Background and Objectives: Understanding the relationships between subjective shoulder stiffness, muscle hardness, and various factors is crucial. Our cross-sectional study identified subgroups of shoulder stiffness based on symptoms and muscle hardness and investigated associated factors. Materials and Methods: measures included subjective stiffness, pain, muscle hardness, and factors like physical and psychological conditions, pressure pain threshold, postural alignment, heart rate variability, and electroencephalography in 40 healthy young individuals. Results: Three clusters were identified: Cluster 1 with high stiffness, pain, and muscle hardness; Cluster 2 with low stiffness and pain but high muscle hardness; and Cluster 3 with low levels of all factors. Cluster 1 had significantly higher central sensitization-related symptoms (CSS) scores than Cluster 2. Subjective stiffness is positively correlated with psychological factors. Conclusions: our results suggest that CSS impacts subjective symptom severity among individuals with similar shoulder muscle hardness. In recent years, the prevalence of shoulder stiffness has surged due to extended smartphone use and deskwork, with it being among the top reported symptoms in both men and women . ShouldeMuscle hardness was used as an objective evaluation method for shoulder stiffness. Higher trapezius muscle hardness and tenderness at the symptomatic site have been re-ported in individuals with shoulder stiffness ,7,8. SomThe emotional component of pain in individuals with shoulder stiffness has an association with conscious subjective stress . In addiHigh subjective stiffness and pain with high muscle hardness;Low subjective stiffness and pain with low muscle hardness;High subjective stiffness and pain with low muscle hardness;Low subjective stiffness and pain with high muscle hardness.The current literature reveals an inconsistency in the relationship between objective findings (muscle hardness) and subjective symptoms of shoulder stiffness ,9,10. ToAdditionally, we hypothesized that the differences between subgroups (subgroups 1 and 2) with similar muscle hardness but varying levels of subjective discomfort would be related to daily conscious psychological stress, CSS, or nonconscious psychological stress, which EEG or ANS indicates. On the other hand, we hypothesized that the differences in muscle hardness were associated with physical factors, such as posture alignment. This study aimed to verify the existence of subgroups of shoulder stiffness based on subjective symptoms and muscle hardness and to clarify the comprehensive factors associated with increased subjective symptoms and muscle hardness.This study involved 40 healthy young individuals . Participants with histories of numbness or musculoskeletal disorders of the neck, upper limbs, or back were excluded. This study excluded secondary shoulder stiffness, which was induced by a specific disease. In addition, participants were asked to avoid caffeine and alcohol and maintain their regular sleep duration before the measurement. The study was approved by the Kyoto Tachibana University Ethics Committee , and the participants provided oral consent after being informed about the study. This study was conducted between 1 April and 30 April 2023.Initially, we administered a questionnaire to assess physical and psychological factors related to shoulder stiffness. We then measured the participants’ height and weight and recorded EEG and ANS activity in a relaxed static sitting position. Subsequently, we evaluated postural alignment, muscle hardness, and pressure pain threshold (PPT). The study was conducted in a quiet environment at a room temperature of 23 °C, with participants wearing short-length clothing. For seated measurements, the participants used a chair that allowed their feet to touch the ground and were instructed not to lean on the backrest. All evaluations were performed between 1 pm and 6 pm within one day.We assessed the severity of the participants’ subjective stiffness and pain related to shoulder stiffness using an 11-point Numerical Rating Scale (NRS) ranging from 0 (none) to 10 (extreme).Based on a previous study ,5, we asWe used the Central Sensitization Inventory (CSI) to evaluate the CSS. CSS is a health-related symptom, such as headache, muscle stiffness, sleep disorder, and fatigue, based on central sensitization. The CSI, which consists of 25 items, has been validated for reliability and validity and helps diagnose CSS .We assessed participants daily conscious psychological stress levels using the NRS and participants’ mood at the time of measurement using the Two-Dimensional Mood Scale (TDMS) . The TDMPsychological stress, especially cognitive and emotional states, plays a role in the subjective experience of shoulder stiffness. EEG provides an objective measure of these psychological states. We conducted EEG measurements to use them as objective indicators of psychological stress, including cognitive and emotional states. An EEG  with a sampling frequency of 1000 Hz was used. Electrodes were placed at Fp1 and Fp2, associated with the prefrontal cortex, which is responsible for higher cognitive functions such as thinking, behavior, motivation, attention, and emotional processing ,25,26. TMATLAB R2023a was used to analyze brainwaves. The total measurement time was 5 min and was filtered with a 1–30 Hz bandpass filter. Frequency analysis was performed using the Fast Fourier Transform (FFT) in 1000-point segments every 5 s. The absolute power values of alpha (α) waves (8–12 Hz) and beta (β) waves (13–25 Hz) were calculated. The power values in each frequency band were log-transformed because they exhibited an exponential variation . This stThe ANS, responsible for bodily functions like heart rate, can provide insights into the overall physiological stress levels, potentially offering an understanding of the relationship between shoulder stiffness and systemic responses. ANS activity was measured simultaneously with the EEG using a plethysmography sensor . The sensor was attached to the index finger of the right hand of each participant. Plethysmography captures volume changes in blood vessels caused by the heart pumping blood. The sampling frequency was set to 1000 Hz. OpenSignals software (v. 2. 2. 3a) was used to analyze the volumetric pulse waves. After measuring the waves, frequency analysis was used to calculate sympathetic and parasympathetic nervous system activity using FFT. Absolute power values in the high-frequency band (HF: 0.15–0.40 Hz) and low-frequency band (LF: 0.05–0.15 Hz) were log-transformed as they exhibited exponential variation, and the LF/HF ratio was also calculated for statistical analysis . An incrTo evaluate the impact of postural alignment on shoulder stiffness, we assessed the participants’ posture during quiet sitting. They were instructed to look straight ahead and sit without leaning on a backrest. Markers were placed on the earlobe and seventh cervical vertebra (C7), and photographs were taken from 2.5 m to the participant’s left. Posture alignment was analyzed using the VisionPose application, which employs deep learning for posture estimation. VisionPose is utilized to measure postural alignment . The spiMuscle hardness directly ties to the physical manifestation of shoulder stiffness, offering an objective measure of the condition itself. We used a muscle hardness meter  to measure the hardness of the upper trapezius muscle fibers on both sides. The measurement location was based on a previous study that identified the midpoint of the line connecting the seventh cervical vertebra and the left and right acromions as the upper trapezius muscle at the measurement point . The parShoulder stiffness may result from localized sensitivities, and the PPT provides a means to assess the sensitivity or pain response in the shoulder region. After measuring muscle stiffness, we assessed the participants’ PPT using a digital force gauge . The measurement sites and conditions were the same as those used to measure muscle hardness . A gaugeTo classify the subgroups based on subjective and objective shoulder stiffness indicators, we conducted cluster analysis (Ward’s method) using standardized (Z-scored) values for subjective shoulder stiffness severity, pain severity, and muscle stiffness. A dendrogram was used to classify the patients into three subgroups. We performed logarithmic transformations for non-normally distributed continuous variables to compare variables between clusters, followed by one-way analysis of variance (ANOVA) and multiple comparisons (Bonferroni correction). Chi-square analysis was conducted for categorical variables such as sex. To examine whether factors related to subjective shoulder stiffness, pain, and muscle stiffness differed according to the presence or absence of shoulder stiffness, we used Pearson’s correlation analysis for the entire participant group and subgroups with and without shoulder stiffness. The significance level was set at 5%. We used R version 4.2.3 for statistical analysis.p < 0.01) and Clusters 2 and 3 (p < 0.01). Significant differences were also found in subjective stiffness and pain between Clusters 1 and 2 (p < 0.01) and Clusters 1 and 3 (p < 0.01) ( < 0.01) .These data showed three distinct clusters with varied degrees of subjective and muscle stiffness and pain. Interestingly, Cluster 2 had low subjective readings but still showed high muscle stiffness, indicating that subjective feelings do not always align with objective stiffness measures. The significant differences between Clusters 1 and 3 and Clusters 2 and 3 suggest there might be underlying factors differentiating these groups.The y-axis indicates the Z-score of the muscle hardness. The x-axis indicates the Z-score of subjective stiffness. The bubble size indicates the amount of pain. The orange circle indicates Cluster 1, the blue circle indicates Cluster 2, and the gray circle indicates Cluster 3.p < 0.01) but no significant differences between Clusters 1 and 3 or between Clusters 2 and 3. Furthermore, no significant differences were found across clusters for psychological stress indicators, including daily conscious psychological stress, TDMS comfort, arousal, stability, and activity levels.There were no significant differences in basic demographics  or physical stress indicators  between the clusters according to multiple comparisons and chi-square tests. The CSI scores revealed a significant difference between Clusters 1 and 2  or PPT. Furthermore, no significant differences were observed across all clusters for the ANS and brainwave activity, which were assessed as objective measures of unconscious psychological stress.The findings indicate that posture, PPT, ANS activity, and brainwave activity do not substantially contribute to differentiating the clusters. These results suggest that while these factors might have been suspected contributors, they do not significantly influence the clusters’ perceptions and objective measures of stiffness and pain.p < 0.01), CSI scores , and daily conscious psychological stress . Subjective pain showed significant positive correlations with subjective stiffness  and CSI score  and a significant negative correlation with the Fp1 α/β ratio . No significant correlation was observed with shoulder muscle hardness.p = 0.048), shoulder muscle stiffness , daily conscious psychological stress , and CSI scores . A significant negative correlation was observed with TDMS stability . Subjective pain only showed a significant positive correlation with moderate-load exercise . Shoulder muscle stiffness had a significantly positive correlation only with subjective stiffness . Conversely, participants without shoulder stiffness exhibited a significant negative correlation between muscle stiffness and parasympathetic nervous activity (HF power) .When analyzed according to the presence or absence of shoulder stiffness, participants with shoulder stiffness exhibited significant positive correlations between subjective stiffness and subjective pain (r = 0.41, The strong positive correlation between subjective stiffness and pain indicates that individuals who feel stiffer also tend to feel more pain. This could suggest a common underlying factor or that the sensation of stiffness exacerbates pain perception. The positive correlation of these factors with daily conscious psychological stress and CSI scores may imply that cognitive and psychological