Conventional classification of hand motions and continuous joint angle estimation based on sEMG have been widely studied in recent years. The classification task focuses on discrete motion recognition and shows poor real-time performance, while continuous joint angle estimation evaluates the real-time joint angles by the continuity of the limb. Few researchers have investigated continuous hand action prediction based on hand motion continuity. In our study, we propose the key state transition as a condition for continuous hand action prediction and simulate the prediction process using a sliding window with long-term memory. Firstly, the key state modeled by GMM-HMMs is set as the condition. Then, the sliding window is used to dynamically look for the key state transition. The prediction results are given while finding the key state transition. To extend continuous multigesture action prediction, we use model pruning to improve reusability. Eight subjects participated in the experiment, and the results show that the average accuracy of continuous two-hand actions is 97% with a 70 ms time delay, which is better than LSTM  and GRU . In supplementary experiments with continuous four-hand actions, over 85% prediction accuracy is achieved with an average time delay of 90 ms. The surface electromyography (sEMG) signals, as electrical signals reflecting neuromuscular function, can produce 30–150 ms earlier than the movement of hand action ,3,4. TheTwo main aspects of applications have been implemented: discrete motion classification and continuous joint motion estimation . DiscretRelevant works on motion classification emphasize using appropriate features and classifiers to enhance recognition accuracy. Narayan et al.  used a dAlthough discrete motion classification is widely investigated in the field of sEMG-based pattern recognition, it is still hard to satisfy the real-time requirements efficiently. One reason is that it does not consider the factors which affect realistic scenarios, such as the discontinuity of the discrete motion. Generally, human-hand action is a continuous process for practical applications, and continuous motion estimation is more applicable for hand action recognition.Currently, continuous joint motion estimation is the primary research direction in continuous motion estimation, which realizes intention recognition by establishing a mapping relationship between sEMG signals and a continuous variable such as joint angle or joint torque. Motion estimation plays an essential role in stable control for robots ,23,24,25These methods for continuous joint motion estimation only consider the continuous motion process of the upper and lower limbs. Similar to the upper and lower limb movements, hand actions are also continuous, and it is necessary to explore the continuous process mechanism of hand actions. Furthermore, the accurate prediction of continuous hand actions based on this process mechanism can recognize the subsequent gesture in advance, which is conducive to the coherent execution of continuous hand actions.In our study, a method for sEMG-based continuous hand action prediction is proposed. The overview of the motion prediction is described in A new supplement to the research of sEMG-based motion intention recognition.A modified Viterbi algorithm of GMM-HMMs which can build long-term memory for the prediction process.A Model pruning which can expand the number of participating hand gestures for continuous multihand action prediction.The main contributions of this paper are as follows:A is the transition probability matrix, B is the observation probability matrix, and s = [N is the number of hidden states. In our work, N = 6, and A = i at time t to state j at time t + 1. B = i generates t, where t. The initial state distribution, i at the start time point. The states of HMMs were modeled by a Gaussian mixture model.Different from discrete motion classification, the prediction of continuous hand actions needs more information on time dependence. The hidden Markov model is applicable for time series modeling . By assuThe results should be achieved before hand actions are completed for the ideal prediction of continuous hand actions. Typically, a robust and reliable judging condition for prediction is essential. Continuous two-hand actions were divided into six states, and the fourth state, which played a vital role in linking the front and rear gestures, could be used as a key state transition for prediction. During continuous hand actions, the period of hand transition is much shorter than that of hand stabilization, and the overall real-time performance can be improved if the correct prediction results can be obtained during the period of hand transition. Relevant research has shown that prediction results can be obtained during hand transition . It indii at time t; t; and N denotes the number of states.After providing the judging condition for continuous hand action prediction, RMS values were sequentially input into the trained models, and then the path of the key state transition could be backtracked by maximum state probability . Many faTo derive the global state path, the state recursion was expressed as follows: With the description of the Viterbi algorithm, we provide a modified Viterbi algorithm to realize the marginalization; the specific marginalization based on the sliding windows was expressed as: The maximum probability of the state in the first sliding window was calculated as: The maximum probability of the state in the subsequent sliding windows was modified as: t is equal to 1, the last state probability distribution of the previous sliding windows was used to initialize the subsequent sliding windows.When i at time t; j to state i; T is the length of the sliding window.The following formula could backtrack the path of the state transition: Generally, the marginalization is based on the inheritance of constraints on the state probability distribution of previous sliding windows. By utilizing The number of models is proportional to that of hand gestures in GMM-HMMs-based gesture recognition . For reaAfter constructing a set of GMM-HMMs on continuous two-hand actions, we could find the most probable model by using the maximum likelihood criterion with the distribution of the observation sequence . During Firstly, RMS values in the sliding window were input into multiple models, and then the state path with the maximum probability could be derived. It was noted that the maximum likelihood varied cumulatively using the marginalization of sliding windows. With the movement of the sliding window, the maximum likelihood would vary based on the degree of model-fitting. We searched for the key state transition in the model which had the maximum likelihood.Secondly, the next hand action Finally, the key state, which was the fourth state of the Algorithm 1: Model PruningThe datasets from previous works ,41,42 onEight right-handed subjects (aged 24 to 30) participated in the experiment. Four distinct hand gestures were chosen to form twelve types of continuous two-hand actions, and each type of continuous two-hand action was formed by the continuous action of two different hand gestures. The GForcePro+ armband system was used to record sEMG signals. Every continuous two-hand action was performed 100 times per person. Every continuous two-hand action lasted about 5 s, of which the front and rear gestures each lasted 2.5 s. In order to reduce the negative impact on muscle fatigue, participants were given a 5 s rest between every two repetitions. Besides, participants had to relax for 5 min after completing one continuous two-hand action with twenty repetitions. The recorded sEMG signals were collected at a frequency of 1000 Hz and analyzed using MATLAB 2020b. The configuration of the dataset is shown in Collected sEMG signals were preprocessed using a bandpass filter (20 Hz–150 Hz) to remove unusable noise and a notch filter to remove 50 Hz power disturbances. An energy-based active segmentation algorithm was used to segment sEMG signals for accurate data acquisition . On the In the experiment, a six-state fully connected left-to-right structure HMM was used. According to the setting of continuous two-hand actions, six states were divided into two parts, while each of them contained three states: transitive state (rising), steady state, and transitive state (declining). Since all the hand actions started from state one, the initial state probability of state one was set to 1, and those of other states were uniformly set to 0. We adopted a single-component GMM to represent the substate of HMMs, as Yang et al.  indicateIn order to validate the feasibility of the setting on the key state transition, the data before states 4 and 5 were split for prediction. A sliding window with a length of 20 frames and an increment of 20 frames was used. As part of continuous multi-gesture actions, continuous four-hand actions were selected as being representative for prediction. Since continuous four-hand actions had plenty of combinations, we chose four different types for the experiment. For continuous four-hand actions, we still used RMS values as the input of the models. It was noted that the acquisition time needed to be limited to 10 s. We collected 30 samples for each type of continuous four-hand actions to predict.The proposed method was used to predict continuous hand actions in advance; the special partitioning and modeling of continuous states made it better than other methods. To show the robustness of our method, we used conventional LSTM and GRU models, which have been widely utilized in discrete gesture prediction for comparison. In order to ensure the uniformity of the comparison, we also used the sliding window strategy on LSTM and GRU to simulate the prediction process. A sliding window with a length of 20 frames and an increment of 20 frames was used.We investigated the effect of different sliding window settings on the prediction results. The normalized confusion matrix of our method is shown in Continuous four-hand action prediction was tested to evaluate the performance of model pruning. The prediction results could also validate the feasibility of continuous multigesture action prediction. Since there were many combinations of continuous four-hand actions, we selected four types of them which are listed as follows: (1): making a fist–fingers spread–wrist flexion–making a fist; (2): wrist flexion–wrist extension–making a fist–fingers spread; (3): fingers spread–making a fist–wrist flexion–wrist extension; (4): wrist extension–fingers spread–making a fist–wrist flexion.The prediction accuracy of continuous four-hand actions indicated that model pruning could apply to our work. Notably, as a part of continuous multigesture action prediction, the excellent prediction accuracy of continuous four-hand actions further confirmed the feasibility of continuous multigesture action prediction with our method.Currently, our study focuses on predicting the combinations of four distinctive hand gestures. The prediction task of the combinations of fine-hand actions has not been carried out yet. Moreover, subject-independent problems still exist in our method, and every subject must be modeled separately in the experiment. Our future work will concentrate on predicting continuous fine-hand actions and providing a robust universal model which can solve subject-independent problems.In this paper, we proposed a continuous hand action prediction method based on the continuity of hand actions. In order to predict the subsequent hand gesture in advance, our method set the key state transition as the prediction condition. The GMM-HMMs model was utilized to model the states of continuous two-hand actions, and state four was set as the key state transition. The marginalization of sliding windows was used to build long-term memory for windows, and a dynamic sliding window was used to simulate the prediction process. Compared with LSTM and GRU models, our method achieved a higher prediction accuracy and a lower processing time for continuous two-hand actions. In the extended task of continuous multigesture action prediction, the GMM-HMMs models built by continuous two-hand actions could not predict continuous hand actions with more than two hand gestures. We proposed a model-pruning method which selected the best-fitting models and updated the starting point of state probability accumulation to overcome the limitation. The prediction results also confirmed the feasibility of the method on continuous multigesture action prediction.


