International electives provide a learning platform where interprofessional education and collaborative practice (IPECP) skills can be cultivated. However, hardly any frameworks to guide the implementation of interprofessional education (IPE) during international electives, especially in the context of low-income settings have been published. To address this gap, this study used the modified Delphi approach to develop an IPE framework guide for international electives to be used by health professions training institutions in Sub-Saharan Africa.A rapid literature review and a study among students and faculty in four African health professional training institutions were done to inform the process. This was followed by the modified Delphi technique that used three Delphi rounds with a panel of eight experts to build consensus on the final framework for IPE during international electives. The level of consensus was set at ≥70% on each of the statements in all rounds.n = 37, 71%) reached consensus while  of the statements did not reach consensus and were discussed in round 2. Round 2 led to 42 statements to be utilized for round 3. In round 3, all statements (42) reached a consensus and an IPE framework to guide the implementation of international electives was developed. The framework consists of three sections. Section one highlights the various IPECP competencies to be gained by learners in the areas of teamwork, interprofessional communication, roles and responsibilities of interprofessional collaborative practice, values and ethics of interprofessional collaboration, and reflection and evaluation of oneself and the team. Section two gives guidance on the structuring of the IPE international electives in health professional training institutions. This includes subsections on operational/institutional needs, acculturation considerations, teaching strategies, assessment strategies, mode of delivery, and public health considerations. Section three consists of the various practical guides and approaches that health professional training institutions could use according to what works best in their setting.Out of the 52 statements in round 1 (The modified Delphi technique was an adequate approach to aid the development of an IPE framework to guide implementation during international electives in various health professional training institutions. Interprofessional education (IPE) in health is defined as when two or more professions learn with, from, and about each other to enable effective collaboration and improve health outcomes . A few iThe World Health Organization has supported IPE by developing a framework for action on IPECP that proposed new models of educating healthcare providers . This frThe Interprofessional Education Collaborative (IPEC) defined the key competencies of IPE as; values/ethics for IPECP, roles/responsibilities for IPECP, interprofessional communication practices, interprofessional teamwork, and team-based practice . All theDespite the relevance of all the above frameworks on IPE competencies that also include aspects of respect for culture and uniqueness of other professions, it is challenging to develop guidelines for implementing IPE in various learning contexts including international electives. International electives are defined as the time of learning where students have a choice of where to learn, and the discipline they should be taking across borders from their own country . These mIPE international electives though scarce, have been implemented between Vanderbilt University USA and Nicaragua Eye Hospital and were perceived to be effective to enable students gain IPECP . HoweverMuch of the research on IPE and learning has been informed by psychological and cognitive theories pushing aside the role of the learning environment and cultural context. This study was guided by the social constructivism theory, a social-cultural theory advanced by Vygotsky et al. . This thTo address this gap, the purpose of this study was to develop an IPE framework guide for international electives to be used by health professions training institutions. Though the study was conducted in Sub-Saharan Africa, the framework developed can be adapted to various settings where international electives do occur.This study was conducted in Sub-Saharan Africa utilizing faculty and experts in representative health professions training institutions. The training institutions offer electives as part of their curriculum and conduct training for various health professional disciplines at the undergraduate level. In this study, we employed the modified Delphi technique to develop the IPE implementation framework for international electives in health professions training. In the Delphi approach, the first round could involve unstructured open-ended questions/ statements to guide the discussion with a panel of experts . When a The Delphi process involved a series of stages before coming up with a final framework. These included conducting a rapid literature review, exploring opinions from students and faculty, and finally engaging the panel of experts for consensus building.via Makerere University to identify literature on key concepts related to the topic under study. These included: internationalization in health professions education, international electives, the importance of IPECP in health care, current global disease burden, IPE during international electives, perceptions of faculty and students on IPE during international electives, IPE frameworks and their development, Learning theories for IPE, IPE competencies, the importance of IPE, the value of international electives, operational needs for international elective programs, teaching and assessment methods for IPE in international electives, and delivery approaches to teaching IPECP during international electives. The specific search terms used were: interprofessional education, international electives, interprofessional collaboration, Africa, IPE frameworks, Delphi technique, internationalization in health professions education, multidisciplinary, collaborative, interprofessional, interdisciplinary, teamwork, IPE teaching, assessment, theories, health professions education, and teaching.Firstly, a rapid literature review was conducted to inform the Delphi process for our study. This was done to enable a detailed understanding of IPE and international electives in line with the study objectives and to identify learning theories to be used. The rapid review was conducted using PubMed accessed through HINARI We searched PubMed for manuscripts written in English, and from the years 2000 to 2022 as our inclusion criteria to enable us to have more articles included since there is scanty literature on this topic. The earliest year of the manuscript included was 2000 while the highest was 2021 with the majority being published in the last 10 years as shown in In this process, 84 manuscripts were identified and included see . The refIn addition to the rapid literature to inform the Delphi process, we sought opinions from students and faculty from four African health training institutions that participated in the study to understand their perceptions of IPE during international electives and their suggestions and views on an IPE framework for international electives. The students and faculty were given a brief orientation on what IPE is so that they could give meaningful responses related to IPE. The opinions and perceptions of the students and faculty were further utilized to enable the identification of some key constructs to be added to the draft framework that was used to build consensus. This study was conducted qualitatively among faculty using key informant interviews and quantitatively among students using an online survey. AtlasTI version 8 software was used for qualitative data analysis while SPSS IBM statistics 21 was used for quantitative data analysis. The various training institutions included: Makerere University Uganda, Kenyatta University Kenya, University of Ibadan Nigeria, and the University of Zimbabwe.The specific details of the findings from the students have been published in the Journal of Interprofessional Care . The finThe last stage was engaging the panel of experts to develop a draft framework. The draft framework was used to build consensus from the panel of experts and gain validation of the final framework.Through the rapid review, a Delphi guide developed by Bentley et al.  to develExperts in IPECP, health professions education, and international electives were purposively sampled. Recruitment was from Makerere University and Busitema University in Uganda, Yale University USA, Stellenbosch University South Africa, University of Free state South Africa, University of the Western Cape, South Africa, and the University of Global Health Equity Rwanda. Some of the experts were members of the African Forum for Research and Education in Health, Ghana (one) and the African Interprofessional Network (one) while some (five) did not belong to any of these professional bodies in Africa. Furthermore, multidisciplinary representation was considered, and the experts were from various disciplines, i.e., medicine (three), nursing (two), pharmacy (one), occupational therapy (one), and physiotherapy (one). This enabled us to gain a heterogenous panel, a key requirement for the Delphi approach. In total, eight experts were recruited which is an acceptable number for the Delphi method. All eight members of the panel were academic experts in IPECP, health professions education, and international electives, with more than 5 years of experience as an academic faculty, and have had exposure to guiding and conducting international electives in Africa for