved by PPWC filler. However, the performance of PPWC filler asphalt mastics at low temperatures is less impressive.(4)Considering that PPWC filler will reduce the low-temperature performance of asphalt mastics, it is suggested that the content of PPWC filler should not exceed 30% of asphalt volume.This study aims to evaluate the feasibility of using PPWC filler to replace limestone filler in asphalt mixture. Firstly, limestone and PPWC fillers’ chemical and physical characteristics were examined. Then, asphalt mastics with various filler-to-asphalt volume ratios were prepared using two different types of fillers, and their physical and rheological characteristics were examined. Based on the test results, the main conclusions are as follows:The above conclusions show that PPWC filler can be used to replace limestone filler in asphalt mixtures. Replacing limestone filler with PPWC filler can maximize resource utilization, thereby providing a new approach to the development of eco-friendly road infrastructure. In the ongoing study, PPWC filler will be used to replace limestone filler to prepare asphalt concrete and study its road performance.


The production of bio-based composites with enhanced characteristics constitutes a strategic action to minimize the use of fossil fuel resources. The mechanical performances of these materials are related to the specific properties of their components, as well as to the quality of the interface between the matrix and the fibers. In a previous research study, it was shown that the polarity of the matrix played a key role in the mechanisms of fiber breakage during processing, as well as on the final properties of the composite. However, some key questions remained unanswered, and new investigations were necessary to improve the knowledge of the interactions between a lignocellulosic material and a polar matrix. In this work, for the first time, atomic force microscopy based on force spectroscopy measurements was carried out using functionalized tips to characterize the intermolecular interactions at the single molecule level, taking place between poly(butylene succinate) and four different plant fibers. The efficiency of the tip functionalization was checked out by scanning electron microscopy and energy-dispersive X-ray spectroscopy, whereas the fibers chemistry was characterized by Fourier-transform infrared spectroscopy. Larger interactions at the nanoscale level were found between the matrix and hypolignified fibers compared to lignified ones, as in control experiments on single lignocellulosic polymer films. These results could significantly aid in the design of the most appropriate composite composition depending on its final use. The production of bio-based composites with enhanced characteristics has gained attention since governments make strong efforts to minimize the scarce fossil fuels being consumed . In thisAmong plant fibers, bast fibers are thick-wall sclerenchyma cells located in the bark tissue of plants such as hemp, flax, jute, or kenaf. Despite some common morphological traits, chemical composition shows large variations depending on the developmental stage and botanical origin ,18,19. FIn the present study, we hypothesized that variations in the surface chemistry of the fibers impact on their adhesion properties with composite matrix. AFM tips were functionalized with the PBS matrix by chemical coupling prior to the quantitative characterization of intermolecular adhesion forces with lignocellulosic polymer films and plant fibers longitudinal surfaces at the nanoscale. This comparison performed both on single lignocellulosic polymer films and on fibers with various lignin contents confirms that PBS exhibits the best adhesion on hypolignified materials.−1), or CNCs (18 g L−1) were cast on Petri dish (43 cm2) and dried at ambient air for 24 h. Films thickness was measured using a dial thickness gauge , and the mean value was approximately 30 µm ± 2 µm for each film. Lignin-coated films were prepared from organosolv lignin solution  at 5 g L−1 in aqueous dioxane , and 30 μL of this solution was spin-coated onto silicon wafers (area of 1 cm2), using a commercial spin-coater  at 4000 rpm, as detailed in [Lignocellulosic films were obtained as a single-polymer system, using glucomannan , xylan (XYL) extracted from oat spelt , celluloailed in . Prior tPlant fibers  were provided by Fibres Recherche Développement . These fibers were stored in room conditions prior the AFM-FS measurements.