The effectiveness of protective coatings based on 3-propyltriethoxysilane (OFTES) in protecting concrete surfaces against water was tested. For the synthesis of OFTES, 2,2,3,3,4,4,5,5-octafluoropentanol, which is a by-product in the synthesis of poly(tetrafluoroethylene), was used. The proposed silane is a cheaper alternative to the fluorinated organosilicon compounds currently used. The coatings were deposited by the sol-gel method. As a result of the creation of chemical bonds between the concrete surface and the silane, a coating was created that permanently increases the hydrophobicity of the concrete. Fluorine chains attached to silicon atoms are an effective barrier that prevents access to water and limits its impact on the concrete surface. As a result of the proposed silanization, the concrete surface obtained a hydrophobic character at contact angles of up to 126°, and the water absorption of the concrete decreased by up to 96%. Reinforced concrete is a strong and hard material widely used in modern construction. Moisture is the determining factor in most of the processes that damage concrete structures. As many aggressive substances enter the interior of concrete structures along with water, the absorption of surface water by concrete is an important factor determining the stability of concrete structures ,2,3,4. IOne of the groups of compounds most commonly used in hydrophobic preparations is alkoxysilanes . OrganofThe hydrophobic nature of the coating is due to the presence of alkyl groups ,11,12,132. The triethoxyoctylsilane without any solvent was applied by Zhan et al. [2). Christodoulou et al. [Dai et al.  used a tn et al.  to proteu et al.  presenteFluorine-containing organosilicon derivatives have recently attracted a great deal of attention because of their possible use in the production of modern materials. Fluoroalkylsilanes used in the production of surfaces resistant to oil, dirt, and water are particularly attractive ,22,23,242 particles were applied to the concrete surface and then functionalized with fluorinated alkoxysilane. The authors focused on the self-cleaning properties of the obtained coatings, without examining the barrier effects. Unfortunately, limited access to substrates and complicated synthesis limit the use of fluoroalkylsilanes. In the case of the modification of building materials, various fluorinated derivatives are used mainly for the preparation of superhydrophobic protective coatings. Husni et al.  used 1H,As mentioned before, despite the excellent effects that can be obtained by surface modification with fluorofunctional organosilicon compounds, their use is not very widespread. This is mainly due to the high price and poor availability of raw materials, as well as the complex and difficult technology of their synthesis. Our experience allowed us to design and synthesize fluorinated derivatives of organofunctional silanes . Therefo2(CF2)3CH2O(CH2)3Si(OCH2CH3)3 was synthesized according to the procedure described in the literature [The chemicals were obtained from Sigma-Aldrich  and used without any additional preparation. Concrete samples were made of Portland type 32.5 concrete  according to EN 197-1 , gravel terature . The Sil3 coarse aggregate (gravel), 595 kg/m3 fine aggregate (sand), 380 kg/m3 cement, and 190 kg/m3 water. The free water-to-cement ratio (w/c) was set at 0.5. Fresh concrete was poured and vibrated in the form of 100 × 100 × 100 mm3 cubes. The cubic samples were removed from the mold after 24 h of curing and then placed in an oven  ≥ 90%) for 28 days.Concrete samples were made from a mixture containing 1295 kg/mThe cured concrete samples were washed with water to remove all loose parts and dried for 5 days, after which the samples were silanized. A series of alcoholic OFTES and tetraethoxysilane (TEOS) solutions were prepared. The prepared solutions were added to ethanol with 0.5 mL of concentrated hydrochloric acid (pH ≈ 2) and 5 mL of water, and then, while vigorously stirring, the appropriate amounts of silanes (OFTES and TEOS) were added to the solutions. The amount of ethanol was chosen to obtain 500 mL of solution each time. The amounts of silanes added to the solutions are shown in The solutions were mixed for 1 h, after which time the concrete samples were completely immersed in the solutions for 1 h. After removal from the solution, the samples were dried at room temperature for 24 h.2).The consumption of the silane solution was calculated from the weight loss of the OFTES solution (g) by the silanized surface area  with an ammonium hydroxide solution. A concrete sample was placed in the solution immediately after being neutralized. The previous procedure was then followed.Scanning electron microscopy (SEM)  images were taken on an FEI Quanta 250 FEG microscope equipped with an EDAX Energy Dispersive Spectroscopy detector (EDS) . The images were taken in high vacuum mode with 10 kV accelerating voltage. EDS mapping was performed with an electron beam energy of 20 keV using the EDS Octane SDD detector (EDAX). The SEM images were recorded for concrete samples cut to the size of a cube of 10 × 10 × 10 mm and subjected to silanization. The concrete samples were prepared for SEM imaging by gluing them onto standard SEM carbon adhesive tape. Static water contact angle (WCA) measurements  on all samples were made using a Krüss GmbH DSA 100 Expert Drop Shape Analyzer equipped with a software-controlled (DAS4 2.0): x, y, z-axis table, a quadruple-dosing unit with zoom and focus adjustment, illumination, and a camera with 780 × 580 px resolution. All the data presented are arithmetic means of measurements made for 5 drops per sample. The measurements of contact angles were performed immediately after the deposition of a drop on a studied surface. In an additional experiment on the surface of the concrete sample, a drop of water containing a dye was placed to observe the hydrophobic character of the surface; the drop containing a 0.1% solution of methyl orange had a volume of 20 µL.m is the mass variation before and after submersion (kg); A is the surface area of the sample (m2).To determine the water absorption of concrete cubes, a liquid water permeability test was used. The test was carried out according to the EN 1062-3 standard . Before The penetration depth of the silanization solution was determined according to the EN-1504-2 standard [29Si MNR analysis, it was found that after 1 h, total hydrolyzed silane and small amounts of partially condensed siloxanes are present in the system [Fluorocarbosilane (OFTES) has been used to create protective coatings that reduce the penetration of water into concrete. These coatings were made on the basis of the sol-gel process. As a result of the hydrolysis and condensation reactions taking place in the system, chemically bonded to the hydroxyl groups present on the concrete surface, a siloxane coating was obtained. According to current knowledge , silanese system . In the 2, calculated for the OPTES solutions.The protective coatings were obtained by immersing concrete samples in silanizing solutions. The solutions used can be divided into two groups: solutions containing only one silane (OFTES) and solutions prepared with the addition of tetraethoxysilane (TEOS). A series of solutions containing 1 to 5% of the discussed silanes was prepared. The prepared solutions were additionally acidified with hydrochloric acid to accelerate the hydrolysis reaction. Before the concrete samples were immersed, the solutions were mixed for 1 h. This time was selected on the basis of previous studies of sol-gel processes that take place in the presence of OFTES ,34. The These observations are also confirmed by the EDS mapping images of silicon and fluorine atoms presented in The EDS mapping images of the silicon and fluorine atoms also confirm the dominant role of these elements on the surface of concrete subjected to silanization with solutions containing the TEOS additive. The surface of sample F5_5 silanized in a solution containing 5% OFTES and 5% TEOS is characterized by the highest content of fluorine b. Howeve2 to 20.42 g/m2) reduced water absorption by almost 96% compared to unmodified concrete. As expected, it was observed that the barrier properties increased with increasing OFTES concentration. The addition of TEOS does not improve the barrier properties of the coatings. In systems containing identical amounts of OFTES, higher water absorption was obtained for systems containing TEOS. It is more advantageous to use systems that contain only OFTES.The penetration depth of silane (silanizing solution) into the concrete is presented in As mentioned above, the formation of KCl crystals on the surface of silanized concrete is due to the presence of relatively large amounts of HCl in the solution. Concrete silanization with a neutral 5% OFTES solution was also carried out. After 1 h of stirring, the acidic solution was neutralized with ammonium hydroxide solution to pH ≈ 7. A concrete sample was immediately introduced into the neutralized solution, followed by the original procedure. The SEM and EDS images of the sample containing 5% OFTES after neutralization (F5*) are shown in The stability of the obtained coatings was tested on the basis of immersing and drying concrete samples. Five cycles of immersing and drying were performed. The samples were first immersed in water for 24 h and then dried for 5 days. In addition, studies were also carried out with the commercially available Silicone L6 in order to compare the results. The results are summarized in The effectiveness of OFTES-based protective coatings in protecting concrete surfaces against water was tested. As a result of the sol-gel process, chemical bonds between the concrete surface and the silane are created; the obtained coatings increase the hydrophobicity of concrete. As a result of the silanization, the water absorption of concrete decreased by up to 96%. Only a small amount of OFTES (2.5 and 5%) reduced the water absorption of concrete by more than 90%. It was also shown that OFTES creates stable coatings and the water absorption did not change in the subsequent cycles of the immersion and drying of concrete samples. It has been shown that the neutralization of silanizing solutions has a negative effect on the produced coatings.