states influence or are influenced by stiffness and pain perceptions. Additionally, the fact that subjective pain had a negative correlation with the Fp1 α/β ratio might indicate that certain brain activities are inversely related to pain perception. The difference in correlations among participants with and without shoulder stiffness might also suggest different underlying processes or influences for these two groups. Overall, the results emphasize the complexity of shoulder stiffness and pain perceptions and their interaction with cognitive and psychological states.In this study, we hypothesized that the participants could be classified into four subgroups based on subjective stiffness, pain, and muscle hardness. However, the analysis resulted in three subgroups: Cluster 1 had high levels of subjective stiffness, pain, and muscle hardness; Cluster 2 had low levels of subjective stiffness and pain but high muscle hardness; and Cluster 3 had low levels of all three factors. Multiple comparisons between subgroups revealed significant differences only in CSI scores, with Cluster 1 showing higher values than Cluster 2. No significant differences were observed in other conscious, physical, and psychological stress-related factors. Correlation analysis investigating factors related to subjective stiffness, pain, and muscle hardness showed associations between subjective stiffness, pain, the TDMS stability score, daily conscious psychological stress, and the CSI score in participants with shoulder stiffness. Subjective pain was suggested to be related to the Fp1 α/β ratio. Moreover, muscle hardness tended to be lower in participants without subjective shoulder stiffness when parasympathetic nervous activity was high.In the present study, significant differences were observed in subjective shoulder stiffness and pain. Among the comprehensive factors related to conscious physical and psychological stress, only CSI scores showed significant differences, with no significant differences found in other factors. The CSI is a CSS questionnaire that assesses subjective symptoms, consisting of subfactors such as physical symptoms, emotional distress, headache, temporomandibular joint symptoms, and urological symptoms . The CSIBetween Clusters 2 and 3, there were no significant differences in subjective shoulder stiffness or pain, both of which were low. However, there was a significant difference in muscle hardness. Previous studies reported that the hardness of the trapezius muscle does not directly reflect subjective shoulder stiffness, and the findings of this study are consistent with those of previous studies. Clusters 2 and 3 had low subjective shoulder stiffness and pain scores, suggesting that shoulder stiffness caused minimal conscious psychological stress. No significant differences were observed in factors related to psychological stress. The differences between clusters 2 and 3 were presumed to be influenced by conscious and unconscious physical stress factors. Previous studies have reported that forward head posture  and VDT For Clusters 1 and 3, Cluster 1 showed significantly higher values for subjective shoulder stiffness, pain, and muscle hardness. Although there was no significant difference in CSI, Cluster 1 had more severe cases and higher CSI values than Cluster 3. No significant differences were found in other factors, suggesting that even if subjective shoulder stiffness, pain, and muscle hardness differed, there might be no differences in conscious/unconscious physical and psychological stress factors. Furthermore, previous studies have reported associations between blood circulation and shoulder muscle fatigue . FactorsIn this study, we found that CSS severity was associated with subjective symptoms. This study observed significant positive correlations among CSI scores, subjective stiffness, and pain. It has been reported that CSI scores are related to pain due to the influence of conscious psychological stress . AdditioTo our knowledge, this is the first study to analyze unconscious psychological stress using brainwaves and ANS activity. Our results revealed that individuals with similar muscle hardness experienced different subjective symptoms, with CSS emerging as a related factor. Moreover, individuals who did not report subjective shoulder stiffness exhibited lower muscle hardness when the parasympathetic nervous system activity was high. Regarding brain wave activity, the entire study population demonstrated a tendency for subjective pain to decrease as relaxation increased owing to alpha wave activity in the left hemisphere, which indicates pleasure. Traditionally, interventions for shoulder stiffness have focused on physical factors such as posture . HoweverThis study had some limitations. First, we assessed the muscle condition using a muscle hardness meter. Previous studies have suggested that impaired blood flow to the shoulder muscles due to circulatory disorders could be a factor in shoulder stiffness . Using mOur study established a connection between CSS and variations in the severity of subjective symptoms, even among participants with comparable shoulder muscle hardness. Furthermore, elevated conscious psychological stress and diminished brain wave activity indicating positive emotions, are notably linked with subjective symptoms in those suffering from shoulder stiffness. Our findings may help in the management of shoulder stiffness.


The effects of ultrasonic treatment on the structure and physicochemical properties of pea starch were investigated in this study. The results showed that ultrasonic treatment increased the hydrolysis rate and particle size of pea starch. In the process of treatment, there were some depressions and pores on the surface of pea starch granules. Although the crystallization type of starch was retained, its crystallinity decreased. The pasting temperature of pea starch remained stable after ultrasonic treatment, but its peak viscosity, trough viscosity, cold viscosity, breakdown viscosity and setback viscosity all declined significantly. The transparency of starch paste decreased, but proper ultrasonic treatment could improve the strength of starch gel. The obtained results can provide a reference for the physical modification of pea starch. As the second largest edible legume in the world, the pea is rich in nutrients such as starch and protein, and its starch content is about 45% to 55%. Compared with corn starch and potato starch, pea starch is easy to extract and cheap ,2. HowevUltrasonic treatment mainly promotes the movement of starch molecular chains through mechanical effect, cavitation effect and thermal effect, changes the original hydrogen bond and double helix structure in starch granules and regulates the physicochemical properties of starch through the influence on the surface and internal structure of starch granules ,8. Hu etIn this study, the effects of ultrasonic treatment time on the structure and physicochemical properties of pea starch were systematically investigated, in order to provide a theoretical basis for the development of modified pea starch.Pea starch, glucose, phenol and sulfuric acid were purchased from Shanghai Yuanye Biotechnology Co., Ltd. . Ultra-pure water was produced using a Thermo Genpure UV/UF water system .w/w) and loaded on a SCIENTZ JY99-IIDN ultrasonic homogenizer . The sample vessel was cooled using an ice bath. The sample was treated at 680 W for 10, 20 and 30 min. The action time and interval time of ultrasound were set to 2 s. After ultrasonic treatment, the starch was washed with distilled water and dried at 40 °C.Starch was suspended in 100 mL distilled water with a concentration of 20% . By comparing it with the standard curve of glucose, the amount of glucose produced via hydrolysis was determined and the hydrolysis rate was calculated .Native and treated pea starch were evenly coated on the conductive adhesive attached to the loading platform, and then sent to the sample compartment after vacuum gold spraying. An FEI Quanta 200 environmental scanning electron microscope  was used to observe the starch particles under the magnification of 2000 times, and the representative pictures were recorded via microscope .An appropriate amount of 1% starch solution was dropped on the slide, covered with the cover slide and placed on the loading platform. Polarizing characteristics of starch were observed through the use of a Hengping BH200P polarizing microscope  200 times .After the appropriate dilution of starch solution, particle size distribution was determined using a BETTER BT-9300H laser particle analyzer .An appropriate amount of starch was taken into the sample tank, the excess sample was scraped off and the surface of the sample was kept smooth. A Rigaku Smartlab SE X-ray diffractometer  was used to determine the crystalline structure of starch. Test parameters: scanning rate, 8°/min; scanning range, 5–70°; step size, 0.02°. The crystallinity of the sample was calculated using JADE6.0 software .A total of 20 mL of 1% starch suspension was heated in a boiling water bath for 15 min. The light transmittance of starch paste at 620 nm after cooling was measured through the use of a Persee TU-1810 spectrophotometer  with water as the control .A 3.0 g starch sample was mixed with 25 mL distilled water and placed in a Perten RVA 4500 rapid visco analyzer  . The samThe starch sample (3.2 g) was put into a 50 mL beaker and distilled water was added until the total mass was 40.0 g. The starch solution was heated at 95 °C and stirred for 30 min until the starch had gelatinized completely. The starch that had been cooled to room temperature was transferred to a refrigerator and kept at 4 °C for 16 h to form a stable starch gel sample. The strength of starch gel was determined using a TA-XT Plus texture analyzer  with a P/0.5 probe. The measurement parameters were set as follows: trigger force, 2 g; compression distance, 10 mm; descending speed, measuring speed and rising speed of the probe: 1.5, 1.0 and 1.0 mm/s, respectively .All experiments were repeated three times and expressed as mean ± standard deviation. The statistical comparison was based on Duncan’s test with a confidence level of 95%. Origin 8.0 software  was used for statistical processing and plotting.Studies on the modification of pea starch via heat–moisture treatment , amylomaUltrasonic treatment will change the morphology of starch particles, such as cracks, collapses, holes, etc., whereby the extent of which depends on the intensity of the ultrasonic treatment . Figure The formation of a polarized cross originates from the spherulite structure of starch. When polarized light passes through starch particles, an obvious polarized cross is observed, which can reflect changes in the molecular chain arrangement and crystal structure of starch . Figure Starch granules from different cereals have their own unique shapes and sizes, and the size of starch granules has an important influence on the properties of starch. There are differences in gel properties, gelatinization, retrogradation and enzymatic hydrolysis properties of starch particles with different sizes . The effThe common XRD patterns of starch are A-type, B-type and C-type . As showThe transparency of starch paste is reflected by the transmittance of starch paste , and theThe gelatinization of starch is the thermal disorder of the crystal structure of starch granules, which is not only related to the type of starch, system temperature and pH, but is also affected by the crystal structure of starch and the ratio of amylose to amylopectin . In TablUltrasonic treatment has an obvious effect on the texture properties of starch gel. Song et al. found that the gel strength of Pickering emulsion gels developed by sorghum flour was proportional to the amount of sorghum flour added . In thisUltrasonic treatment had significant effects on the structure and physicochemical properties of pea starch. Due to the hydrolysis caused via ultrasound, the surfaces of the starch particles were sunken, meaning they could easily absorb water, expand and aggregate, resulting in an increase in particle size. With the extension of treatment time, the crystallinity of starch slightly decreased, and the peak viscosity, trough viscosity, cold viscosity, breakdown viscosity and setback viscosity declined significantly, but the pasting temperature remained stable. And, suitable ultrasonic treatment could improve the strength of starch gel.