more than 5 years.The Delphi method lends itself to the concept of the researcher assessing the scope of the problem and the available resources to determine the panel size that would be appropriate . FurtherEngagement with the experts was done virtually due to their various geographical locations. The Delphi process involved three rounds. We began engagement with the panel of experts jointly as one group through email to generate interest and commitment. An email introduction to the 8 experts was done detailing the study aims, the process of participation, the time frame, etc. To ensure we capture full commitment to participate, a doodle poll was sent for all the experts to indicate their time of availability for Rounds 1 and 2. They were given 2 weeks to have this completed. All data collection and rounds were done online using Zoom meeting software for Rounds 1 and 2 (that were recorded), and email sharing for Round 3. Rounds 1 and 2 lasted one and a half hours while Round 3 lasted 3 weeks.For capturing the ratings of each expert on all 52 statements, the study tool was built in Microsoft Forms on an online survey platform. During the Zoom session, each of the participants was sent the online link. The researcher (FN) led the sessions and shared her screen to enable the experts to see the statements but also jointly go through them one by one as each of them submitted their responses using the online survey link as per the draft framework that was developed . This waper se or Likert scale, we used a percentage level of agreement to establish consensus. A score of ≥70% on a particular statement was deemed as relevant and important to be included in the framework while a ≤ 69% score on any statement meant that there was a lack of consensus and thus needed to be discussed and taken up for Round 2. This percentage level of agreement and disagreement was adopted from existing literature from studies that have used the Delphi process for health issues consensus building.Globally, there is a lack of uniform guidelines on what constitutes consensus in a Delphi study . DiffereDuring Round 2, participants were given the scores on all statements, i.e., those that achieved consensus and those that did not. Participants were invited to comment, discuss, and rate the 15 statements that did not reach consensus, i.e., statements with scores of ≤69%. Each of the participants was given a chance to discuss and give their views on these statements and all the suggestions were captured by the researcher. An agreement was reached to leave out the section on the importance of international electives in Africa. This is because the framework is meant to guide the implementation of IPE during international electives and the importance of IPE is already well elaborated from the various existing frameworks and literature  has been used in other studies to build consensus in various health professions education research including IPE . Many tiIn this study, we had representation from various geographical locations, disciplines, and expertise which enabled triangulation and adaptability in various training institutions including Africa . AlthougThe IPE core competencies of this framework were tailored toward gaining IPECP skills that can be applied in all areas of healthcare practice for the students in their future practice. These competencies in our framework were adapted as a combination of various IPECP competencies developed by the European Interprofessional Practice and Education Network , the IntStructuring any new approach in health professions education involves various levels of preparation. This includes the leadership, administration, teaching faculty, training facilities, teaching and assessment methods, public health safety considerations, and modes of delivery among others. Section 2 of the framework covers this in detail. It gives guidance and approaches on how to enable leadership support for IPE during international electives. For international electives, this should be done through institutional partnerships with home and host institutions. Our framework emphasizes reciprocity to enable equity which is key in building equitable partnerships for training, especially in international electives where there is often an imbalance in opportunities for students from various African training institutions . In thisThe teaching and learning approaches put across as options in this framework have been described in the literature that was used to inform this process. In particular small group learning  has been efficient in various approaches to group training . This, tUtilization of bedside teaching , clinicaClinical observerships as a form of training commonly used in international electives in developed countries  did not Assessment of acquisition of IPECP competencies during international electives should be formative