Antibiotics guard us against bacterial infections and are among the most commonly used medicines. The immediate consequence of their large-scale production and prescription is the development of antibiotic resistance. Therefore, rapid detection of antibiotic susceptibility is required for efficient antimicrobial therapy. One of the promising methods for rapid antibiotic susceptibility testing is Raman spectroscopy. Raman spectroscopy combines fast and contactless acquisition of spectra with good selectivity towards bacterial cells. The antibiotic-induced changes in bacterial cell physiology are detected as distinct features in Raman spectra and can be associated with antibiotic susceptibility. Therefore, the Raman-based approach may be beneficial in designing therapy against multidrug-resistant infections. The surface-enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy (RRS) additionally provide excellent sensitivity. In this review, we present an analysis of the Raman spectroscopy–based optical biosensing approaches aimed at antibiotic susceptibility testing. Antibiotics remain the most essential drugs in the treatment of bacterial infections . RecentlSurface-enhanced Raman spectroscopy (SERS) is a powerful analytical method having single-molecule sensitivity ,11. As aThe practical applications of SERS include detection of various trace chemicals, such as pesticides ,20, drugIn this review, we present an analysis of the Raman spectroscopy–based optical biosensing approaches aimed at antibiotic susceptibility testing and prospective antibiotic detection.Staphylococci have different susceptibility to available antibiotics [Antibiotic susceptibility studies have been published since 1945, when Neter found that various strains of ibiotics . The numActive bacterial cells differ from resting cells by certain features in their Raman spectra, as was noted by Webb and Stoneham in 1977 . ResonanAntibiotics are strikingly efficient against bacteria because of the significant differences in the biochemistry of prokaryotic and eukaryotic cells. The common biochemical targets of antibiotics are shown in Bacteria develop resistance to antibiotics due to spontaneous genetic mutations. The key process involved is the horizontal gene transfer, i.e., transfer of mobile genetic elements, such as integrons and plasmids, that contain antibiotic-resistant genes . This prSeveral important antibiotic classes disrupt the microbial cell wall biosynthesis. Beta-lactam antibiotics  have structural similarity to the D-Ala-D-Ala fragment of the peptidoglycan building blocks. Highly reactive carbonyl in the β-lactam system ensures the acetylation of the serine residue in the active center of transpeptidases involved in the final steps of the peptidoglycan synthesis . The widThe resistance to β-lactams drove the development of glycopeptide antibiotics (such as vancomycin) that bind to the terminal D-Ala-D-Ala fragment and thus disrupt the proper synthesis of peptidoglycans . ResistaSeveral polypeptide antibiotics (such as polymyxins and gramicidins), being structurally similar to glycopeptides, target bacterial membranes. As one may expect, these antibiotics are less selective than their counterparts targeting cell wall biosynthesis, and have considerable toxicity ,56. GramThe lipopeptide antibiotic daptomycin has attracted much attention due to its unusual mode of action. As shown by combined proteomic and fluorescent lipid probe studies, daptomycin affects bacterial membrane fluidity, consequently blocking cell wall synthesis and altering phospholipid synthesis, and thus affecting respiration, membrane potential, and cell division . This inProkaryotic nucleic acids are another obvious target for antibiotics. The topology of circular bacterial DNA is controlled by the crucial type II topoisomerases: gyrase and topoisomerase IV. Quinolones, synthetic antibiotics introduced in clinical practice about five decades ago, exploit these enzymes for targeting the bacterial DNA, forming non-covalent complexes at the enzyme–DNA interface and converting type II topoisomerases into cellular poisons . ResistaBacterial protein synthesis machinery differs from its eukaryotic counterpart, making it a target for several types of drugs. Aminoglycoside antibiotics bind into or close to the A-site in the 16S rRNA , causing misreading of mRNA, leading to synthesis of faulty proteins . The priThe functioning of bacterial ribosomes can also be affected by the macrolides—small molecules inducing dissociation of the peptidyl-tRNA complex from ribosome and the premature interruption of the protein synthesis . The resTetracyclines bind to RNA, including bacterial rRNA. Although the precise mechanism of action remains elusive, the broad activity spectrum and correlation of 16S rRNA mutations with tetracycline resistance suggest the direct binding to 16S rRNA . Recent E. coli SERS spectrum is shown in Inelastic light scattering, occurring as a result of the interaction of photons with vibrations of atoms or molecules, was predicted by Smekal  and thenThe key question of using Raman spectroscopy is whether there are significant differences between the spectra of susceptible/resistant bacteria treated/untreated with antibiotics. In other words, are there any features in Raman spectra that can be exploited as biomarker signals to identify the effect of antibiotics on bacteria?The Raman spectra of bacteria are strongly affected by laser wavelength, bacterial species, growth phase of bacteria, substrate, etc., and the interpretation of spectra is a challenging task. The proliferation state of bacteria and its susceptibility to antibiotics can be identified by observing dynamic changes occurring in the SERS spectra of living bacteria. In addition, the effect of antibiotics and/or antibiotic susceptibility can be recognized by the altered spectra of metabolites secreted by bacteria. Therefore, there are two approaches to study the antibiotic effect on bacteria—direct investigation of bacterial cells (either in suspension or on the solid support) and detection of secreted metabolites  .S. aureus, E. coli, P. aeruginosa, S. pneumonia, etc., can be effectively detected by Raman spectra [−1 [Raman shift)  . The difE. coli were revealed after 20 min of exposure to ampicillin, indicating the inhibition of microbial proliferation. When S. aureus was treated with cell wall targeting antibiotics such as ampicillin, vancomycin, and cefotaxime, the spectral changes were observed within an hour. Noticeably, the SERS spectrum of S. aureus after oxacillin treatment was temporarily restored, and the authors consider that the effect of the cell wall targeting antibiotic is compensated for by the thick layer of peptidoglycan found in Gram-positive bacteria. When treated with antibiotics that inhibit protein biosynthesis like gentamicin and tetracycline, the discernible SERS changes occurred only after 9–12 h [The obvious changes in SERS spectra of r 9–12 h .P. aeruginosa decrease, while the intensity of nucleic acid bands increases [−1, apparently, because aminoglycosides inhibit protein biosynthesis.Features in the spectra of amikacin-treated bacteria show that as the concentration of amikacin increases, the protein-related peaks of ncreases . SeveralE. coli via antibiotic concentration achieves the maximum, with the maximum intensity being achieved at the minimum inhibitory concentration (MIC), while the Raman signal of antibiotic resistant strain slightly depends on the antibiotic concentration [The SERS signal of sensitive bacteria responds more dramatically to antibiotic treatment than that of their resistant counterparts ,94. The ntration .E. coli O157:H7 susceptibility to ampicillin, were studied in [Several SERS-based approaches, including the conventional drop method, extracellular matrix analysis, and filter mapping for investigating udied in . Bacteri−1 at MIC and 2×MIC concentrations were lower than that of untreated bacteria; in contrast, the highest intensity was observed at subMIC concentrations. This behavior was characteristic of E. coli treated with amikacin, ciprofloxacin, polymyxin, and tigecycline and S. aureus treated with ciprofloxacin, chloramphenicol, erythromycin, and vancomycin. Raman data were consistent with the standard medium method. The authors supposed that antibiotics at subMIC concentrations promoted bacteria reproduction, as revealed in the Raman spectra [Different concentrations of antibiotics influence the Raman spectra of bacteria treated with the antibiotics. The intensity of Raman spectra peaks at 735 cm spectra .S. aureus resistance to oxacillin/cefazolin was detected based on the changes in I734/I867, I1163/I959 and I1372/I1349 ratios [734/I867 showed a negative correlation with MIC values, while I1163/I959 and I1372/I1349 were increased with MIC values. When studying the development of S. typhimurium resistivity, the I990/I1348, I1165/I1205, and I958/I1017 ratios were monitored in dynamics [SERS also allows monitoring the development of bacterial resistance to the antibiotics by examining the changes in the signal intensity ratios characterizing molecular targets in bacteria. dynamics . In bothAs antibiotics are often employed to fight life-threatening infections, emerging antibiotic resistance demands rapid testing in the clinical conditions. Surface-enhanced Raman spectroscopy provides high sensitivity and robustness to the antibiotic susceptibility assays. Studies on antibiotic susceptibility testing are listed in Raman spectroscopy enables contactless and very fast (often in the range of seconds) signal acquisition, with a sensitivity down to single-cell level. With the appropriate signal-processing framework, this approach offers express study of the antibiotic–microbe interaction. We discuss below various the Raman-based techniques suitable for antibiotic susceptibility testing.E. coli cells in the presence or absence of cefazolin, penicillin, and streptomycin. Unexposed and treated E. coli cells form separate clusters, with minimal overlap between groups. Antibiotic-sensitive and -resistant strains of S. mutans were recognized as fast as in 30 min [The ability to distinguish individual cells by their spectra is essential for the development of clinical diagnostic tools, as it allows rapidly detecting minor subpopulations in a heterogeneous population of cells. Laser-tweezer Raman spectroscopy combined with principal component analysis (PCA) has made it possible to identify various metabolic states of bacterial cells ,106,107.n 30 min .P. aeruginosa and agrees well with its presumed mode of action [It was shown that UV resonance Raman spectroscopy can be used as a reliable tool for quantitative assay of the amikacin action against f action . The cluE. faecalis were revealed this way. The MICs of vancomycin against E. faecalis, E. coli, K. pneumoniae, and S. aureus were determined and were two times higher than the MIC values obtained by the conventional cultured-based method. This discrepancy may be caused by the too-short incubation time (0.5 h) used in metabolic imaging. Mismatch in the inhibition of bacterial growth and inhibition of the metabolic activity could be another possible reason. Nevertheless, the antibiotic susceptibility defined at the single-cell level might be invaluable for studying non-cultured or fastidious bacteria.Stimulated Raman scattering (SRC) metabolic imaging can be employed to visualize the glucose metabolic activity of living bacteria at the single-cell level . Differe2O labeling were applied to perform antibiotic susceptibility testing of cefotaxime on 103 E. coli strains. Metabolic activity was monitored using signals of C-D (carbon-deuterium) bonds of deuterated biomolecules. The morphological changes in bacteria were quantified based on the bacterium area of cefotaxime-susceptible/cefotaxime-resistant strains treated or not treated with antibiotics. Interestingly, C-D intensity or morphological deformation alone led to erroneous MIC estimation. Only the combination of signals from C-D and morphological deformation of bacteria was sufficient for MIC determination, with reported 93.2% essential agreement with the standard reference method.Stimulated Raman scattering metabolic imaging allows measuring both metabolic activity and morphological changes in antibiotic-treated bacteria . SRS andS. aureus and E. coli and their susceptibility to antibiotics can be quickly identified