−1. Each record corresponds to an average of 32 scans with a spectral resolution of 4 cm−1. The baseline of the spectra was corrected by using OMNIC software and normalized by applying a correction factor 1000/(A1800–780 cm−1), where A1800–780 cm−1 is the area of the spectra between 1800 and 780 cm−1.Plant fibers and lignocellulosic polymer films were analyzed by mid-infrared spectroscopy, using a Nicolet 6700 Thermo Electron FTIR spectrometer in attenuated total reflection (ATR-IR) mode equipped with diamond crystal that allows for the surface analysis of sample through a 2 µm effective depth. For each type of sample, 4 records were performed in the range of 1800–780 cm−1 and nominal frequency of 65 kHz) were initially washed in chloroform and dried with a nitrogen flux. Then 3-aminopropyltriethoxysilane (APTES) and Hunnig’s base (N-diisopropylamine)  were added in a ratio of 3:1 in volume for the amino functionalization of the AFM tip (−1) for 2 h at room temperature inside a polytetrafluoroethylene (PTFE) beaker . The amine groups from the AFM tip reacted to the maleimide end from the linker molecule, forming stable amide bonds  setup. EDXS was used to identify the elemental composition of the AFM tip apex after the functionalization procedure with PBS.The efficiency of the AFM tip functionalization with PBS was checked by scanning electron microscopy (SEM), using a JEOL  JSM-7900F setup. SEM images were recorded from the secondary electrons at primary beam energies of 1.5 keV. SEM setup was coupled with UltiMax 170 mm−1, a nominal frequency of 70 kHz, and a tip radius of 2 nm were used to acquire topography information necessary to calculate the root mean square average (Rq) roughness parameter of the lignocellulosic films and the plant fibers, as indicated elsewhere [The topography of lignocellulosic films  and plant fibers was acquired by PeakForce tapping mode in air to check the integrity and the homogeneity before spectroscopy force experiments. AFM imaging was carried out at 25 °C and 45 ± 0.3% relative humidity (RH), using a Multimode-8 AFM setup , which was coupled with a Wetsys system RH generator . The value of the relative humidity was monitored during image acquisition by a Tinytag device . ScanAsyst Air probes  with a nominal spring constant of 0.4 N mlsewhere . At leas−1 as the load rate, 500 nm as ramp size, 0 s as the retract delay, and 5 μm × 5 μm as the scan size for all samples, with the exception of CNCs film, where the scan size was defined as 1 μm × 1 μm. Then 48 ramps per line (2304 force curves) and 60 ramps per line (3600 force curves) were acquired for the studied single lignocellulosic polymer films before applying this methodology on the longitudinal surfaces of plant fibers. The topography of the functionalized AFM tips with PBS was recorded before and after the F−V acquisition to ensure proper PBS attachment. Histograms from the adhesion data were fitted by a Gaussian distribution. Histogram bin size was defined at 0.5 nN for all force distributions.The intermolecular forces between the functionalized tips with PBS and lignocellulosic materials (films and plant fibers) were investigated using the AFM equipment, as described in the previous section, but operating with force−volume (F−V) mode in air. This mode measures the real-time mapping of local areas, which allows for the quantification of adhesion forces extracted from force–distance curves, which were analyzed as indicated elsewhere . Longitup < 0.05).One-way analysis of variance (ANOVA) calculations were used for the adhesion forces recorded between PBS functionalized tips with respect to the studied lignocellulosic films and the plant fibers. Additionally, to ascertain the significantly differences of the groups, a Turkey–Kramer post hoc test was carried out in combination with the r-Pearson test. Significant differences were assumed with a significance level greater than 95% (−1 (corresponding to acetyl group (C=O) for hemicellulose) and at 1160 and 1100 cm−1 corresponding to the glycosidic bonds (C-O-C) and secondary alcoholgroups (C-O stretching), respectively, for cellulose. The main differences between lignin and polysaccharidic films were observed at 1515 cm−1  and at 1231 cm−1, corresponding to the C-O stretching in aromatic rings and in xylan [−1, 1506 cm−1, and 1231 cm−1 between (flax and hemp) and (kenaf and jute) fibers. No significant band characteristic of lignin (1506 cm−1) was found for flax and hemp (hypolignified) fibers in contrast to jute and kenaf. Likewise, the band intensity at 1231 cm−1 for jute and kenaf was approximately twice that of flax and hemp. Regarding the low intensity of polysaccharide regions, albeit the high intensity for cellulose band (1100 cm−1), other differences in intensity of bands were related to non-cellulosic polysaccharides (1730 cm−1), as jute and kenaf contain more hemicelluloses (as mainly xylan) than flax or hemp. In consequence, the