Autologous fat grafting (AFG) is the most prevailing tool for soft tissue regeneration in clinics, although efficiency is limited to unpredictable volume resorption due to poor vascularization and eventual necrosis. This study sought to improve the AFG efficiency using a hydrogel as a carrier for human fat graft (F) with and without platelet-rich plasma (PRP). PRP is clinically well known for the local release of several endogenous growth factors and has been in clinical use already. A human-fat-graft-encapsulated pectin–alginate hydrogel (FG) was developed and characterized. PRP was added to F to develop a human fat graft with PRP (FP). FP was admixed with a pectin–alginate hydrogel to develop FGP. FG and FGP showed the smooth injectable, elastic, and shear-thinning properties. FG and FGP groups showed enhanced cell viability and proliferation compared to the control F in vitro. We also investigated the in vivo angiogenesis and neo-adipogenesis ability of F, FG, FGP, and FP in nude mice after subcutaneous injection. After 2 and 4 weeks, an MRI of the mice was conducted, followed by graft explantation. The explanted grafts were also assessed histologically and with immunohistochemistry (IHC) studies. MRI and histology results revealed better vascularity of the FG and FGP system compared to fat graft alone. Further, the IHC studies, CD 31, and perilipin staining also revealed better vasculature and adipogenesis of FG and FGP systems. These results indicate the enhanced angiogenesis and adipogenesis of FG and FGP. Thus, developed pectin–alginate hydrogel-based fat graft systems FG and FGP replenish the native microenvironment by mediating angiogenesis and adipogenesis, thereby maximizing the clinical outcomes of autologous fat grafting. Autologous fat grafting (AFG) is an inevitable revolutionizing technique employed for soft tissue augmentation following congenital, traumatic, and post-surgical loss ,6,7,8,9.The regained popularity of fat grafting following liposuction has led to a search for various aspects to improve fat graft survival in adipose tissue engineering (ATE). This includes technical aspects of harvesting, processing, and preparation of sterile fat graft  and incoVarious strategies have been tried to improve fat graft survivability by adding of adipocyte-derived stem cells, scaffolds, hydrogels, and growth factors ,17,24. TThe addition of PRP, commonly employed in clinical situations along with fat graft, tends to favor the viability by reducing the resorption rate ,38,39,40Based on the above aspects, we focused on improving the cell survival, adipogenesis and, vascularization of fat graft by encapsulating it in an injectable pectin–alginate hydrogel as shown in The fat graft and PRP were obtained from a consenting human volunteer undergoing liposuction for lipodystrophy. Institutional ethical committee approval was obtained. Alginic acid sodium salt from brown algae , pectin derived from citrus peel , calcium chloride, Oil Red O, Harri’s Hematoxylin, and Eosin solution were procured from Sigma. Anti-human CD 31, anti-human perilipin, and anti-human vimentin antibodies were purchased from Abcam. α-MEM, Pen-strep, FBS, and Trypsin-EDTA were obtained from Invitrogen. Live staining kit and Quanti-iT Picogreen dsDNA assay kit were procured from Thermo Fisher Scientific.After infiltrating the tumescent solution, fat graft was harvested from a healthy human donor undergoing abdominal liposuction by syringe technique using a 10 mL syringe and a 3 mm blunt tip Mercedes cannula. The harvested fat graft was centrifuged at 3000 rpm for 3 min to remove the upper oily layer while the decanting lower layer of blood and tumescent solution. The middle layer containing viable adipocytes was used as the fat graft (F) as shown in A total of 4% alginate and 4% pectin were dissolved in cell culture media  at a volume ratio of 3:5. Both the polymeric solutions were allowed to mix thoroughly for 30 min, followed by the addition of 2 wt% calcium chloride as a cross-linking agent. The homogenous, smooth hydrogel obtained was termed pectin–alginate hydrogel (G). The fat-graft-encapsulated hydrogel (FG) was prepared by blending the fat and hydrogel at a volumetric ratio of 60:40, as illustrated in About 20 mL of whole blood was withdrawn from the same patient undergoing liposuction. Platelet-rich plasma (P) was prepared in our blood bank using a standard protocol. Fat graft with PRP (FP) was prepared at a volumetric ratio of 9:1. Fat graft with PRP-encapsulated hydrogel (FGP) was prepared by fat with PRP in the above-mentioned ratio by blending hydrogel at a volumetric ratio of 60:40, which is shown in The developed hydrogel was loaded into a 1 mL syringe and injected through an 18 G needle to understand its flow behavior. The fat-graft-encapsulated hydrogel was kept in a beaker, inverted, and undisturbed to assess its solid behavior.−1) to analyze the functional groups of alginate, pectin, and fat graft present in the hydrogel. The lyophilized fat-graft-encapsulated hydrogel (FG), fat graft (F), hydrogel (G), alginate, and pectin were pelletized with potassium bromide and analyzed using the FTIR instrument.FTIR spectrometer  was performed  and viscous modulus (G″). The change in elastic modulus, viscous modulus, and phase angle (δ) was compared among groups.The viscoelastic property of F, G, FP, FG, and FGP was evaluated using Malvern Kinexus Pro Rheometer armed with Peltier system for regulating the temperature. All the studies were performed using 20 mm parallel-plate geometry with a 0.5 mm gap at 37 °C. The injectable property of the prepared hydrogel was analyzed by flow curve in the range of 0.1 s2 for 24 and 48 h. After the specific time points, dsDNA was extracted from cells by incubating in lysate buffer (1% Triton X-100 in PBS) for 15 min. The dsDNA content of the cell lysate was then quantified using a fluorometric picogreen assay kit at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The DNA content of the HUVECs seeded in contact with various systems  was quantified. The dsDNA standards and samples were used in triplicate. Calcein AM staining and Oil Red O staining was carried out to assess the viability of fat cells and lipid accumulation of the developed FG system.The cytocompatibility of F, FP, FG, and FGP was performed using human umbilical vein endothelial cells (HUVECs). The cells were seeded onto the above groups with a seeding density of 10,000 cells per well of a 96-well plate. This was cultured at 37 °C in an incubator complemented with 5% COThe hydrogel (G), fat graft, and its various combinations  were injected onto the dorsum of nude mice in the subcutaneous region (1 mL) at three sites, one in the upper back and two in the lower back region using a 2 mL syringe and an 18 G needle. MRI was carried out to assess vascularity using a Bruker 7-tesla small animal magnetic resonance image coiler with Gadolinium as contrast (0.4 microliter/g of 0.5 mmol/mL of meglumine gadoterate). Nude mice were euthanized by an overdose of anesthesia  at the end of the 2nd and 4th weeks. Injected fat graft groups were carefully dissected, explanted, and incubated in neutral 10% formalin solution, followed by paraffin embedding for further histological evaluation.Paraffin-embedded sections of 5 μm thickness were deparaffinized and dehydrated using graded series of ethanol and were stained using hematoxylin, followed by Eosin.Vascularity was assessed by antibodies against CD 31 as an endothelial marker, and the viability of the adipocytes was assessed by perilipin staining of injected fat graft groups . VimentiThe optimal concentration of the fat graft and injectable hydrogel at a ratio of 60:40 was used to form a dense porous network to encapsulate the pectin–alginate hydrogel. Divalent calcium ions act as a cross-linker forming a stable hydrogel, enhancing the smooth flow of hydrogel through the syringe of an 18G needle as depicted in −1 due to asymmetric and symmetric stretching of carboxylate vibrations, respectively. Pectin showed peaks at 1017 to 1103 cm−1, which correspond to carboxyl groups. The pectin–alginate hydrogel (G) confirmed the presence of both pectin and alginate. The peaks of control fat (F) were observed at 2922, 1746, and 1466 cm−1, indicating the asymmetric stretching of –CH2 group, fatty acid ester, and –CH2 bending, respectively. The final system of the fat-graft-encapsulated pectin–alginate hydrogel (FG) speculated that all peaks corresponded to individual components of the hydrogel, confirming the encapsulation of fat graft into the hydrogel.An inversion test was performed to confirm the gelation property of FG, and it was found to be stable B. The smViscoelastic behavior was studied to understand the injectability and strength of the prepared hydrogels. The elastic and viscous modulus was analyzed against frequency, as shown in Accumulation of lipid droplets in FG was observed using Oil Red O staining, indicating the retained adipogenic property of fat graft after encapsulation A. CalceiThere was no inflammation, seroma, or abscess formation of grafts or adjacent tissue during this study. The specimens were harvested for histological examination A. The reHistological analysis using H&E staining in Blood vessel formation and the endothelialization of de novo explants were analyzed using CD 31 antibody staining. FP, FG, and FGP had higher ingrowth of numerous capillaries compared with the F group at 4 weeks A. The F To confirm the origin of the newly formed adipocytes, vimentin staining was carried out. The human adipocytes stained positive, whereas nude mice adipocyte did not, thus ensuring the neo-adipogenesis of the human fat graft .The in vivo study of the developed fat-graft-encapsulated pectin–alginate hydrogel showed improved outcomes in fat grafting. This dynamic system can improve the current autologous fat grafting (AFG) scenario by minimizing undesirable effects, such as necrosis, and resorption. Though several studies focused on increasing cell survival, there exists a void on a simpler and mechanistic approach towards AFG.Low methoxy pectin and alginate undergo sol–gel phase transition via classical scalar percolation theory, and their synergistic effect tends to mold the cellular behavior within hydrogels ,43,44. TThe