by studying the dynamic changes occurring in the single-cell SERS profiles on Ag/AAO substrates [Lactococcus lactis to antibiotics was observed in 60–90 min using SERS and PCA [S. aureus and E. coli study, the changes in SERS spectra were evident after 120 min of antibiotic treatment [5 CFU/mL. The quantitative SERS assay with silver nanoparticles reported in [5 CFU/mL, classifying bacteria pathogens with ~94% accuracy, and determining the antibiotic sensitivity of bacteria with 81–100% accuracy).The reproduction of bstrates . The res and PCA . The disreatment . SERS-baorted in  was suitE. coli cells in the presence of antibiotics revealed the coexistence of different spectral populations with ratios varying depending on the concentration of the antibiotic. The test procedure, overcoming single-cell heterogeneities, was devised to estimate the MIC and determinate the susceptibility phenotype of the tested bacteria using only a few single-cell spectra in 4 h, including the preculture step [2O allowed determination of the resistance of E. coli, P. vulgaris, S. entérica, S. flexneri, K. variicola, E. fergusonii, and P. rettgeriprofiles to ampicillin, chloramphenicol, kanamycin, and meropenem [The study of the Raman spectra of sensitive and resistant ure step . Isotopeeropenem . Spectraeropenem . FRAST a5 [3 CFU/mL [E. coli. The microfluidic microwell device with an automated microfluidic control system as demonstrated in [A microwell method with SERS detection was successfully used for rapid antibiotic susceptibility tests. The microwell device can significantly reduce the required bacterial concentration for a detectable SERS signal. The bacterial LOD (limit of detection), while using the microwell platform, is much lower than conventional SERS-AST, and according to some reports, is 5 × 105  and 103 3 CFU/mL  for E. crated in  can perfManipulation of bacterial cells with dielectrophoresis (DEP) improves the identification, as it takes into account the dielectric properties of bacteria as well as their size and shape. The use of a combined DEP-Raman substrate significantly reduces the analysis time compared with the standard cultural method and reduces errors due to the differences in cultivation conditions and the standardization of differences between batches. Changes in the spectra occur in 90–120 min after the start of detection, making it possible to distinguish antibiotic-resistant strains from susceptible ones ,115,121.Whole-cell biosensing provides numerous advantages over the traditional analytical chemistry methods and immunoassays. Bacteria are the natural target organisms for most antibiotics. Sensitive bacterial cultures can be easily maintained and scaled up, making them good candidates to be used in biosensing devices.E. coli cells were employed for tetracycline detection by Korpela et al. [Bioluminescent, genetically modified a et al. . This apa et al. .E. coli bacterial biosensor with a response reflecting the mode of action of the sensed antibiotic was demonstrated by Bianchi and Baneyx as early as 1999 [Bacillus subtilis and enabled the rapid screening of 14,000 natural products with a bactericidal action, elucidating the mode of action of several natural antibiotic candidates [A whole-cell multifunctional  as 1999 . Introdundidates . These bDespite the recent efforts in the direct SERS-based detection of antibiotics  and infeThe decades of antibiotic research have provided highly efficient bactericidal weapons aimed at specific prokaryotic targets: cell wall synthesis, cellular membrane, protein synthesis, and nucleic acids. However, the development of novel antibiotics is a costly and lengthy process, and for the foreseeable future, we will be limited in the choice of antibacterial drugs.Widespread antibiotic pollution can pose a risk of multidrug resistance development in bacteria. Mechanisms of antibiotic resistance are known and sometimes circumvented by suitable inhibitors. However, rapid antibiotic susceptibility testing is still highly desirable in the clinical setting. Surface-enhanced Raman spectroscopy provides fast and contactless acquisition of antibiotic-induced changes in bacterial cells. Therefore, a SERS-based approach may serve as the analytical method for personalized medical treatment, especially when multidrug-resistant infection is suspected.Finally, SERS-based monitoring of living bacterial cultures promises the creation of whole-cell biosensors for sensitive antibiotic detection. Interestingly, the same SERS setup may be further employed for direct the SERS detection of antibiotics, provided there is a suitable sample preparation kit. Thus, high sensitivity of bacterial sensors can be coupled with a high information capacity of SERS spectra.


Oncorhynchus mykiss), which were fed diets supplemented with five different forms of Met: Met-Met, L-Met, HMTBa, DL-Met, and Co DL-Met. No differences in growth performance were found in trout fed with different Met forms, but changes in the whole-body composition were found. In particular, Met-Met and L-Met promoted a significant body lipid reduction, whereas the protein retention was significantly increased in fish fed with HMTBa and Co DL-Met. The latter affected the hepatic Met metabolism promoting the trans-sulfuration pathway through the upregulation of CBS gene expression. Similarly, the L-Met enhanced the remethylation pathway through an increase in BHMT gene expression to maintain the cellular demand for Met. Altogether, our findings suggest an optimal dietary intake of all tested Met sources with similar promoting effects on fish growth and hepatic Met metabolism. Nevertheless, the mechanisms underlying these effects warrant further investigation.In aquafeeds in which plant proteins are used to replace fishmeal, exogenous methionine (Met) sources are demanded to balance the amino acid composition of diets and meet the metabolic fish requirements. Nonetheless, since different synthetic Met sources are commercially available, it is important to determine their bioavailability and efficacy. To address this issue, we conducted a two-month feeding trial with rainbow trout ( Aquafeeds supply all the nutrients  necessary for the optimal growth and health of cultured fish. Among nutrients, protein is the most expensive part of fish diets and supplies amino acids (AA) for energy and growth, and substrates for key metabolic pathways.In aquafeed formulations, fishmeal (FM) has traditionally been chosen as the primary natural protein source for farmed fish due to its high nutritional value and digestibility. Nevertheless, in recent decades, the inclusion of FM in compound feeds has shown a clear downward trend, mainly due to the limited supplies and price fluctuations . In ordeAmong EAAs, methionine (Met) is notably under-represented in vegetable meal compared to FM representing the main limiting AA in fish diets, especially in those containing high levels of soybean meal . A cost-Coating, encapsulation, microencapsulation, or polymerization are technological processes developed to increase the feed stability, overcome the loss due to leaching, and delay the intestinal absorption of CAAs ,10.Currently, the Met sources used to fortify aquafeeds include L-Met and its derivative forms, such as DL-Met (the racemic mixture of D- and L-isomer of Met), and Methionine Hydroxy Analogue (MHA). The main MHAs are 2-hydroxy-4-methylthio butanoic acid (HMTBa) and its calcium salt, HMTBa-Ca, both precursors of L-Met. HMTBa is a racemic mixture of Met D- and L-isomer and consists of about 65% in monomeric form, 23% in the dimer/oligomeric form, and the remaining 12% being water. Because HMTBa bears a hydroxyl group instead of an amino group, it is classified as an organic acid . HMTBa aIn fish species, the bioavailability of different Met forms is different as it is mainly related to the leaching rate in the water, the intestinal uptake, and the bioconversion to L-Met. Indeed, in the study by Vázquez-Añón et al. , the cheThe metabolic conversion of L- and D-HMTBa and D-Met to L-Met is a two-step process, with each compound being converted first to a keto-methionine intermediate called ketomethylthio-butanoic acid (KMB). The second step of conversion is a transamination of the KMB to form L-Met ,12,13. TIn fish, knowledge on the efficacy–efficiency of different Met sources on growth performance and metabolism is scarce. In species such as rainbow trout, common carp, Nile tilapia, channel catfish, and cobia, DL-Met resulted a better source than MHA with regard to the growth performance ,15,16,17By contrast, turbot fed HMTBa and channel catfish fed MHA, MHA-Ca, and microcapsulated DL- Met grew more than fish fed L-Met or DL-Met ,19. In tDifferently from DL-Met and HMTBa products, L-Met is seldom used as an exogenous feed supplement, as it is obtained from DL-Met purification with high production costs. However, recently, a feed-grade L-Met product has become commercially available in some countries. The feed-grade L-Met (99% purity) can be produced from chemical synthesis, microbial fermentation of plant-based raw materials, or a combination of both .DL-methionyl-DL-methionine (Met-Met) is a new dipeptide formed by the dehydration and condensation of two DL-Met molecules. Met-Met has some advantages such as better intestinal absorption than crystalline Met and lower water solubility. The latter characteristic overcomes the leaching challenges, which benefit diet efficiency, fish growth, and a healthy environment .Ictalurus punctatus), as reported by Zhao et al.  was characterized by high level of FM. BAS+ contained 1% of Met that only derived from the raw ingredients . In contAt DISAFA facility, the two basal diets were ground. Ground BAS+ was mixed with fish oil  to obtain 1 kg of finished mixture (CTRL+ diet). Water was added to the mixture and pellets were obtained using a 1 mm die meat grinder. Then, pellets were dried at 50 °C for 48 h. Similarly, ground BAS− (857 g) was mixed with fish oil (143 g) and pellets were prepared as aforementioned to obtain a CTRL− diet.To prepare the five experimental diets, 5 g of each Met form−  was firstly mixed with fish oil and then added to BAS− diet following the same protocol used to prepare CTRL− diet. Oncorhynchus mykiss) fingerlings. Fish were lightly anesthetized using MS-222; PHARMAQ Ltd., UK and then individually weighted . The mean initial body weight (iIBW) was 3.40 ± 0.40 g. After weighting, trout were randomly distributed into 21 fiberglass tanks of 100 L volume connected to a flow-through open system supplied with artesian well water at a constant temperature of 13 ± 1 °C, and flow rate of 8 L min−1. Dissolved oxygen was measured once a week and it ranged between 7.6 and 8.7 mg L−1.A 59-day feeding trial was performed using 567 rainbow trout  three times a day, seven days per week. Each of the seven experimental diets was fed to 3 tanks of trout. Fish mortality was checked daily. To measure growth, fish were weighed in bulk every 2 weeks. At the end of the trial, after 24 h of fasting, fish were individually weighted and mean individual final body weight–(iFBW) was calculated.Mortality (%) (M%) = [(number of dead fish/number of fish at the beginning) × 100];Individual weight gain  = iFBW  − iIBW ;Feed conversion ratio (FCR) = total feed supplied  × 100;(lnFBW+lnIBW)×0.5];Feeding rate  = [ × 100/number of feeding day)]/[e1/3 − IBW1/3)/Ʃ(T × d) × 100 .Thermal growth coefficient =  and analyzed for DM (#934.01), CP (#984.13), and ash (#942.05) contents according to AOAC International (2000). EE (#2003.05) was analyzed according to AOAC International (2003). To perform the assessment of the fish whole-body composition (WBC), at the end of the trial and after 24 h of fasting, 3 fish per tank (9 fish per diet) were sampled, euthanized by overdose of MS-222 (300 mg/L), ground with a knife mill , and frozen at −80 °C until analyses. For the analysis, the same methods described for feed sample analysis were used.