calculated lignin/cellulose intensity ratio, 1506/1100, varied from 0.01 to 0.03 (flax and hemp) and from 0.14 to 0.22 (jute and kenaf), and the ratio 1231/1100 corresponding to aromatic and hemicelluloses bands increased in the following order: 0.18 (flax), 0.31 (hemp), 0.56 (jute), and 0.60 (kenaf)  show large variations in intensity at specific bands according to their surface composition . Betweenin xylan . For the (kenaf) . These r−1, i.e., 71.4%; 3 O = 48 g moL−1, i.e., 28.6%) [3N4 bare tips. As it was shown that the beam penetration depth on gold surfaces ranged from 20 nm at 2.0 keV to 3 nm at 0.5 keV, respectively [The efficiency of AFM tip functionalization with PBS was checked by SEM and EDXS after the functionalization procedure. Looking at the SEM image, it seems that the procedure leads to the coating of the tips with a homogeneous layer a. To be , 28.6%)  and confectively , we coulDifferent single-polymer films  were characterized. The AFM images were acquired in several areas of each film to ensure the homogeneity of the exposed external surface. The mean values of the height and roughness were analogous to those of previous works, 6.2 ± 0.2 nm, 9.1 ± 0.7 nm, 23.6 ± 1.8 nm, and 10.0 ± 0.5 nm, respectively . Then foThus, the adhesion forces are nearly three times lower when the PBS interacts with lignified surfaces. This finding is not unexpected since lignin and PBS molecules present a large amount of hydrophobicity due to their phenolic groups  and the The aim of this set of experiments is to gather further insights into the extension of the forces involved in the binding of PBS functionalized tip with single lignocellulosic polymer films. This information will significantly aid in our understanding of the interactions taking place between the same PBS tip and the longitudinal surfaces of native blast fibers. The upcoming subsection concerns F–V experiments with PBS tips on the four tested bast fibers.The interaction forces of four different bast fibers  were measured by AFM-FS, using PBS functionalized tip . The zenRepresentative force curves are shown in For the different fibers, the data are in good agreement with the results obtained with single lignocellulosic polymer films. Such variation between lignified and hypolignified fibers can be explained by the polarity of PBS. The nature of this material is partially hydrophilic after its polymerization synthesis process. Indeed, PBS polymer chain extension is formed by the polycondensation reactions of the direct esterification of a dicarboxylic acid and a diol, which are very polar components. For this reason, a polar material such as PBS can establish strong interactions with other high-polar surfaces, such as hypolignified fibers (such as flax and hemp), which mostly contain cellulose and non-cellulosic polysaccharides (pectins and hemicelluloses), as shown by ATR-IR .These results make a significant contribution to improving our understanding of how the PBS matrix interacts with different plant fillers. Playing with the plant fiber sources and their lignin content, it may be possible to design composites with their mechanical performance tuned to the intended use. Some previous efforts have been made in this field, such as the continuous attempt of improving the interfacial adhesion properties between PBS and lignin , which mThe adhesion properties between a polar matrix (PBS) and four relevant bast fibers  were addressed for the first time at the single-molecule level. For it, AFM-FS measurements were carried out with coated PBS tips where the apex contacts the longitudinal external portion of the fibers, thus recording the force interaction between these surfaces under controlled conditions. The obtained results showed that PBS has higher adhesion forces with hypolignified fibers (flax and hemp) than with lignified ones (jute and kenaf). These findings were corroborated by AFM-FS control experiments on single lignocellulosic polymer films  and ATR-IR measurements of the surface chemical composition. Whereas cellulose and hemicelluloses, which mainly consist of hydroxyl groups, can establish more affinity with polar materials such as PBS, lignin is more hydrophobic than polysaccharides, thus significantly limiting the extension of bond formation with the matrix of interest. This fact evidences the strong impact of surface chemistry on matrix–filler adhesion properties.Thus, future research on the adhesive properties between polymer matrices such as PLA, PHA, or PBAT and natural plant fibers could be extended by using this methodology. This will provide a more comprehensive view of the interfacial forces exerted between the polymer matrix and green-sourced biobased materials.