and summative as exhibited in this framework to capture the various IPECP skills gained by the students as they participate in an IPE international elective. Pre and Post-elective surveys have been popularly used to establish the IPECP gained . In the Globally various modes of delivery have been used to deliver IPE. These include the physical or in-person approaches that involve face-to-face interaction of interprofessional teams of students with faculty , and theThe modified Delphi technique was an adequate approach to aid the development of an IPE framework to guide implementation during international electives in various health professional training institutions. The IPE framework developed is adaptable and can be implemented in various health professional training institutions. The framework developed enables effective structuring of IPE international electives given that it has all domains required in implementing international electives, i.e., the core competencies to be gained, the operational institutional needs, teaching and assessment methods, modes of delivery, and public health considerations, especially for the fact that international electives require mobility to another country. Unlike other frameworks, this framework also provides some practical guidance on various approaches to implementing IPE during international electives thus creating a benchmark for new approaches to learning during international electives, especially in low-income settings.IPE is one of the key health professions education research priorities for sub-Saharan Africa . Our worThe modified Delphi approach is an adequate approach to be used to develop IPE frameworks to support IPE training in various learning environments including international electives. With the framework now in place to guide the implementation of IPE in international electives, a pilot has been done in the four institutions that participated in this study’s Delphi information process. The results have been published by BMC Medical Education . HoweverGiven the qualitative nature of the Delphi approach, trustworthiness and rigor were observed. For credibility, prolonged engagement with the experts, various rounds to enable consensus, having a cut-off point for consensus, review of the final framework by the experts were done. To ensure triangulation the use of both a research study and rapid literature review to inform the process, experts from various training institutions, and disciplines was done. A detailed description of the methods used was done to enable transferability in similar contexts. To ensure the dependability of the findings various rounds with scores to establish consensus were done. Lastly, confirmability was observed by having the final framework reviewed by the study team and the panel of experts for accuracy and alignment with the study objectives.The panel of experts was in different countries and time zones thus leading to the use of online options to conduct all the other rounds. However, despite the online virtual approach, we were able to record the sessions thus enabling the replay of the sessions for point articulation. Furthermore, the online approach enabled timely rating of the statements which allowed a quick turnaround time. The number of experts involved may be seen as a limitation to some however, this was still within the acceptable number for a Delphi panel recommended  . Four inThe original contributions presented in the study are included in the article/This study followed all the required regulations and guidelines by the Helsinki Declaration. Ethical approval to conduct the study was granted by the Makerere University School of Medicine Research Ethics Committee (SOMREC) Mak-SOMREC-2021-96 and the Uganda National Council for Science and Technology (UNCST) HS2078ES. Administrative clearance was obtained from the University of Ibadan, the University of Zimbabwe, and Kenyatta University. Written Informed consent was sought from all participants of the study.FN, IM, AV, SK, and AM conceptualized and developed the study and jointly drafted the manuscript. FN implemented the study. FN, IM, and AM cleaned and analyzed the data. TR and FW contributed to the final review and development of the framework developed in this study.