® 16 Instrument, Promega, Milan, Italy), and a total RNA purification kit (Maxwell® 16 Tissue LEV). Briefly, 125 mg of each tissue was mixed with ice-cold Lysis Solution containing 1-thioglycerol and then homogenized using GentleMACS Dissociator  until no visible tissue fragments remained. The RNA Dilution Buffer was added to the lysate; then, the whole volume was vortexed and transferred into Maxwell® 16 LEV Cartridges for RNA isolation.Total RNA was extracted from each sample of rainbow trout liver using an automatic system , whereas RNA integrity was verified by agarose gel electrophoresis. The purity of RNA was assessed at 260/280 nm and averaged 2.10 ± 0.01.After extraction, 3 µg of total RNA was reverse-transcribed into cDNA in a mix of 20 µL volume containing oligo dT16 primers, as described in the SuperScript III reverse transcriptase kit .BHMT, SAHH, and CBS genes, liver cDNA was amplified via PCR, using for each gene a forward primer engineered to contain a T7 phage polymerase promoter sequence at its 5′ end and a reverse primer  and then sequenced in both SP6 and M13 directions.Two sets of forward and reverse primers were designed based on Atlantic salmon BHMT (betaine-homocysteine methyltransferase), and SAHH (S-adenosylhomocysteine hydrolase) cDNA sequences available in the NCBI database . Another set of primers was designed for cystathionine-beta-synthase (CBS) based on the rainbow trout cDNA sequence available in the NCBI database (GenBank acc. nr.: NM_001124686). To generate the in vitro-transcribed mRNAs for e primer . PCR proIn vitro transcription was performed using T7 RNA polymerase and other reagents supplied in the RiboProbe In Vitro Transcription System kit  following the manufacturer’s protocol.TM Universal Probes One-Step kit  as reported in Terova et al. [The synthetic mRNAs obtained by in vitro transcription were used as quantitative standards in the real-time PCR analysis of the biological samples. For this, defined amounts of mRNAs at 10-fold dilutions were amplified via real-time PCR using iTaqa et al. . The Ct ® quantitative real-time RT-PCR. Biological samples were loaded in the same 48-well plate with standard mRNAs and run under the same aforementioned real-time RT-PCR conditions.One hundred nanograms of total RNA extracted from each trout liver was subjected to One-step TaqmanSM PCR primers and gene-specific fluorogenic probes were designed by Invitrogen, Milan, Italy.Real-time Assays-by-Design® probes used for each target gene amplification are shown in ® PCR reactions were performed on a Bio-Rad® CFX96. System.Primer sequences and TaqMan4). To ensure a good calibration curve, five concentrations were prepared.For the HPLC analysis, SAM and SAH standards  were first prepared in HPLC water at 1 mM concentration and then sequentially diluted with 0.4 M perchloric acid  was mixed with 800 µL of perchloric acid (0.4 M) and homogenized for 4 min using a TissueLyser II , at 4 °C with stainless steel beads of 5 mm diameter. After centrifugation at 10,000× The HPLC system consisted of a PU-2089 quaternary pump connected to a degasser and a MD-2015 diode array, both obtained from Jasco-Europe S.r.l Company . Separation was carried out in a Kinetex XB-C18 reversed-phase analytical column  with a particle size of 5 µm  equipped with an HPLC universal guard column . Solvent A and B formed the gradient elution.2PO4, and it was adjusted to pH 3 with H3PO4. Solvent B consisted of 100% methanol. Solvent A was filtered through a 0.2 µm membrane filter. The chromatographic conditions are reported inTerova et al. [Solvent A was an aqueous buffer containing 8 mM octanesulfonic acid sodium salt and 50 mM NaHa et al. . Data prFor the validation, each biological sample and each standard were run three times. A blank sample was injected daily at the beginning of analysis to check the baseline and to monitor the matrix effect due to sample injection. A blank was also run between each sample to ensure there was no memory.2 = 0.998 for SAM, and r2 = 0.998 for SAH.The equation of the calibration curves showed rThe growth performance and fish whole-body composition were analyzed by one-way ANOVA (IBM SPSS Statistics v. 25.0 for Windows) using the following model:The assumption of normality was checked using the Shapiro–Wilk test. Levene’s homogeneity of variance test was used to assess homoscedasticity. If such an assumption did not hold, the Brown–Forsythe statistic was applied to test the equality of group means instead of the F one. Pairwise multiple comparisons were performed to test the difference between each pair of means .Met metabolism and the gene expression data were analyzed by one-way ANOVA using GraphPad Prism 8 software . If there were significant differences between dietary groups, Tukey’s test was applied as post hoc analysis.p ≤ 0.05.The results were expressed as the mean and pooled standard error of the mean (SEM). Significance was set at The mortality and growth performance parameters are reported in p < 0.05); Met-Met (p < 0.05); HMTBa (p < 0.05); DL-Met (p < 0.05); and Co DL-Met (p < 0.05). A similar trend was reported for SAH (p < 0.05). Moreover, the L-Met dietary group displayed a significantly higher level of SAH with respect to the HMTBa (p < 0.05), Met-Met (p < 0.05), and Co DL-Met (p < 0.05) dietary groups. The CTRL− dietary group had significantly lower levels of SAH with respect to CTRL+ (p < 0.05) and DL-Met (p < 0.05). The significance level of 0.05 was also detected for the comparisons of the CTRL− vs. Met-Met dietary group and CTRL− vs. Co DL-Met dietary group, respectively. As shown in  for SAH B, whose The SAM/SAH ratio showed no statistical differences among the diets C.BHMT transcript level (p < 0.05) was found in fish fed with L-Met in comparison to fish fed with a Met deficient diet (CTRL−) and with DL-Met, and HMTBa-supplemented diets.As shown in CBS gene (p < 0.05) than in the HMTBa and Co DL-Met groups.The mRNA copies of the CBS gene B were siSAHH mRNA copy number did not show differences related to diets being at similar levels in all the dietary groups (p > 0.05).The The use of synthetic Met sources is a prerequisite to correct the Met deficiency in plant-based diets and allow the normal and healthy growth of fish. There is limited information and debates still exist on the bioavailability of different Met sources, which mainly depends on their intestinal absorption, gut microbial metabolism, and the conversion of isomers into the biologically active form of L-Met . For insAccordingly, our research focused on the bioefficacy of five dietary Met sources in a commercially relevant species, rainbow trout. Five experimental diets supplemented with different forms of Met  were tested in a two-month feeding trial. In addition, a CTRL+ diet with a high level of FM and 1% of Met deriving only from the raw ingredients, and a CTRL− diet characterized by a high level of vegetable meal and deficiency in Met, were tested too.With regard to fish performance indexes, at the end of the feeding trial, no differences were found between the Met-supplemented dietary groups and negative and positive control groups. Mortality ranged from 3.70% (CTRL+) to 9.88% (CTRL−) and, even if it was numerically higher in the CRTL− group, no statistical differences between groups were recorded. At the end of the trial, fish of all dietary groups reached an FBW about 6-fold higher than the IBW, suggesting an optimal nutrient utilization. This is also sustained by the low FCR, which was found to be less than 0.9 in all dietary groups, as well as by the high PER and SGR values.® (MHA liquid form)-, and DL-Met-supplemented diets were statistically not different from fish fed with L-Met-supplemented diets [In rainbow trout, several trials have reported differences in bioefficacy between MHA and DL-Met based on the growth parameters. Powell et al.  found a ed diets . The raied diets . Howevered diets , dietaryAs known, Tau is an amino sulfonic acid widely distributed in animal tissues, but very low in plants . For thiBody composition, specifically crude protein, moisture, ash, and lipid content of carcasses, was significantly affected by Met sources. Fish fed the CTRL+ diet had a lower crude protein content than fish fed the Met analogue (HMTBa) and Co DL-Met. It seems that these two Met forms promoted protein accretion more efficiently than a diet in which Met was supplied as intact protein. In common carp  and in rIn our feeding trial, the coated form of DL-Met performed better than DL-Met. The coating technique most likely delayed the leaching of AA and allowed Met to be slowly released in the intestine. In this way, free Met supplemented to the feed was absorbed synchronously with Met derived from dietary protein digestion . The coaWith regard to the lipids, the highest body lipid content in our study was found in trout that received the Met-deficient diet, whereas both protein-bound and synthetic Met  promoted a significant body lipid reduction as a result of an efficient utilization of these forms by fish.Nearly half of the Met metabolism (48%) and up to 85% of all methylation reactions take place in the liver . TherefoThe activated Met, namely SAM, is an important methyl donor and it is involved in several reactions, such as the methylation of DNA, RNA, and proteins. Following the transfer of a methyl group to an acceptor molecule, SAM is converted to SAH, which is a potent competitive inhibitor. Consequently, elevated levels of SAH suppress SAM-dependent transmethylations and have an effect upon the ratio of SAM/SAH within hepatic tissue. The SAM/SAH ratio is influenced by the activity of S-adenosylhomocysteine hydrolase (SAHH), which catalyzes the reversible hydrolysis of SAH to Hcy and adenosine.Hcy is an important intermediate of Met metabolism and by the action of BHMT enzyme, the methyl group of betaine molecule can be transferred to Hcy and regenerate Met. The Met pool can also be used in the trans-sulfuration pathway producing Cys and Tau in reactions requiring cystathionine β-synthase (CBS) .CBS gene was downregulated in fish fed the CTRL+ diet, in which Met was supplied by raw ingredients. The CTRL− diet did not promote any significant changes in CBS gene expression, but when CTRL− to CTRL+ dietary groups are compared, although not statistically significant, the low-Met diet numerically promoted an increase in CBS mRNA copies. This trend was also found in salmonids fed a Met-deficient diet; in those fish, the CBS gene expression increased [In our study, the ncreased ,35.CBS gene in the liver tissue. In agreement with our results, Zuo et al. [CBS in hepatic cells from porcine (IPEC-J2 cells), thus favoring the trans-sulfuration. In chicken small intestine, HMTBa (DL-HMTBa) was preferentially diverted to the trans-sulfuration pathway, thus leading to an increase in Cys and Tau content in the enterocytes [The two Met sources, HMTBa and Co DL-Met, increased the mRNA copies of o et al.  found therocytes . As alreBHMT mRNA copies in trout liver. The same downregulation was found in both whole fry and broodstock liver of rainbow trout by Fontagné-Dicharry et al. [Our data show that the Met-deficient diet reduced significantly y et al. . On the MAT gene encoding for the corresponding enzyme. Nevertheless, we can postulate that the liver of fish fed the L-Met-supplemented diet had a higher methyl transfer capacity due to the increased SAM levels. Furthermore, SAH hepatic concentration significantly increased in trout fed L-Met with respect to fish fed other Met sources. A similar metabolic response was found in Atlantic salmon liver, in which Met intake affected the methylation capacity [In this regard, feeding rats with increasing levels of Met increased hepatic SAM levels, as a result of higher hepatic methionine adenosyl transferase (MAT) activity . This encapacity . As a macapacity  did not capacity  concludeCBS gene over the levels found in fish fed with the CTRL+ diet, maintaining a positive health status of rainbow trout. This was also highlighted by the high CP content recorded by the Co-DL Met and HMTBa feeding groups with respect to CTRL+.The Co DL-Met and HMTBa affected the trans-sulfur pathway more than the other Met sources, favoring the production of antioxidants. Indeed, the Co-DL Met and HMTBa diets increased the transcript levels of the SAHH gene in fish fed with different diets. We can hypothesize an Hcy accumulation at the hepatic level without compromising fish health. Future studies should quantify the levels of Met and Hcy in liver.In general, all tested Met forms did not influence the methylation ratio (SAM/SAH) as confirmed by the similar expression of the In summary, the present study showed an optimal dietary intake of all tested Met sources with similar promoting effects on fish growth, and hepatic Met metabolism. Nevertheless, the mechanisms underlying these effects warrant further investigation.