injectable nature of the developed system is of utmost importance in this study as the intention is to deliver in a minimally invasive fashion. The shear-thinning ability of a hydrogel determines the injectable nature of the material for filling defective sites. It was found that all the hydrogel groups retained their strength with minimal variations over a fixed frequency range, even under constant strain C. Fat grIn vivo vascularity and adipogenic ability followed the same trend as in vitro cell proliferation. The origin of neo-adipogenesis when injecting the human fat graft into the immune-deficient nude mice was questionable as to whether it is of human or mice origin. The neo-adipogenesis could be of either pre-existing preadipocytes of human fat graft or due to recruitment of endogenous precursors from the host, i.e., nude mice. Contrary to the study conducted by Stillaert et al., vimentin staining, as shown in Haug et al. studied the effect of HUVECs on neovascularization in an adipogenic mouse model for 6 months. They found that HUVECs play a significant role in stabilizing vessel formation . PRP-conThe developed FG hydrogel is speculated to provide a conducive microenvironment that encourages cell migration, proliferation, and differentiation. Adrian et al. demonstrated the effect of angiogenic growth factors with the commercially available extracellular matrix during adipogenesis. The combination of growth factors significantly fortified the adipogenic ability of ADSCs ,52. The Autologous fat graft being a rich source of ADSCs, preadipocytes, and adipocytes, with the hydrogel, serves as the next-generation candidate for carrying bioactive factors necessary for adipogenesis. In the conventional method of AFG, the fat graft survives by nutrient and oxygen diffusion during the initial 48–72 h until neovascularization occurs but is restricted to peripheral sites, resulting in necrosis of the inner core of the fat graft . As neovThe pectin–alginate hydrogel system provides the hydrophilic environment, facilitating the diffusion of the nutrients and thereby extending adipocyte viability. The hydrophilic nature of the hydrogel has the potential to absorb nutrients from the surrounding tissues and to diffuse gases and metabolites, enriching the core of the fat graft. The admixing of the hydrogel with fat graft encapsulates the cells, protecting them from inflammatory cells . It alsoIn this study, a human fat graft encapsulated into a pectin–alginate hydrogel was developed to enhance the adipogenesis and angiogenesis of fat graft. The prepared hydrogel is stable, injectable, and has a shear-thinning property. Biocompatibility of the prepared F-containing hydrogel system showed enhanced cell growth. In vivo MRI imaging showed enhanced vascularity of the fat-graft-containing gel system compared to F alone. Histology and immunohistochemistry also revealed better angiogenesis and adipogenesis of the fat-graft-encapsulated hydrogel system. Vimentin staining proved that neo-adipogenesis is of human origin. Hence, this dynamic hydrogel system can act as a carrier for fat graft in AFG to overcome the current drawback of resorption. This fat-graft-encapsulated pectin–alginate hydrogel system has potential applications in soft tissue regeneration.


Lactococcus lactis lactis subspecies 1.2472, Streptococcus thermophilus 1.2718, and thermostable Lactobacillus rhamnosus HCUL 1.1901–1912 were used to ferment rice flour for preparing rice bread. The characteristics of fermented rice bread were studied to elucidate the mechanism by which fermentation improves the anti-staling ability of rice bread. The amylose content of rice flour increased after fermentation. The peak viscosity, attenuation value, final viscosity, recovery value, and gelatinization temperature decreased. Amylopectin was partially hydrolyzed, and the amylose content decreased. The crystallinity of starch decreased, and the minimum crystallinity of Lactococcus lactis subsp. lactis fermented rice starch (LRS) was 11.64%. The thermal characteristics of fermented rice starch, including To, Tp, Tc, and ΔH, were lower than RS (rice starch), and the △H of LRS was the lowest. Meanwhile, LRS exhibited the best anti-staling ability, and with a staling degree of 43.22%. The T22 of the LRF rice flour dough was lower, and its moisture fluidity was the weakest, indicating that moisture was more closely combined with other components. The texture characteristics of fermented rice bread were improved; among these, LRF was the best: the hardness change value was 1.421 times, the elasticity decrease was 2.35%, and the chewability change was 47.07%. There, it provides a theoretical basis for improving the shelf life of bread.In this study,  Rice is the most widely consumed cereal in the world, with nearly 4 billion people considering it as a staple food, accounting for 23% of their dietary energy intake . Rice isThe processing technology and composition of starch-based foods are significant factors that influence the staling of such products ,10. CurrLactococcus lactis subsp. lactis and Streptococcus thermophilus is not reported yet. Therefore, this study investigated the anti-staling mechanisms of three types of lactic acid bacteria-fermented rice flour and rice starch and concurrently produced bread from them to examine the anti-staling capacity of bread. The aim of this study was to provide a theoretical foundation for extending the shelf life of bread.In recent years, numerous researchers worldwide have explored the anti-staling mechanisms of rice-based foods ,16,17. MLactobacillus rhamnosus HCUL 1.1901–1912 , Lactococcus lactis subsp. lactis (strain: 1.2472), and Streptococcus thermophilus (strain: 1.2718) were purchased from China General Microbiological Culture Collection Center (CGMCC); Japonica rice  was purchased from Heilongjiang Yifeng Commodity Trading Co., Ltd. .; wheat flour  was purchased from Qingdao Bailemai Food Co., Ltd. . Milk powder (food grade) was purchased from Bright Dairy Co., Ltd. ; butter (food grade) was purchased Shanghai DeYang Co., Ltd. ; angel yeast, sugar, and salt (food grade) were all purchased from a local market; MRS broth medium, sodium hydroxide, hydrogen chloride, ethanol, and sodium chloride were all analytically pure reagents.Lactobacillus rhamnosus (strain HCUL 1.1901–1912), Lactococcus lactis subsp. lactis (strain 1.2472), and Streptococcus thermophilus (strain 1.2718) were stored at −80 °C in 25% glycerol. Prior to use, cultures were aseptically thawed and activated for 24 h in sterile MRS broth at 37 °C till the viable count reached about 108 cfu/mL, and then these were used in the following analyses [analyses ,22.Streptococcus thermophilus fermentation suspension (5%), Lactococcus lactis subsp. lactis fermentation suspension, and thermostable Lactobacillus rhamnosus fermentation suspensions, all prepared using distilled water as the baseline. The japonica rice to distilled water ratio was 1:4. Specifically, the flask was then incubated at 37 °C for 1 d. The prepared fermented rice broth of Lactococcus lactis subsp. lactis rice flour (LRF) was homogenized, centrifuged at 4000 r/min for 15 min, and the supernatant was discarded. The obtained precipitate was dried at 45 °C in a hot-air dryer for 24 h, and then crushed with a pulverizer and sieved using an 80-mesh screen to obtain the LRF [Lactobacillus rhamnosus-fermented rice flour (TRF) and Streptococcus thermophilus-fermented rice flour (SRF) were prepared using the same method.A conical flask was charged with a mixture of  the LRF . ThermosLactococcus lactis subsp. lactis fermented rice starch (LRS), Streptococcus thermophilus fermented rice starch (SRS), and thermostable Lactobacillus rhamnosus fermented rice starch (TRS) were prepared using the same method. Rice flour (RF) was used as a control and did not require further treatment.Rice starch (RS) was prepared as follows: Rice flour was added into 0.2 g/100 mL NaOH solution at a solid–liquid ratio of 1:3 (g/mL). The mixture was extracted for 3 h and centrifuged at 3000 r/min for 10 min. The supernatant was discarded and the upper layer of the brownish material in the sediment zone was removed. The residue was washed with water and centrifuged four times until the starch paste turned white. The slurry was adjusted to pH 7.0 with 1 mol/L HCl, centrifuged, dried at 30 °C, and sieved through an 80-mesh screen, and thus rice starch was obtained . LactocoThe bread ingredients  were mixTo analyze the content of the three types of starches, the methods described by Li et al.  were useThe viscosity of the samples was determined using a rapid viscosity analyzer (RVA) according to the AACC Approved Method 61–02 (2000). Each RVA sample measurement container comprised 3 g of the sample and 25 mL of distilled water. The sample measurement container was placed in an RVA testing instrument equipped with a paddle, and stirred at a constant speed of 160 rpm. The temperature control software was activated, raising the starch slurry temperature to 50 °C to start the timing. The temperature was held at 50 °C for 1 min, then increased to 95 °C (reached at 4.75 min), followed by a 2.5 min holding period at 95 °C (until 7.25 min). Subsequently, the temperature was decreased to 50 °C (by 11 min) and maintained until 16 min ,27,28. T−1 and scanning range of 4000–400 cm−1. Absorbance ratios of 1047/1020 cm−1 and 995/1020 cm−1 were calculated from the deconvoluted spectra [A mixture of the three different rice starches (6.0 mg) and 200 mg of KBr (spectroscopic grade) was ground into a fine powder and pressed into KBr pellets. Infrared spectra were recorded using a FT-IR spectrometer  with a resolution of 4.0 cm spectra ,30.v/v), gelatinized in a boiling water bath for 1 h, and stirred at 30 °C for 48 h. Thereafter, the samples were passed through a 0.45 μm filter and the filtrate (200 μL) was injected into a high-performance liquid exclusion chromatography system, equipped with a multi-angle laser light scattering (MALLS) detector , a refractive index (RI) detector , and Ultra hydrogel TMLinear 300 mm × 7.8 mmid × 2 (×2 means two columns in series). The mobile phase was a 0.1 mol/L NaNO3 solution containing 0.02% NaN3. The temperature of the column was set as 30 °C.The molecular