Mycobacterium tuberculosis, representing the second leading cause of death by an infectious agent worldwide. The available vaccine against this disease has insufficient coverage and variable efficacy, accounting for a high number of cases worldwide. In fact, an estimated third of the world’s population has a latent infection. Therefore, developing new vaccines is crucial to preventing it. In this study, the highly antigenic PE_PGRS49 and PE_PGRS56 proteins were analyzed. These proteins were used for predicting T- and B-cell epitopes and for human leukocyte antigen (HLA) protein binding efficiency. Epitopes GGAGGNGSLSS, FAGAGGQGGLGG, GIGGGTQSATGLG (PE_PGRS49), and GTGWNGGKGDTG (PE_PGRS56) were selected based on their best physicochemical, antigenic, non-allergenic, and non-toxic properties and coupled to HLA I and HLA II structures for in silico assays. A construct with an adjuvant (RS09) plus each epitope joined by GPGPG linkers was designed, and the stability of the HLA-coupled construct was further evaluated by molecular dynamics simulations. Although experimental and in vivo studies are still necessary to ensure its protective effect against the disease, this study shows that the vaccine construct is dynamically stable and potentially effective against tuberculosis.Tuberculosis is a disease caused by  The World Health Organization (WHO) estimated that in 2021, 10.6 million people acquired tuberculosis (TB), an increase of 4.5% over cases in 2020, and 1.6 million people died of TB . TherefoThe TB etiologic agent is Mycobacterium tuberculosis (MTB), which belongs to the Mycobacterium tuberculosis complex (MTBC) or clade “Tuberculosis-Simiae” ,5. It isReverse vaccinology approaches use computational methods and tools to identify antigens and epitopes that are likely to be recognized by the immune system and induce a protective response. Compared to in vitro studies, reverse vaccinology is performed in less time, saving economic resources and experimental work to support successful vaccine development ,17.In a previous study by our group, proteins PE-PGRS49 and PE-PGRS56 from the MTB H37Rv proteome were identified as highly antigenic molecules . PE-PGRShttps://www.ncbi.nlm.nih.gov/ (accessed on 1 February 2023) with reference numbers YP_177961.2 and Rv3512, respectively.The amino acid sequences of the PE-PGRS49 and PE-PGRS56 proteins from MTB H37Rv were downloaded in FASTA format from the National Center for Biotechnology Information  was used to predict B-cell epitopes. This program is developed to predict antigenic determinants and it is based on a semi-empirical method which use physicochemical properties of amino acid residues and experimental data. The antigenic peptides are determined by the Kolaskar and Tongaonkar method [The Immunomedicine tools server  was employed for the prediction of MHC epitopes, where frequencies of alleles, haplotypes, and genotypes of different world populations are selected to choose the most frequent alleles in the population [The Allele Frequency Net Database   [http://imed.med.ucm.es/Tools/rankpep.html (accessed on 15 February 2023) [http://tools.iedb.org/mhcii/ (accessed on 16 February 2023) [The servers used to predict peptide binding to HLA Class I and II molecules were Propred1, which is an online service for identifying the MHC Class-I binding regions in antigen. ProPred1 also allows the prediction of the standard proteasome and immunoproteasome cleavage sites in an antigenic sequence. Thus, the prediction of MHC binders and proteasome cleavage sites in an antigenic sequence leads to the identification of potential T-cell epitopes (ry 2023) , RANKPEPry 2023) , and IEDry 2023) . A windohttp://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html (accessed on 22 February 2023) using the default values [The antigenicity of the epitopes obtained previously was predicted by the VaxiJen v2.0 server  [http://ddg-pharmfac.net/AllergenFP/ (accessed on 23 February 2023) [https://webs.iiitd.edu.in/raghava/toxinpred/index.html (accessed on 23 February 2023). The server uses amino acid composition-based models, considering that some patterns/motifs could be associated with peptide toxicity [All selected epitopes were analyzed by predicting various physical and chemical parameters. The molecular weight, theoretical pI, amino acid composition, atomic composition, estimated half-life time, and instability index were estimated with the ExPASy server (ry 2023) . The allry 2023) , based otoxicity .https://www.rcsb.org (accessed on 24 February 2023). Proteins were prepared before docking by removing water molecules and each crystallized ligand with PyMOL (https://www.charmm.org/ (accessed on 27 February 2023). Additionally, energy minimization was performed on each structure. The epitopes were docked with the following MHC alleles: HLA-A0101 (PDB ID: 3BO8), HLA-A0201 (PDB ID: 4UQ3), HLA-A0301 (PDB ID: 2XPG), HLA-A2402 (PDB ID: 5HGH), HLA-B5801 (PDB ID: 5IM7), DRB1-0401 (PDB ID: 2SEB), and DRB1-1501 (PDB ID: 1BX2). Peptide-protein docking was performed using the HPEPDOCK server (http://huanglab.phys.hust.edu.cn/hpepdock/ (accessed on 1 March 2023) The server implements a hierarchical docking protocol with fast conformational sampling of peptide conformations and ensemble docking of generated peptide conformations against the protein. The docking server accepts both sequence and structure as input for proteins/peptides [http://pymol.org (accessed on 6 March 2023) was