The main objective of this study is to better understand the performance changes of naturally aged glass fiber-reinforced concrete (GFRC) and polypropylene fiber-reinforced concrete (PPFRC), especially the degradation of fibers, which is of great significance for evaluating the durability of structures using these two types of composite materials. The mechanical properties, water absorption, and microstructures of GFRC and PPFRC at a curing age of three years, including their compressive strength, full curves of water absorption, fiber-matrix interaction, and fiber degradation, were systematically studied, and the related properties were compared with those at the curing age of 28 days. The degradation of fibers after freeze-thaw cycles was also studied. The results revealed the following. The water/binder ratio (w/b) affects the rate of increase of the long-term compressive strength of naturally aged concrete. In general, the water absorption of fiber-reinforced concrete (FRC) at the curing age of three years was found to be significantly reduced, but with the increases of w/b and the fiber content to the maximum values, the water absorption of the specimens cured for three years was higher than that of the specimens cured for 28 days. Moreover, with the increase of the curing age, the optimal glass fiber (GF) contents for reducing the water absorption decreased from 1.35% to 0.90% (w/b = 0.30), and from 0.90% to 0.45% (w/b = 0.35), respectively. The GF surface was degraded into continuous pits with diameters of about 200 to 600 nm, and the surface of the pits was attached with spherical granular C-S-H gel products with diameters of about 30 to 44 nm. The freeze-thaw cycles were found to have no significant effect on the pits on the GF surface and the granular C-S-H gel products attached to the pits, but caused a portion of the cement matrix covering the GF to fall off. The interfacial bonding between the polypropylene fiber (PPF) and the cement matrix exhibited almost no change in the PPFRC after three years of curing as compared with that after 28 days of curing. Furthermore, the cement hydration gel on the PPF surface was not significantly damaged by 150 freeze-thaw cycles. Concrete is the most commonly used building material worldwide, but in the service process of concrete structures, deterioration caused by physical and/or chemical factors often occurs; for example, chloride attack, sulfate attack, freeze-thawing, shrinkage, carbonization, alkali-silicon reaction, and steel corrosion, etc., seriously threaten the durability of concrete. Via in-depth research, it has been found that water is usually involved in various degradation processes. As a carrier of aggressive ion migration, water is a direct participant in physical degradation and an indirect participant in chemical degradation ,4,5,6,7.2, and a denser interfacial transition zone [Glass fiber (GF) and polypropylene fiber (PPF) are two excellent materials for the micro-reinforcement of concrete. GF is characterized by a low specific gravity, low water absorption, a high elastic modulus, and high tensile strength, thereby providing great potential for concrete reinforcement. PPF has attracted the attention of researchers due to its low cost, excellent toughness, and high resistance to shrinkage and cracking. The addition of short-cut GF or PPF forms a three-dimensional staggered support network inside concrete, thereby compacting its internal structure ,13,14 anion zone ,16,17, wion zone .Although GF and PPF have excellent engineering properties, the durability of fiber-reinforced concrete (FRC), especially the degradation of GF in concrete, limits its engineering applications to a certain extent ,25,26,27It takes a long amount of time to study the natural degradation of GF in GF-reinforced concrete (GFRC) via natural curing. Due to the lack of long-term performance data, researchers often use accelerated aging tests in lab conditions that simulate aging to study the durability of GFRC composites ,32,33,34Based on the current research situation, the following problems are identified. First, while the compressive strength is an important index for concrete structure design, most studies on the compressive strength of GFRC and PPFRC have been conducted after curing for 28, 56, or 90 days, while there have been few studies on the long-term (more than 1 year) compressive strength. More importantly, the influences of the water/binder ratio (w/b) and fiber content on the development of the FRC strength with the increase of age have not been considered. Second, most studies on the water absorption of FRC only tested the water absorption at curing ages between 28 and 90 days ,14,18,21Research on the property changes of concrete naturally aged for several years, especially investigations of fiber degradation, would contribute to the durability design of FRC structures. In the present work, the properties of GFRC and PPFRC naturally aged for three years at room temperature, including the compressive strength, full curves of water absorption, fiber-matrix interaction, micro-morphology of the interfacial transition zone, and fiber degradation, were systematically studied. The test results were compared with those of samples cured for 28 days. Moreover, a rapid freeze-thaw cycle test was conducted to study the fiber degradation of the FRC aged for 3 years under freeze-thaw cycles. These investigations provide references for the design and evaluation of the structural durability of GFRC and PPFRC.The P.O 42.5 ordinary Portland cement was used in this study, and its physical and mechanical properties are shown in The fly ash is Class I fly ash produced locally in Harbin, China. The chemical compositions of fly ash are shown in The chemical compositions of silica fume are shown in 3.The chemical compositions of slag are shown in The strength grade of the slag is S95, and the density is 2.89 g/cmThe fibers used in the test are chopped GF and PPF with a length of 12 mm. The morphology and main parameters of the fibers used are shown in Natural river sand was selected as fine aggregate and its fineness modulus was Mx = 2.66. Coarse aggregate with 5–20 mm continually graded macadam and a 14.87% crushing value index was used.The water used in the test is the municipal water supply for the city.A polycarboxylate-type superplasticizer was adopted to meet the requirement of a slump for fresh concrete.The mixture designs were carried out according to the JGJ 55-2011 standard , and the3 in size. After 28 days of water curing under standard conditions, some of the samples were subjected to the corresponding performance test, and the others were cured at room temperature for three years. The room temperature was 24 ± 2 °C, and the humidity was 40% ± 3%.The samples were prepared in accordance with the CECS13-2009  and GB/TA total of 84 cubic samples were used for the compressive strength tests. According to the GB/T 50081-2002 standard , a YAW-2The freeze-thaw cycle test was conducted according to the GB/T 50082-2009 standard . A KDR-VThe 100 × 100 × 100-mm specimens were immersed in water for water absorption tests, and the test procedure was conducted according to the ASTM C642-2013 standard  were obtThe scanning electron microscopy (SEM) test procedure was conducted according to the ASTM C1723-2010 standard . A QuantAccording to 3y-28d represents the rate of increase of the three-year compressive strength of concrete relative to the 28-day compressive strength. It is evident that when w/b = 0.30 and 0.35, the I3y-28d value of the GFRC was significantly lower than those of specimens PC-30 and PC-35. At the same GF content, the I3y-28d value of the GFRC with w/b = 0.30 was higher than that with w/b = 0.35. For the PPFRC, excluding the increase of the strength of specimen PPF-30-135 from 18 to 35.4 MPa (an increase of 96.67%), the other I3y-28d values were lower than that of plain concrete, and the I3y-28d values of the PPFRC were higher than those of the GFRC at the same fiber content. In other words, w/b and the fiber content affected the rate of increase of the long-term compressive strength of the naturally aged concrete; after w/b increased, as the contents of GF and PPF increased, the compressive strength of concrete increased more slowly.In 3y-28d, which reflects the increase of water absorption compared to the specimens cured for 28 days [3y-28d is negative, it indicates that the water absorption of the concrete aged for three years was lower than that of the concrete aged for 28 days; thus, the smaller the value of W3y-28d, the greater the rate of reduction of water absorption, and the more compact the inner structure of the concrete. Water absorption is a good index to measure the porosity of concrete. The durability of concrete can be estimated by porosity, because higher porosity will make it easier for harmful chemicals to penetrate into the concrete. These chemicals can react with its components and change the performance of concrete materials ,15,21. T 28 days . When thIt can be seen from When w/b = 0.30, after adding PPF, the water absorption of the concrete cured for three years increased, and the water absorption increased with the increase of the PPF content. The water absorption of specimen PPF-30-135 was 4.66%, which was 127.3% higher than that of specimen PC-30.When w/b = 0.35, the water absorption of the GFRC increased with the increase of the GF content, and GF contents of 0.45% and 0.90% respectively reduced the water absorption by 37.6% and 15.0% as compared with that of specimen PC-35. In contrast, the GF content of 1.35% increased the water absorption by 47.9%. Thus, 0.45% is the optimal content of GF. In other words, with the increase of w/b to 0.35, when the effect of GF on the water absorption of the specimen aged for 28 days was considered, and 0.90% was found to be the optimal content . HoweverWhen w/b = 0.35, the influence of PPF on water absorption was the same as that when w/b = 0.30, and the water absorption increased with the increase of the PPF content. The water absorption of specimen PPF-35-135 increased from 2.34% (the water absorption of specimen PC-35) to 7.12%, thereby reflecting an increase of 204.3%.3y-28d values of GFRC and PPFRC ranged from -3.0% to -35.0%, and the W3y-28d value of PC-30 was −41.6%. This means that for the GFRC and PPFRC, the water absorption of the same samples cured for three years exhibited different degrees of reduction as compared to that of the samples cured for 28 days, but the water absorption of plain concrete had the highest rate of decrease. When w/b = 0.35, the W3y-28d values of specimens GF-35-45 and PPF-35-45 were less than that of PC-35; the values were 9.1% and 10.9%, respectively, which means that when the fiber content increased to 1.35%, the water absorption rates of these two samples after curing for three years did not decrease, but increased. The reason for this is as follows. The water absorption test used the same sample at two different curing ages. During the first water absorption test and that after three years, the unreacted cementitious material inside the concrete continued to react with water to make the structure more compact, thereby reducing the water absorption. In reference [According to