weight distribution of the three rice starches was measured as per the method of . SamplesDiffraction patterns of the samples were acquired using XRD  at 30 kV. The samples were scanned at 10 mA in a range of 4–40° (2θ) using Cu-Kα radiation. The relative crystallinity of the starch granules was calculated as the ratio of the sample peak area to the total diffractogram area using JADE software  ,33.Differential scanning calorimetry was used to measure the thermal properties of rice starch including the gelatinization onset temperature (To), gelatinization peak temperature (Tp), gelatinization completion temperature (Tc), and gelatinization enthalpy (ΔHgel). A measure of 3 mg rice starch was mixed with 7 μL distilled water in an aluminum pan, and was sealed hermetically and equilibrated overnight at 4 °C. The heating range was 20−100 °C at a rate of 10 °C/min. An empty pan was used as the reference. All samples were analyzed in triplicate.Completely gelatinized rice starch was stored at 4 °C for 7 days, and the To, Tp, Tc, and staling enthalpy (ΔHret) of the crystals formed by staling were measured. The degree of staling was calculated using the following equation:In the formula: ΔHret is the staling enthalpy/(J/g); ΔHgel is the gelatinization enthalpy (J/g).2) relaxation time and the fraction of signal amplitude of protons in each part of T2. Three grams of breadcrumb  were placed in a 40 mm NMR tube, which was then sealed with Parafilm®, to prevent moisture loss during the NMR experiment. The parameters used in the CPMG sequence: the proton resonance frequency was 22.6 MHz, the number of echoes C0 = 3000, the number of repeated scans NS = 8, and the half-echo time TE = 0.3 ms. Using CONTIN software 20 to call CPMG sequence inversion, each peak time constant T2i (peak top time) and its area fraction A2i were recorded for subsequent analysis [A low-field (21 MHz) 1H nuclear magic resonance spectrometer  operating at 32.0 ± 0.1 °C was used to study the range of proton molecular mobility by measuring the transverse  and compressed (trigger force = 5 g) to 50% deformation with a cylindrical probe (P/35 DiaCylinder Aluminum) and the following parameters: pre-test speed (2 mm/s), test speed (1 mm/s), and post-test speed (2 mm/s). Hardness (height of the first compression peak) and elasticity (ratio of second/first compression peaks) were determined [The hardness and elasticity of the breadcrumbs were measured using a TA.new+plus texture analyzer . The central part of the bread was cut into a cube  of the zone I starch was slightly lower than that of rice starch, indicating that the molecular chain length of zone I rice starch was altered by fermentation. The molecular weight distribution of rice starch in zone II narrowed, indicating that fermentation made the amylose component in zone II singular and played a role in purifying the starch. At the same time, the retro gradation value of starch decreases, playing an anti-staling role. It has been shown that fermentation reduces the molecular weight of millet starch, resulting in the partial hydrolysis of amylopectin in region I and a decrease in amylose content in region II [egion II .−1, -CH absorption peaks appeared in the range of 2800–3100 cm−1, and -OH absorption peaks were seen in the range of 3200–3500 cm−1.Fourier transform infrared spectroscopy is widely used because of its sensitivity to changes in starch molecular conformation, enabling the analysis of the molecular structure of starch . InfrareThe infrared absorption spectra of rice starch fermented by the three strains and the unfermented rice starch showed no significant differences in peak shape at −1 represented the structural characteristics of the amorphous region of starch, whereas the infrared absorption at 1047 cm−1 represented the structural features of the crystalline region of starch molecules. The absorbance ratio represented the degree of order of the starch double helix arrangement [−1 and 995/1022 cm−1 represent changes in the internal order (DO) and double helix content (DD) of the starch molecules, respectively. A greater transmittance at 995 and 1047 cm−1 or a lower transmittance at 1022 cm−1 indicated an increase in the crystallinity of the starch structure [Through infrared structural analysis, the deconvolution of the absorption peaks was performed to analyze the short-range ordered structure of the starch molecules. Park et al.  found thangement . The tratructure .From Starch is a highly ordered macromolecule comprising crystalline and amorphous regions. X-ray diffraction (XRD) patterns were used to analyze the long-range order of starch. The crystalline structure represents a long-range order and is characterized by distinct diffraction peaks. In the crystalline region, the grain size is large, and the crystal form is well ordered. In contrast, the amorphous region  is characterized by short-range order and long-range disorder with obvious diffuse scattering features .As indicated in Thermal properties can be employed to determine starch gelatinization, and in general, the thermal properties of starch are related to the content of linear starch in the crystalline region of starch granules, the distribution of branched starch chains, and the double helical structure of amylopectin chains .As shown in After gelatinization, rice starch undergoes transformation from a disordered to an ordered structure during storage, with rearrangements leading to recrystallization. The higher the staling enthalpy, the more pronounced is the recrystallization, indicating a more severe level of staling. Rice starch was assessed using DSC at different storage time intervals to analyze changes in its thermodynamic properties over the storage period.After staling, completely gelatinized fermented rice starch exhibited recrystallization between molecular chains, resulting in a single exothermic peak with higher symmetry during the heating process. As shown in 21 corresponds to bound water, primarily water tightly bound to starch or gluten proteins; T22 represents weakly bound water, flowing between bound water and free water, primarily associated with interactions among macromolecules such as proteins and starch; and T23 represents free water [21 signal in the fermented rice flour dough was greater than that in RF, with SRF > LRF > TRF. The T22 peak was the main peak, accounting for approximately 80% of the total signal, indicating that in dough with well-formed gluten, the primary form of water was weakly bound. Differences were observed in the moisture forms and distributions of the four types of rice flour dough. The T22 peak times for the three fermented rice flour doughs were shorter than those for RF (7.10 ms), with TRF (5.34 ms) > LRF (4.64 ms) ≈ SRF (4.60 ms). From the above analysis, it can be concluded that dough made from RF had the highest water flowability, with water being loosely associated with other components, compared to dough made from fermented rice flour. Among the three types of fermented rice flour dough, LRF had the highest amplitude of the T21 signal and the shortest T22 peak time, indicating that the water flow ability was the weakest in the LRF dough, and that water was more tightly bound to other components.In ee water ,58,59. A2 and corresponding peak area A2 of the fermented rice flour dough are shown in 21 represents water within starch granules or tightly bound to proteins, whereas A22 represents water outside the starch granules or within the protein network [21 in the fermented rice flour dough was consistently higher than that in the RF dough, whereas A22 was lower, indicating a tighter association between water and other components of the fermented rice flour dough, resulting in lower water mobility. The interaction between water and starch is pivotal in determining the fluidity of water in the dough, as proposed by Doona et al. [The effects of different bacterial strains on the T network . A21 in a et al. . In thisa et al. . This coTexture parameters of bread include hardness, elasticity, chewiness, adhesiveness, cohesiveness, and viscosity, which are used to evaluate bread quality . In thisHardness is the most important textural parameter of bread and serves as a crucial indicator to assess the level of bread staling. This represents the peak pressure during the first penetration of the sample . Figure Elasticity reflects the extent of structural damage to the bread network during the first compression in TPA testing, and is an important indicator for evaluating bread staling . As showAs shown in In this study, lactic acid bacteria fermentation hydrolyzed short amylopectin, increased the proportion of longer amylopectin and amylose in rice flour, and degraded amylose. This resulted in a subsequent reduction in peak viscosity, setback viscosity, final viscosity, setback value, and gelatinization temperature of fermented starch. FT-IR and XRD analyses indicated a decrease in short- and long-range order for fermented rice flour, with LRS exhibiting the lowest crystallinity. DSC thermal analysis showed a general decrease in thermal parameters of rice starch, along with lowered staling degrees, with LRS having the smallest at 44.71%. Low-field NMR measurements revealed that water mobility in the fermented rice flour dough was limited, with tighter water interactions with other components, which was particularly pronounced in LRF. Texture analysis demonstrated that during storage, the hardness of non-fermented rice flour bread increased by a factor of 1.441, while fermented rice flour bread showed an increase by a factor of 1.421 for LRF, 1.426 for SRF, and 1.428 for TRF.By investigating the physicochemical properties of fermented rice flour and rice starch, as well as the quality characteristics of dough and bread products, this study provides scientific evidence for the anti-staling properties of lactic acid bacteria-fermented rice flour and lays a theoretical foundation for extending the shelf life of rice bread. In this study, the anti-staling mechanism of rice bread prepared from fermented rice flour was analyzed only from the perspective of starch. However, further studies are required to investigate the mechanism of action of proteins and the interactions between proteins and starch in bread anti-staling.