used to visualize and analyze docked complexes.For molecular docking of epitopes, the crystal structure of molecules in complex with their self-peptides was obtained from the PDB (Protein Data Bank) database  in the molecular docking assays. These epitopes were thus inferred to be antigenic given the prediction results, and therefore used to construct the vaccine candidate, with the linker GPGPG connecting them. Additionally, the adjuvant RS09, which is a short peptide (APPHALS) that mimics bacterial lipopolysaccharide , was conhttps://www.rcsb.org (accessed on 5 April 2023). Protein was prepared before docking by removing water molecules and each crystallized ligand with PyMOL (https://www.charmm.org/ (accessed on 7 April 2023). Additionally, energy minimization was performed on the structure. The vaccine candidate was docked using the HDOCK server (http://hdock.phys.hust.edu.cn/ (accessed on 9 April 2023) [http://pymol.org (accessed on 25 April 2023) was used to visualize and analyze docked complexes.For molecular docking of the vaccine candidate, the crystal structure of TLR-4 (PDB ID: 4G8A) was obtained from the PDB (Protein Data Bank) database (il 2023)  using deThe stability of peptides bound to MHC proteins was assessed by molecular dynamics simulations. Peptide structures were predicted with HPEPDOCK and then docked into the MHC I and II structures. The protein-peptide complexes were prepared with the Charmm-GUI solution builder, and each system was solvated using the TIP3P water model and neutralized with KCl at a final concentration of 0.15 M. Then, these structures were employed for molecular dynamics simulation performed with Gromacs v2018.3 and the CHARMM36m force field. Molecular dynamics simulation was performed at 310 K in three stages: 5000 steps of energy minimization, 125,000 steps of equilibration (NVT ensemble), and 50 ns of molecular dynamics production (NPT ensemble). Trajectory analysis was performed with Gromacs, and plots were generated with Grace 5.1.25 to visualize the stability of bound epitopes or vaccine candidates to MHC proteins, respectively.Twelve linear epitopes with a range from 7 to 15 amino acids were identified in the PE_PGRS49 protein using the IEDB server, while in the PE-PGRS56 protein, 33 linear epitopes with a range from 7 to 19 amino acids were identified. Epitopes predicted in both proteins are shown in the For MHC Class I, alleles HLA-A0101, HLA-A0201, HLA-A0301, HLA-A2402, HLA-B0702, HLA-B0801, HLA-B1501, HLA-B5801, and HLA-B3901 were selected as representatives in the population, and for the prediction of MHC Class II epitopes, DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501 alleles were chosen.A total of 16 MHC Class I linear epitopes were predicted for the PE_PGRS49 protein by using the ProPred I server and one epitope with the Rankpep server. The amino acid residues that were most frequently recognized were located at positions 61–73, 110–124, 128–136, 222–233, and 274–286. In the PE_PGRS56 protein, we identified one epitope with ProPred I in the region 832–839; however, no epitopes were predicted with Rankpep.Regarding the prediction of MHC Class II epitopes for the PE_PGRS49 protein, 10 epitopes were identified with the Rankpep server. The regions most frequently recognized were 177–187, 222–233, 262–271, and 274–286. On the other hand, 26 epitopes were predicted in the PE_PGRS56 protein. The regions most frequently recognized were 102–112, 338–347, 579–587, 780–789, and 1034–1044. The allele DRB1*0401 showed more matches on the RANKPEP and IEDB servers. All epitopes predicted in both proteins are shown in the Epitopes that were predicted to be recognized by both T and B cells were the first selection criteria. Six epitopes of the PE_PGRS49 protein and five of the PE_PGRS56 protein were thus selected .The prediction of peptide antigenicity was performed with the VaxiJen server; the higher the value calculated with the server, the higher the antigenicity level (a score greater than 0.4 is considered antigenic by the VaxiJen server). For the PE_PGRS49 protein, the scores ranged from 0.0638 to 2.2969, of which peptide 177–187 does not have antigenic qualities according to the prediction. Similarly, in the prediction of peptide antigenicity for the PE_PGRS56 protein, scores ranged from 0.9916 to 2.2536, all having antigenic qualities .Some chemical and physical properties were investigated with the Expasy server: molecular weight, instability index, theoretical pI, and half-life time. Peptides predicted in PE_PGRS49 had a molecular weight between 919.90 and 1365.46, with a pI in the range of 5.52–8.47. The half-life time estimated in hours ranged from 1.9 h to 30 h. In addition, almost all peptides were predicted to be stable in vitro, according to the predictor. Predicted epitopes in PE_PGRS56 had a range of 10 to 15 amino acids, their molecular