the data presented in eference , similareference , even ifR2 is much larger than 0.9).It can be seen from In specimen GF-30-135 shown in It can be seen from Based on the preceding analysis, the freeze-thaw cycles did not cause significant damage to the GF surface, and the deterioration  of the gel layer of cement hydration products surrounding the GF was related to the thickness and density of the gel layer; the denser the gel structure and the thicker the structural layer, the less likely it was to fall off or be damaged after the freeze-thaw cycles.This study systematically investigated the mechanical properties, water absorption properties, and fiber degradation of GFRC and PPFRC after natural aging for three years. The following conclusions were reached.3y-28d values of plain concrete, GFRC, and PPFRC with different fiber contents all decreased, and the range of decrease was between 6.58% and 51.96%. In other words, after the increase of w/b, as the contents of GF and PPF increased, the compressive strength of concrete increased more slowly.The water/binder ratio (w/b) was found to affect the rate of increase of the long-term compressive strength of naturally aged concrete. With the increase of w/b from 0.30 to 0.35, the I3y-28d value of the FRC was generally lower than that of plain concrete. The I3y-28d value of the GFRC was lower than that of the PPFRC with the same fiber content. Excluding that of specimen PPF-30-135, the I3y-28d values of the GFRC and PPFRC were lower than those of plain concrete.The type and content of fiber were found to affect the rate of increase of the long-term compressive strength of naturally aged concrete, and the ICompared with the water absorption of the specimens cured for 28 days, that of the FRC specimens cured for three years was significantly reduced, with a range of decrease between 3% and 52.4%. This is due to the subsequent hydration reaction that compacted the internal structure of the concrete. However, with the increases of w/b and the fiber content, the water absorption of specimens GF-35-135 and PPF-35-135 cured for three years increased by 9.1% and 10.9%, respectively, as compared to those of the specimens cured for 28 days. This was due to the large internal pore size and numbers of pores in these two groups of concrete at the initial stage (28 days), and the products produced by the hydration reaction in the later stage (after 28 days) were not sufficient to effectively improve the internal pore structure.With the increase of the curing age, the optimal GF content to reduce the water absorption of concrete was found to decrease. Considering the effect of GF on the water absorption of concrete, 1.35% (w/b = 0.30) and 0.90% (w/b = 0.35) were found to be the optimal GF contents if the water absorption of the specimens cured for 28 days is used to measure the GF effect; however, if the water absorption of the specimens cured for three years is considered, the optimum GF contents were found to be reduced to 0.90% (w/b = 0.30) and 0.45% (w/b = 0.35), respectively.2 and a large amount of flocculent or granular C-S-H gel. The GF surface was degraded into continuous pits with diameters of about 200 to 600 nm, and the surfaces of the pits were attached with spherical granular C-S-H gel products with diameters of about 30 to 44 nm. The cement hydration products in the interfacial transition zone and the products attached to the GF surface were highly consistent in both morphology and size.When the natural curing age was three years, the interfacial transition zone between the GF and cement matrix consisted of a small amount of Ca(OH)The freeze-thaw cycles were found to have no significant effect on the pits on the GF surface and the granular C-S-H gel products attached to the pits, but caused a portion of the cement matrix covering the GF to fall off. The factors affecting the shedding area and form of the coating mainly included the compactness and thickness of the coating.SEM observation revealed that the interfacial bonding between the PPF and the cement matrix in the PPFRC exhibited almost no change after curing for three years as compared with that after curing for 28 days. Moreover, the cement hydration products on the PPF surface were not significantly damaged by 150 freeze-thaw cycles.Compared with the diameter of GF, the degradation pits on the GF surface were relatively shallow after curing for three years, from which it can be inferred that the degree of adverse effects on the compressive strength, water absorption, and durability of concrete was lower. However, it is unclear whether the depth of the degradation pits will further increase with the increase of the curing age. If so, the degrees of influence on the deterioration of the strength and durability of concrete require further study.


To study the effect of SAP incorporation on the early shrinkage of SAP internally cured concrete with the aim to solve the problems of early shrinkage and cracking of bridge leveling-layer concrete, using the SAP incorporation method as a parameter, the mechanical properties test of internally cured concrete, the shrinkage performance test of ring restraint and the scanning electron microscope observation test were carried out. The effects of the SAP content and incorporation method on the flowability, mechanical properties, shrinkage performance and microstructure of internally cured concrete were analyzed. The experimental results show that when the content of the SAP in concrete is 0.2% of the mass of cementitious materials, it has the least influence on the compressive strength of concrete. The addition of preabsorbed water to the SAP can delay early cement hydration, increase the later cement hydration rate and final hydration degree, and improve the concrete strength. Preabsorbed water mixed with an SAP can effectively improve the shrinkage of concrete, and the shrinkage reduction effect is more obvious than that from the dry addition of the SAP; the early cracking risk of concrete without an SAP is high, and it will crack before day 28. The addition of an SAP can strengthen the microstructure of concrete and improve its density and crack resistance, effectively avoiding concrete cracking. It is recommended that the water-absorbent resin be incorporated in a preabsorbent manner, and the SAP preabsorbent ratio is equal to the maximum water storage rate of the SAP. Cracking caused by concrete shrinkage is an important problem affecting the durability of concrete ,2,3. ModConcrete cracking is affected by many parameters, including the water-cement ratio, cement type, admixture, structural geometry and external conditions ,6,7. On The concept of internal curing was first proposed by Bentz  in 1991,The shrinkage of concrete is obviously reduced after adding a water-absorbing material ,19,20, aAt present, there is much research on the shrinkage cracking of concrete. Most scholars have studied the effect of SAP incorporation on mechanical property and concrete shrinkage performance ,19. Yu eIn view of the influence of different incorporation methods of the SAP, the main direction of existing research is focus on the SAP’s influence on the rheological properties of concrete. Liu et al.  evaluateIn summary, most of the studies on the influence of an SAP on the concrete shrinkage performance at present focus on the influence of the SAP type and content on concrete shrinkage cracking. In view of the influence of different SAP incorporation methods on concrete properties, most of the research focuses on how to determine the additional water content of the SAP to improve the rheological properties of cement-based materials. It can be seen that, at present, under different SAP incorporation methods, and especially under the condition of SAP partial pre-water absorption, the influence on concrete mechanics and shrinkage properties is still small.Based on the above reasons, this paper takes the SAP incorporation method as the parameter and considers the change law of water absorption over time in the concrete mixing stage to carry out the flow performance test, compressive strength test and constrained shrinkage performance test of internally cured concrete. The effects of different SAP incorporation methods on the fluidity, mechanical properties and ring shrinkage strain of internally cured concrete were studied. The degree of hydration of concrete under different SAP incorporation methods was analyzed by using scanning electron microscopy experiments, and the internal hydration mechanism of concrete under different SAP incorporation methods was explained in order to provide a reference for the application of SAP internally cured concrete.The cement used was P • O 32.5 ordinary Portland cement, according to the “general Portland cement” GB 175-2007 [The particle size of the coarse aggregate used in the test was 4.75~19.5 mm, and the mixing ratio of 4.75~9.5 mm and 9.5~19.5 mm was 4:6. The crushing value of the crushed stone was 17.5%, the needle flake content was 4.3% and the mud content was 0.12%.The fine aggregate used in the test was machine-made sand, and the fineness modulus was 2.5. According to the interval of the cumulative sieve residue curve, this kind of fine aggregate is a medium sand with good gradation.The internal curing agent used in the test was a super-absorbent resin (SAP), which is a highly crosslinked sodium acrylate produced by aqueous solution polymerization (crosslinking agent content of 0.06%), and the average particle size was 150 μm.The water used in the test was tap water. The admixture used in the test was a polycarboxylate powder concrete superplasticizer, the water reduction rate was 30% and the bleeding rate ratio was ≤20%.Because of the inability to accurately measure the amount of water released by the SAP inside concrete, researchers have measured the water absorption kinetics curve of the SAP in cement-based centrifugal fluids. The results show that the development law of SAP water absorption in a cement slurry centrifugal fluid is divided into three stages: first, it rises rapidly to the maximum, then gradually decreases, and finally, tends to be stable ,34. Stud1 = 146.02) and the water storage rate of the preabsorbed SAP when the concentration of the pre-water absorption SAP in the sodium chloride solution was balanced (u3 = 35.00). The u2 (u2 = 111.02) is the water release rate of the SAP in the concrete mixing stage. Considering the change in the water absorption rate of the SAP in the concrete mixing stage, the SAP pre-absorption water and concrete unit water consumption rates of each test group were calculated. The mix proportion design results of internally cured concrete are shown in 0 is the pre-water absorption of the SAP, and the content of the SAP is 0.2% of the mass of cementitious materials). Six kinds of mix proportions were designed: the J0 group without an SAP, the G0 group with a dry SAP, the G1 group with a dry SAP and additional m × u3 water, the Y0 group with a pre-saturated SAP and reduced water consumption of m × u2, the Y1 group with a pre-saturated SAP and reduced water consumption of m × u2, and the Y2 group with m × u3 pre-water absorption.Firstly, the presaturated water-absorbing SAP was placed in a 0.7 mol/L sodium chloride solution