Cassava chips (CSC) have typically been used as an energy source for ruminant rations in tropical regions due to their high starch content and higher degradability rate in the rumen, which is higher than 90%. However, inconsistencies in production and fluctuations in prices encourage animal nutritionists to search for alternatives to CSC. The introduction of new tuberous plant species may help improve local feed diversity while also reducing feed shortages in particular areas. The winged bean tuber (WBT) has a high proportion of starch that might be able to replace CSC as an energy source. Moreover, it was discovered that steamed WBT might be useful for improving feed efficiency, which could lower rapid starch degradability and maintain rumen pH when compared to the WBT raw material.2 increased the ash content (p < 0.05), whereas the crude protein (CP) content was lower after treatment with NaOH (p < 0.05). Steam reduced the soluble fraction (a) and effective dry matter degradability of WBT in situ (p < 0.05). In addition, the WBT steaming methods result in a lower degradation rate constant in situ (p < 0.05). The degradation rate constants for the insoluble fraction (c) in the untreated CSC were higher than those of the other groups. Starch modification with LA reduced in vitro dry matter degradability at 12 and 24 h of incubation (p < 0.05). The starch modification method of the raw material showed the lowest pH value at 4 h (p < 0.05). The source of starch and starch modification methods did not influence the in vitro ammonia nitrogen concentrations, or in vitro volatile fatty acids. In conclusion, compared to the CSC group and untreated treatment, treating WBT with steam might be a more effective strategy for enhancing feed efficiency by decreasing or retarding ruminal starch degradability and maintaining ruminal pH.This research assessed the impact of cassava chips (CSC) and winged bean tubers (WBT) with various starch modification methods on the chemical composition, ruminal degradation, gas production, in vitro degradability, and ruminal fermentation of feed using an in situ and in vitro gas production technique. Experimental treatments were arranged for a 2 × 5 factorial, a completely randomized design with two sources of starch and five levels of modification treatments. Two sources of starch were CSC and WBT, while five modification treatments of starch were: no modification treatment, steam treatment, sodium hydroxide (NaOH) treatment, calcium hydroxide (CaOH2) treatment, and lactic acid (LA) treatment. The starch modification methods with NaOH and CaOH Manihot esculenta) is a commonly grown tuberous tropical field crop, especially in the northeast of Thailand [Cassava  be used more widely, particularly in tropical areas [It has been recommended that al areas . The WB al areas . The WB al areas ,7, whichal areas .Usually, high proportions of carbohydrate sources are often used for high-producing ruminants to support high milk production and quick weight gain . However2), as well as lactic acid (LA) [Steaming is a physical method for modifying starch that improves starch digestion in the rumen ,13. In acid (LA) ,14.Although BWT contains a lot of starch, it can rapidly break down in the rumen, resulting in inadequate utilization. The novelty of the current work is due to the lack of knowledge used to control starch digestion in the rumen for BWT and the appropriate tests for in vitro investigation. This study tested the hypothesis that a modified starch product obtained from WBT could be used as a potential energy source, decrease the rapid ruminal degradation rate, and maintain ruminal pH. The aim was to evaluate the chemical composition, ruminal degradation, gas kinetics, and ruminal fermentation of CSC and WBT with various modified starches using an in situ degradability and in vitro gas production technique.All of the methodology and procedures involved in this research were approved by the Ethical Committee under the Institutional Guidelines of Khon Kaen University, National Research Council of Thailand (record no. IACUC-KKU-46/65) to ensure animal welfare.2 in diameter) and sun-drying for 48 h until the moisture content was less than 10%. The NaOH treatment method followed the process of Srakaew et al. [2 treatment method was taken from the procedure of Wanapat et al. [2 solution at a 1:1 (vol/wt) ratio, followed by 48 h of anaerobic fermentation in bottles and then 48 h of sun drying.The cassava chips (CSC) were obtained from Khon Kaen University’s animal feed factory, Department of Animal Science, Faculty of Agriculture, and WBT was provided by the Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Thailand . The steam treatment method followed the procedure of Srakaew et al. [t et al. . CSC andThe LA treatment method was performed using the method of Pilachai et al.  with 1.02-treated, and LA-treated.The present study was performed with different incubation intervals and two sub-experiments using a nylon bag measurement and gas production technique. In both sub-experiments, a 2 × 5 factorial experiment design was carried out and set up using a completely randomized design (CRD) with three replication runs. The diets used in the experiments had two starch energy sources, CSC and WBT, with five modified starch treatment methods: untreated, steam-treated, NaOH-treated, CaOHTo prepare the experimental diet for the chemical composition test, the nylon bag test, and the gas production test, all of the dietary samples were oven-dried for 48 h at 60 °C, and ground and passed through a 1-mm sieve . Experimental dietary samples were analyzed for their chemical compositions with the standard method of the AOAC , includiUsing an adiabatic calorimeter bomb, gross energy (GE) was calculated .2 cages within an evaporative barn and freely watered with individual nipple cups. All animals were fed the diet for the adaptation period of 14 days prior to the experiment.Three male ruminally fistulated Thai native beef cattle (White Lamphun) with body weights (BW) of 325 ± 25.0 kg were used in this experiment. All cattle received a concentrate diet of 1.0% BW (16.0% CP and 10.46 MJ of ME/kg DM) and rice straw twice daily ad libitum. All animals were housed in individual 2 × 5 m2, 40 m average pore size). All of the nylon bags were fastened to an iron chain with nylon threads and then left hanging in the rumens of three fistulated cattle for 2, 4, 8, 12, 16, 24, and 48 h. Using triplicates based on each incubation time point, the samples of each treatment were incubated concurrently in three animals.The in situ nylon bag approach was used to determine DM disappearance, according to Ørskov and McDonald . After 4All sample test bags were taken out of the rumen after each incubation period and handwashed in lukewarm water for 10 min or until the wash water was clear. The following step involved drying nylon bags holding mixed test feed leftovers in an air oven for 48 h at 60 °C. We gathered all bag feed samples for their appropriate blanks, and a blank bag containing no sample for each removal time. The samples at 0 h of incubation underwent a similar procedure for washing and drying in an oven. Then, the difference in weight before and after incubation was used to calculate the DM loss.t (%) p was calculated as follows:−1), and t is the time of incubation (h).Data for DM ruminal disappearance features were fitted to an exponential equation using the NEWAY program, following the method outlined by Ørskov and McDonald . The dis−1) [The following equation was used to determine the effective degradability (ED) of dry matter (EDDM) and the effective degradability of organic matter (EDOM). Drying at 100 °C for 24 h yielded the DM, and ashing at 550 °C for 4 h yielded the OM..05 h−1) .In order to obtain a representative rumen fluid, four native Thai beef cows were considered. Four native Thai beef cows weighing 350 ± 20 kg each were employed as donors for ruminal liquor. To obtain ruminal liquor, the animals were fed a concentrate diet ad libitum at 0.5% of BW daily , along with roughage (rice straw) (6:30 a.m. and 4:30 p.m.). Individual enclosures were employed to house the cattle, and free access to mineral blocks and clean water was provided. For 21 days, the cattle were fed a diet before obtaining rumen liquor. Before morning feeding, ruminal liquor from each animal was collected using a stomach tube connected to a vacuum pump. The ruminal liquor was passed through five layers of cheesecloth before being placed in pre-heated thermos bottles at 39 °C and transported to the laboratory.3-N, and volatile fatty acid (VFA) analyses. For the NH3-N and VFAs analyses, a total of 10 mL of liquid samples were employed. Another set of 60 bottles was used for a digestibility analysis (3 bottles per treatment × 10 treatments × 2 sample times: 12 and 24 h incubation).A sample containing 0.5 g of substrate  was weighed out and put into 40 mL bottles. The rumen fluid was taken to the laboratory and combined with freshly made artificial saliva at a 2:1 ratio to form an inoculum. The process described by Sommart et al.  was usedt (mL), a is the immediate production of gas from the soluble fraction (mL), b is the production of gas from the insoluble fraction (mL), c is the gas production rate constant for the insoluble fraction (mL h−1), t is the incubation time (h), and a + b = is the potential extent of gas production (ml).The first set was used for the evaluation of gas production. A time series of incubations was conducted at 0, 1, 2, 4, 6, 8, 10, 12, 18, 24, 48, 72, and 96 h. The observed gas data were then entered into the following equation :Y = a + 2SO4 and held at 20 °C for ammonia nitrogen (NH3-N) analysis using a spectrophotometer  based on the method by Fawcett and Scott [Using the second set, and a digital pH meter (HANNA Instrument (HI) 8424 microcomputer, Singapore), the ruminal pH was measured after 4 h of incubation. The 20 mL was maintained in 5 mL of 1 M Hnd Scott . Then, ind Scott . This wand Scott .The in vitro digestibility of dry matter (IVDMD) was evaluated after 12 and 24 h of incubation when the contents were filtered through pre-weighed Gooch crucibles, and residual DM was quantified. The percent weight loss was calculated and displayed as IVDMD. The IVDMD (%) was computed as follows:2 treated, and LA treated), Abij is the interaction between factor A and B, and Ɛijk is the residual effect. Tukey’s multiple comparison test was used to determine whether there were significant differences between treatments [p ≤ 0.05.All data were subjected to an analysis of variance (ANOVA), which was carried out using SAS PROC GLM . The dateatments , and stap < 0.05) than those of the CSC group , while the NDF and NFC contents were lower (p < 0.05). Nevertheless, the source of starch had no effect on the ash or GE content.The chemical composition comparison of CSC- and WBT-modified starch is presented in p < 0.05) when the starch was treated with NaOH and CaOH2. However, the CP content decreased after treatment with NaOH, and there was also a lower NFC content in the CaOH2 group (p < 0.05). Furthermore, after steam treatments, GE in CSC and WBT increased (p < 0.05).As a result of the starch modification methods, the ash content increased (p < 0.05). Untreated CSC had the highest soluble fraction (p < 0.01) (a), EDDM, and EDOM, which had averages of 49.79 ± 0.13, 94.44 ± 3.34, and 95.69 ± 2.46%, respectively. In contrast, the untreated CSC (raw) had a lower soluble fraction than the potential degradable fraction (b), with an average of 57.17 ± 1.08%. However, there was no interaction effect (p > 0.05) on the rate constant (c). There was an effect (p < 0.05) of the source of starch on the rate constant (c), with CSC having a higher rate constant than the WBT groups. The modification of starch with the steam method reduced the rate constant (c) by 36.36% when compared with the untreated group (p < 0.05).The effects of rumen incubation on the kinetics of degradation of the DM and OM characteristics of CSC- and WBT-modified starch are presented in (−ct)). There was an interaction effect (p < 0.05) between the source of starch and the starch modification methods in the immediately soluble fraction (a) and the degradation rate constants for the insoluble fraction (c). The WBT treated with the steam process showed lower (p < 0.05) than the other groups on the immediately soluble fraction (a). In addition, the results showed that the degradation rate constants for the insoluble fraction (c) in the untreated CSC were significantly higher (p < 0.05) than those of the other groups. However, there were no interaction effects on the insoluble fraction of gas production (b) or the potential extent of gas production (|a| + b). The steam method showed greater values of b and |a| + b with 164.00 and 172.42 mL/0.5 g DM, respectively. Furthermore, the cumulative gas production was higher in the untreated group (p < 0.01) than in the other groups.The gas kinetic profiles after 96 h of incubation are shown in p > 0.05). There were no differences in IVDMD and IVOMD according to the source of starch. The starch modification methods with LA significantly reduced IVDMD at 12 and 24 h of incubation (p < 0.05) compared with the untreated group . IVOMD showed the highest value (p < 0.05) in the untreated group (679.62 g/kg DM). However, there were no changes in the mean values of IVDMD and IVOMD at 24 h, or in the mean value of IVOMD.3-N concentrations are shown in p < 0.05) than the other groups. In contrast, no interactions were found between pH at 4 h after incubation and NH3-N concentrations. The ruminal pH at 4 h of incubation was significantly different (p < 0.05) between the starch modification methods, with the lowest value occurring in the untreated group. However, there were no changes in the NH3-N concentration. The mean concentrations of NH3-N varied from 16.96 ± 1.65 to 18.01 ± 1.40 mg/dL, respectively.The effects of CSC- and WBT-modified starch on ruminal pH, and NHCassava chips (CSC) have been used as an energy source in tropical ruminant rations due to their high starch content and more than 90% immediate rumen degradability. However, WBT may eventually replace CSC as a source of energy due to its high starch content. All parameters had no interaction effects between the starch source and the starch modification procedures. This study shows that the source of starch is different in the CSC group, which had lower CP, EE, and ADF values and higher NDF and NFC values than in the untreated WBT group, which is similar to previous reports ,27. GeneIn this study, the CP content was significantly different based on the modification methods with NaOH treatment, which had negative side effects and resulted in a lower CP value . This co2. The NaOH and CaOH2 treatments increased the amount of ash. This is probably due to treatment with alkaline chemicals that contain inorganic components, causing the untreated materials to have more inorganic components [2 group as well. From the table, it can be seen that the GE increased after treatment with steam due to the sample’s rich starch gelatinization and amylopectin concentration. This result is consistent with reports by Pu et al. [Furthermore, the results showed that starch modification methods increased the ash content when the starch was treated with NaOH and CaOHmponents . This mau et al. , who disu et al. .This study showed that the interaction between steam treatment and WBT reduced the soluble fractions (a), (b), EDDM, and EDOM in the rumen. Physical modification of starches involves alteration of the starch granules since water and heat only affect them due to the starch-related effects of the increased temperature, which are relatively well documented . By heatNarwojsz et al.  found thIn this study, the source of starch was significantly different. The WBT showed a lower degradation rate constant (c) and starch content (NFC) than the CSC. A cereal grain’s pericarp may cause or reflect variations in the starch’s molecular makeup—specifically, the ratio of amylose to amylopectin, with the availability of additional feed components in the grain, such as fat, fiber, and protein, which can form bonds with starch molecules ,35. ThisIn addition, when considering only the influence of starch modification methods, the steam method reduced the rate constant (c) due to physical treatment affecting the various properties of starch granules, which may have a significant impact on isolated amylolytic enzymes’ capacity to digest starch and, consequently, on the rumen’s ability to degrade starch . FurtherThis study demonstrated a markedly lower soluble fraction (a), (b), EDDM, and EDOM in the steam-treated group than in the untreated group. The results of this experiment showed that steam treatment decreased the solubility (a) of CSC and WBT and their ED in the rumen. As a result of our findings, EDDM and EDDOM significantly reduced DM and OM loss in the rumen when physical treatment was applied to CSC . AccordiThe winged bean tuber (WBT) has a high starch content, making it comparable to cassava chips in terms of producing specific useful gases and digestion. In this study, the immediately soluble fraction (a) had a negative value, which is a deviation from the exponential result of fermentation or a delay in the onset of fermentation. This is due to the fact that when the soluble portion of the substrate has been consumed, but fermentation of the cell walls has not yet begun, there is a lag time before microbial colonization occurs . Thus, tIn the present study, the immediately soluble fraction (a) and the degradation rate constants for the insoluble fraction (c) decreased in WBT treated by steaming, but there was greater gas production from the insoluble fraction (b) and the potential extent of gas production (|a| + b). This was most likely due to the resistant starch of steamed WBT, which also resulted in a lower fermentable soluble fraction that fermented slower . Starch The cumulative gas at 96 h of incubation was lower in the steamed and LA-treated groups. The benefits of physically processing grain have been identified with the use of steam treatments, which are typically accomplished through derivatization processes such as etherification, esterification, and cross-linking . In physSince aqueous starch dispersion has the propensity to thicken upon chilling, Huang et al.  showed tAnother theory is that interactions between gluten and LA can function as barriers to enzymatic breakdown. LA treatment has the ability to slow the enzymatic action of amylases in grains . Moreove2, and LA treatment when compared to no treatment. The modification methods significantly changed the IVDMD and IVOMD, with NaOH, CaOH2, and LA treatments influencing in vitro degradability compared to the untreated group.The WBT and CSC both have a high starch content, which leads to a high degradation rate. Controlling the rate of degradation may provide more opportunities for rumen microbes to use starch for growth. In this study, IVDMD at 12 and 24 h decreased with modified starch produced by NaOH, CaOH2 on reduced in vitro degradability [Rojas et al.  demonstrdability .Treatment with LA impacts starch degradation rates. This probably has to do with the fact that LA causes linearization of the branching amylopectin molecule , which m2 resulted in a lower IVOMD at 12 h. It is possible that the resistant starch treatment effect of LA lowers IVDMD, which may result in lower IVOMD [2) that contain inorganic components, causing the replacement of organic components and possibly making the content of IVOMD lower in the CaOH2 group as well [In this study, treatment with LA and CaOHer IVOMD . In addi as well .The rapid degradation of starch in WBT and CSC resulted in a decrease in ruminal pH. Rapidly lowering rumen pH may harm rumen microbes and reduce their ability to digest feed. Ruminal pH ranged from 6.93–7.16, which is in the normal range of rumen ecology (pH 6.20–7.00) . In the 2 was higher than that of a control group. Deckardt et al. [Humer and Zebeli  reportedt et al.  reportedt et al.  showed t3-N values were within the normal range [3-N concentration. This could be due to the fact that the modified starch did not affect the rate of degradability of protein [In this study, the NHal range ,52. It h protein .WBT may serve as a substrate for the production of VFA, which is comparable to cassava root because of its high starch content ,53. In tThis study demonstrated that WBT has a greater CP and EE content but a lower NDF content when compared with CSC. Treatment with NaOH decreased the CP content. The WBT treated with steam had a lower soluble fraction (a) and EDDM in situ. Furthermore, steaming methods showed a lower degradation rate than in situ methods.Compared to other methods, no treatment resulted in higher cumulative gas production, IVDMD, or IVOMD at 12 h but lowered the pH in vitro. The source of starch (CSC or WBT) and starch modification methods did not influence the in vitro fermentation end-product (VFA). In conclusion, considering that WBT contains more starch and protein than CSC, steaming WBT might be a more successful strategy to enhance feed efficiency, decrease degradability, and maintain rumen pH, resulting in a convenient alternative to CSC. However, further in vivo research needs to be done to determine the actual intestinal availability of steam-treated WBT in animal production.