to simulate the change process of the water absorption rate of the SAP in a concrete mixture. The tea-bag method ,36,37 waThe maximum particle size of the aggregate used in the preparation of the internally cured concrete was 19 mm. The visual slump of concrete was much larger than 10 mm during the trial mixing. Therefore, according to the slump measurement method in the “Ordinary concrete mixture performance test method standard” GB/T 50080-2002 , the floAccording to “the test method of mechanical properties of ordinary concrete” GB/T 50081-2002 , three sAccording to ASTMC 1581-04 , the annAfter the shrinkage test, the concrete specimen was taken out and broken. The cement matrix fragments were screened and soaked in an alcohol solution to terminate the hydration process of the internal cement. After drying with a dryer, the processed dry test block was pasted on conductive glue. After the gold sputtering, the samples were put under the scanning electron microscope  to observe the morphology of cement hydration products and cracks.Due to the high water absorption rate of the SAP, the working performance of the concrete will be greatly affected when it is mixed into concrete with dry or pre-water absorption. Therefore, the flow performance of SAP internally cured concrete is studied. The measured concrete slump under different SAP contents and incorporation methods is shown in As can be seen from Compared with the reference group (J0), the slump of the dry-mixed group (G0) and the dry-mixed water group (G1) decreased with the increase in the amount of absorbent resin, and the decreases were 1.5%, 6% and 10.5% and 1.9%, 3.4% and 3.8%, respectively. When a dry SAP is added to the mixture, the SAP absorbs water from the slurry, reducing the effective water content and w/b, which is equivalent to reducing water . WithoutFor the Y0 group with presaturated water absorption and additional water, the slump increases by 1.9%, 8.1% and 11%. The SAP release of water in the Y0 group increases the water-cement ratio and makes the concrete fluidity increase significantly. Compared with the reference group (J0), the slump of the water-saturated and water-reduced group (Y1) was slightly increased; the reason was that the preabsorbed absorbent resin absorbed less or no longer absorbed the mixed water and cement slurry, and the absorbent resin was evenly dispersed in the concrete, meaning that a small amount of water would be released, and thus, the fluidity of the concrete did not change much. The partial preabsorbent SAP (Y2) did not absorb water or release water after mixing with the mixture; thus, compared with group J0, the slump of the concrete also remained basically unchanged. Some studies  have conThe compressive strength of concrete with different SAP dosages and incorporation methods is shown in According to the analysis of the data in As can be seen from After the SAP content exceeds 0.2%, the strength of each group generally decreases. It is speculated that the main reason may be the uneven distribution of water in the matrix caused by the addition of excessive SAP . SubsequIn summary, when the SAP content is low, its water storage and release function are not obvious, the internal curing effect is not obvious, and the compressive strength of the concrete increases slightly. When the SAP content is too high, the holes left after the shrinkage of the SAP and the release of water become the main factor affecting the compressive strength of the concrete, which reduces the compressive strength of the concrete. Therefore, under the conditions of appropriate dosage, an appropriate water diversion method and appropriate water diversion, the SAP internal curing effect can be the best, and the strength of the concrete can reach its maximum.The compressive strength test was carried out with the optimal dosage of 0.2% . The variation law of compressive strength of internally cured concrete with curing ages under different SAP incorporation methods is studied. The results are shown in As can be seen from The development trend of the compressive strength of group Y1 and group Y2 is relatively consistent. In the early stage, the intensity development was slower, whereas in the later stage, the intensity development speed was faster, but the overall difference was small. The development trend of the compressive strength of the G0 group and G1 group was basically the same, which shows that the early strength developed faster and the late strength developed slower; the 7-d compressive strength reached 90% of the 28-d compressive strength, and the later compressive strength growth was only 2.7 MPa and 2.6 MPa, respectively, which finally stabilized at 39.8 MPa and 35.2 MPa. The compressive strength of the Y0 group was consistently lower than that of the other five groups; the 7-d compressive strength reached 80% of the 28-d compressive strength, and the later compressive strength increased slowly with age.The intensity development trend of the G0 group and G1 group was similar. The main reason is that these two groups of SAPs have the same mechanism of action. After adding water, the water-binder ratio of the G1 group increased and the maximum strength decreased in the later stage. However, the incorporation of the SAP is consistent, and thus, the change trend of strength with age was consistent. Secondly, the dry, mixed SAP absorbed the cement slurry, which is equivalent to reducing the water-binder ratio of the concrete. The hydration heat of the cement was intensified, and the water in the capillary pores of the concrete dissipated faster; therefore, the early strength is high and the later strength is small. Finally, in the cement hardening stage, the SAP water-release shrinkage left holes in the concrete, and the concrete compressive strength decreased, making its 90-d compressive strength lower than that of the J0 group.The compressive strength of the Y0 group was lower. The reason may be due to the fact that in the concrete mixing stage, the ion concentration of the cement slurry is greater than the ion concentration inside the SAP particles of the Y0 group. Therefore, the compressive strength is low in the early hardening stage. At the same time, it also leads to an uneven distribution of water in the concrete, resulting in large pores . HydratiThe strength development trend of group Y1 and group Y2 was similar. In both groups, the SAP was preabsorbed so that sufficient water was stored inside the SAP crosslinked network structure. The water absorbed by the saturated SAP in the Y1 group is quickly released during the concrete mixing process. With the increase in the water-cement ratio, the early compressive strength decreased. After the initial cement setting, the gradual release of the additional water stored in the SAP helps to form a denser and more homogeneous cementing matrix, which acts as an effective filler for the voids near the aggregate and ensures the dense filling of cement particles , thus reFrom the overall test results of compressive strength, it can be concluded that pre-water absorption of the water-absorbing resin has a better effect on the inner curing of concrete than dry incorporation. SAP presaturated water absorption and water consumption reduction have the same internal curing effect as SAP partial pre-water absorption.With 0.2% SAP content, the shrinkage strain of the concrete ring under different SAP incorporation methods was studied. The steel-ring strain test results of different concretes are shown in It can be seen from According to reference , the devThe reason for the above phenomenon is speculated to be that the steel-ring stress gradually increases with the concrete contraction, and the strain values of the three experimental groups are all lower than those of the J0 group, indicating that the incorporation of the SAP effectively reduces the concrete contraction. Studies have shown that the incorporation of an SAP will change the texture of concrete  and imprA scanning electron microscope (SEM) was used to observe the hydration products of cement and the changes in the microstructure of the cement matrix after incorporation of the SAP. The reasons for the change in mechanical properties and the crack resistance of internally cured concrete were analyzed. The results are shown in The microstructure of hardened cement is mainly composed of the following phases: a calcium silicate hydrate phase, calcium hydroxide, an unhydrated binder and an aggregate ,56. It cSAP cement-based materials have been proven to have self-healing properties ,58,59. WTherefore, the incorporation of an SAP slows down the early hydration rate, and the cement is further hydrated after water release; thus, water is released in the later stage of cement hardening and the hydration process is prolonged, which improves the compacting degree of concrete and enhances its crack resistance.As can be seen from When the SAP is dried and incorporated, the cement slurry will be absorbed in the mixing stage, resulting in the reduction in the water-cement ratio of the mixture. However, after the initial setting of concrete, the internal water content of concrete is low, and the SAP releases water prematurely; thus, the hydration process is shorter, and the early strength of the concrete is high and the later strength is low. At the same time, premature SAP water release also weakened the improvement in the concrete shrinkage. When the SAP is preabsorbed and incorporated, the water-binder ratio is reduced during the mixing phase, resulting in an increase in the water-cement ratio of the mixture. In the late curing period, the SAP releases enough water to delay the hydration rate so that the cement is fully hydrated and enough hydration products are generated to make up for the pores produced by SAP shrinkage. Therefore, the early strength of the concrete is low and the late strength is high, and the concrete crack resistance performance is greatly improved.(1)The SAP incorporation method has a significant effect on concrete fluidity. The fluidity of concrete increases when a preabsorbent SAP is added, and is positively correlated with SAP water content and SAP content. The fluidity of concrete decreases when the SAP is dried and is negatively correlated with SAP content.(2)It can be considered that the optimum content of this type of SAP in concrete is 0.2%. The compressive strength of concrete mixed with a preabsorbent SAP has a relatively low development before 28 d, and a relatively large increase after 28 d.(3)The shrinkage property of concrete mixed with an SAP is improved and the shrinkage strain is significantly reduced. The shrinkage strain of concrete mixed with a pre-absorbent SAP decreases the most, and no cracking occurs until the end of the test. The shrinkage-reducing effect of a dry SAP is limited and concrete still cracks.(4)The SAP stores water in the early stage of hydration, and releases water in the later stage to promote hydration. Therefore, the compactness of concrete is improved and the crack resistance is improved. The shrinkage reduction effect of a preabsorbent SAP is better than that of a dry SAP. The pre-water absorption method is recommended, and the pre-water absorption ratio is the maximum water storage rate of the SAP.