Semiconductor cleaning system ultra-clean flow control pumps are critical equipment in the semiconductor industry. Among them, the perfluoroalkoxy alkane (PFA) spring is a pivotal component to control the pump, and its dynamic performance is crucial to ensure the efficient operation of the system. However, the dynamic performance of the spring is often affected by the operating frequency. This paper studied the effect of different working frequencies on the dynamic property of the spring through compression-cycle experiments under uniaxial sinusoidal excitation. The force–displacement curves under different compression frequencies were fitted to obtain the dynamic stiffness of the PFA spring under different cyclic loading frequencies. The variation in the spring’s hysteresis coefficient was evaluated using the hysteresis curves of different cyclic loading conditions. After 2 million compression experiments, the changes in dynamic stiffness, hysteresis coefficient, and spring height were investigated. The obtained results revealed that, as the frequency increases, the dynamic stiffness of the spring increases. The hysteresis coefficient of the PFA spring is the largest at 10 Hz and the smallest at 6 Hz. Upon conducting 2 million compression tests, it was discovered that the dynamic stiffness experiences the greatest attenuation rate of 4.19% at a frequency of 8 Hz, whereas the hysteresis coefficient undergoes the largest attenuation of 42.1% at a frequency of 6 Hz. The results will help to improve the design and application level of PFA springs. The perfluoroalkoxy alkane (PFA) material is highly valued for its exceptional mechanical, chemical, and abrasion resistance ,2. As a Spring stiffness is the most basic performance metric of the spring. As the stiffness increases, the check valve’s leakage rate decreases. However, excessive spring stiffness requires more kinetic energy to open the check valve, impairing its regular operation. Thus, it is crucial to determine an optimal stiffness for the check valve spring that balances the reduction in the leakage rate with the requirement of sufficient kinetic energy for proper valve opening. Jian et al.  conducteGenerally, a spring’s stiffness is designed under the assumption of static conditions. A large amount of research has shown that, in addition to the material and structure of the spring itself affecting its stiffness, changes in working frequency also significantly impact its stiffness. Through uniaxial compression experiments on metal springs, Deng et al.  verifiedWorking frequency changes significantly impact a spring’s service performance. Launay et al.  studied The literature review indicates that extensive research has been conducted on the theoretical stiffness of metal springs and on the changes in the performance of springs under dynamic loads. However, only a few studies have focused on PFA springs. There are very few studies on the dynamic performance of PFA springs. In this paper, a PFA spring working in the ultra-pure pump was designed. The uniaxial cyclic compression test of the PFA spring analyzed whether the spring’s performance met the requirements in a specific frequency range of 6–14 Hz. The spring’s dynamic stiffness, dissipation coefficient, and dynamic relaxation were used to evaluate the PFA spring’s dynamic performance. The changes in the performance of PFA springs with increasing cyclic excitations were also explored. The suitable frequency for PFA spring operation was obtained. Such research is of great significance for the design and application of PFA springs; it can help optimize their performance and reliability and improve their application effectiveness in fields such as semiconductor manufacturing equipment. At the same time, this also provides a specific reference value for researching other material springs.The PFA material selected in this experiment was purchased from DAIKIN . The material trademark was AP-231SH. Its physical properties are summarized in In the early stages of this research, we conducted a systematic comparison and analysis of the advantages and disadvantages of various types of springs. Compared with ordinary springs, rectangular cross-section springs are easier to manufacture and have better stability, which is more suitable for meeting the high precision and strength requirements of ultra-pure pumps. With the guidance of the manufacturer’s technical manual, we successfully designed a spiral spring with a rectangular section based on the valve size and operating conditions.According to the actual performance requirements of the spring, the outer diameter of the spring was D_1 ≤ 28 mm, and the static stiffness of the spring was 95 ≤ k ≤ 160 N/m. The spring’s static stiffness is a key parameter for check valve sealing and flow control, which has an important influence on the working characteristics of the check valve.G is the spring shear modulus.E denotes the bending modulus of the spring; u is the Poisson’s ratio.The static stiffness formula of the rectangular spring is (1)K=4GIs 0.1869 . G is thThe rectangular helical spring’s static stiffness can be obtained asThe specific structure of the spring is shown in As shown in A uniaxial compression-cycle mechanics experiment was conducted on the spring to investigate the effects of the excitation frequency on stiffness, energy consumption levels, and dynamic relaxation. Experimental tests were conducted using the electronic spring fatigue test system, CTM-EF005 , as depicted in The experimental scheme, as outlined in f was the excitation displacement frequency. After the spring vibration is stabilized, the corresponding force response amplitude Stiffness refers to the ability of a structure to resist deformation. It is the most fundamental characteristic of a spring and is also a priority factor when selecting a spring. It is worth emphasizing that the PFA spring studied in this paper is the check valve spring of the pump. It works under repeated loading and unloading excitation conditions. Unlike the performance under static conditions of single compression, the stiffness of the PFA spring will change significantly with the change in excitation frequency. Therefore, it is necessary to use dynamic stiffness instead of static stiffness to describe the spring’s ability to resist deformation under a dynamic load. The dynamic stiffness is the ratio of the response force amplitude to the displacement amplitude. The experiment was to apply different frequency sinusoidal displacement excitations to the PFA spring. And the excitation displacement was The force curves generated from 1000 cycles of excitation with frequencies ranging from 6 to 14 Hz are presented in The spring has a pre-compression amount, and the corresponding spring has an initial force response As shown in The dynamic stiffness can be written as follows:Because the frequency of the force signal and the displacement signal is the same, the phase difference between the two remains constant. From As shown in The dissipation coefficient of the check valve spring in a pump is a critical factor that can significantly influence the fluid flow in the valve. A suitable energy dissipation factor can ensure the system’s normal operation and optimal performance.S(ACE + CEG)) and the sum of the triangle area (S(ABD + DFG)). The hysteresis loop represents the energy dissipated in a cycle, and the sum of the triangles represents the elastic energy stored in a cycle. The dissipation coefficient can be written as follows [The dissipation coefficient of a spring is usually analyzed based on specific hysteresis loops. The hysteretic loop curve refers to the displacement–force response curve during loading that does not coincide with the curve during unloading ,28. The  follows :(6)E=SACAccording to data collected from the experiment, the force–displacement curves of different excitation frequencies were drawn, as shown in To calculate the energy dissipation factor, an orthogonal algorithm and sine fitting were applied to obtain a symmetrical fitted curve, as shown in The results showed that the energy dissipation factor increased from 6 to 10 Hz and decreased from 10 to 14 Hz within the range of 6–14 Hz displacement excitation. The dissipation coefficient was the smallest at 6 Hz and reached the maximum at 10 Hz. In the range of 6 to 14 Hz displacement excitation, under 10 Hz displacement excitation, the energy consumed in one cycle of compression and recovery of spring load was the highest.The energy dissipation of a PFA spring may be caused by the material damping temperature, the air resistance, and the geometry of the spring. In this experiment, the geometry of the spring was the same, and the springs were in a 320 K thermostat for the compression experiments. The excitation frequency will cause a change in the damping of the PFA material. The energy dissipation of the PFA spring is primarily due to material damping. During cyclic loading and deformation, some PFA molecules that make up the spring will rearrange, which requires energy consumption. Not all the energy is released during unloading, and some energy is converted into heat and lost to the surroundings. The frequency of excitation influences the behavior of the PFA molecules. When the frequency is shallow, the chain motion of the polymer can ultimately keep up with the change in the external force; the internal friction is minimal, and the polymer shows the high elasticity of the rubber. When the frequency is very high, the segment motion can keep up with the change in the external force; the internal friction is minimal, and the polymer appears rigid, showing the mechanical properties of the glassy state . Only inWith an increase in the cyclic excitation, the performance of the spring will change. The permanent free-height deformation of the spring is one of the ways of failure of the spring. Therefore, it is necessary to study the dynamic performance and free height of the PFA spring with the increase in cycle times. Let the free height of the spring be It can be seen from the above Equation (7) that when a PFA spring undergoes permanent deformation, such as a decrease in the free height The PFA spring’s dynamic stiffness decay results are presented in The outcomes in These findings indicate that the structure and performance of the PFA spring alter as the number of cycles increases. The PFA spring’s free height decreases, its dynamic stiffness decreases, and its damping internal consumption decreases with increasing displacement excitation cycles. Notably, the most significant degradation in spring performance occurs at 10 Hz, so it is crucial to select a spring operating frequency that avoids this frequency range. Overall, by comprehending how the PFA spring responds to cyclic loading and deformation, it is possible to identify suitable operating conditions to ensure the optimal performance and longevity of the valve system.(1)In the range of experimental tests and operating frequencies, the dynamic stiffness of the PFA spring gradually increases as the excitation frequency increases.(2)By analyzing the hysteretic curve of the PFA spring, the frequency is an essential factor affecting the energy dissipation coefficient of the PFA spring. The ductility coefficient of the PFA spring increases first and then decreases in the range of 6 to 14 Hz and is the smallest at 6 Hz and the largest at 10 Hz.(3)Through the dynamic relaxation analysis of the PFA spring by the hysteresis curve, it is found that the frequency will affect the dynamic relaxation performance of the spring. The spring’s stiffness, free height, and ductility coefficient will decrease with the increase in the number of cyclic excitations. The PFA spring’s performance decays fastest under the excitation frequency of 10 Hz, and the work environment should avoid this frequency band.In this study, the dynamic performance of the spring was investigated by conducting uniaxial cyclic mechanical tests at different frequencies and using indicators such as spring dynamic stiffness, energy dissipation coefficient, and performance attenuation rate. The following conclusion were drawn from the present study:In further studies, tensile spring tests should be performed to measure dynamic performance, and it is necessary to consider the effect of temperature on the spring.