weight was 702.72–1201.26, the isoelectric point ranged between 5.19–5.84, and the estimated half-life time ranged from 1.1 h to 30 h. One epitope in PE_PGRS56 was predicted to be unstable. None of the epitopes were toxic, and most were non-allergenic, according to the ToxinPred and AllergenFP servers, respectively .We performed molecular docking to ascertain if the MHC binding cleft recognizes the epitopes. The selected epitopes were docked with some of the most frequent MHC Class I and II alleles in the population, HLA*A0101, HLA*A0201, HLAA*0301, HLA*A2401, HLA*B5801, DRB1*0401, and DRB1*1501, using an online server, HPEPDOCK. Ten complexes were generated, and the most suitable epitope–MHC allele complex was selected based on correct conformation, binding, and the lowest energy level. Each docked epitope showed different energy values with their respective HLA alleles; the peptides 58–73, 128–138, 222–233, and 274–286 from the PE-PGRS49 protein scored the best binding energies and conformations. Although the 177–187 epitope showed affinity and low energy levels in complex with all HLA alleles, it was discarded because it was not identified as antigenic. In the case of the PE-PGRS56 protein, the epitopes with the best bound conformations were 579–593, 780–789, and 1034–1045 , Table 2Molecular dynamics (MD) simulations were performed to verify the stability of predicted epitopes complexed with MHC II proteins  and for the construction of the vaccine candidate with the same MHC proteins . The rooM. tuberculosis-reported proteins . Of the five markers, only PE_PGRS56 affected T-cell cytokine responses [Because the PE_PGRS protein subfamily contains a high GC content (approximately 80%), its cloning and expression are difficult ,53; howeIt is important to note that the BCG vaccine strain does not secrete PE_PGRS proteins due to the deletion of the RD5 genetic region; this is likely to render BCG incapable of inducing immune responses against all PE_PGRS proteins . There iBy using a reverse vaccinology strategy, a vaccine candidate comprising a molecular adjuvant and epitopes of the highly antigenic and exclusive PE_PGRS49 and PE_PGRS56 proteins was constructed. The results showed that epitopes GGAGGNGSLSS (128–138), FAGAGGQGGLGG (222–233), GIGGGTQSATGLG (274–286), and GTGWNGGKGDTG (1034–1045) are suitable candidates to be used in the development of a vaccine against TB and are expected to elicit humoral and cellular immune responses. Although in vivo and in vitro evaluation is still necessary to test their potential for protection against the disease, the bioinformatic analyses predict a stable and highly immunogenic construct.


Asymptomatic graft failure after coronary bypass grafting surgery (CABG) may have negative impact on the patients’ short- and long-term outcomes. Cardiac computed tomography angiography (CTA) has been proved to be another choice to detect graft failure besides coronary artery angiography in several studies. We aimed to identify the rate and predictors of asymptomatic graft failure detected by CTA before discharge.P > 0.05). The logistic regression at graft-level analysis showed that female , composite grafting , pulse index value  and new postoperative atrial fibrillation (POAF)  were independent risk factors that affect graft failure, while early postoperative dual-antiplatelet treatment with aspirin and clopidogrel was a protective factor .A total of 955 grafts of 346 consecutive asymptomatic patients who received CTA examination after CABGs were included in this retrospective study from July 2017 to Dec 2019. We divided 955 grafts into the patent group and occluded group by CTA results. Logistic regression model at graft-level were established to determine predictors of the early asymptomatic graft occlusion. The overall asymptomatic graft failure rate was 4.71% (45/955), and there was no difference between the arterial and venous conduits in different target territories (Early asymptomatic graft failure is associated with both patient and surgical factors including female gender, high PI value, composite graft strategy and the new POAF. However, the early dual- antiplatelet therapy with aspirin and clopidogrel may be useful for preventing graft failure.The online version contains supplementary material available at 10.1186/s13019-023-02199-0. Coronary artery bypass grafting (CABG) surgery is a widely used revascularization strategy for complex multivessel coronary artery disease (CAD) that provides symptomatic relief and increases the long-term survival in patients with CAD , 2. OwinData for isolated CABG were retrospectively collected from July 1, 2017, to Dec 31, 2019, from the Peking University People’s Hospital database. There were 744 patients who underwent CABGs; we excluded 5 who underwent redo surgeries, 76 who underwent concomitant additional procedures, 259 who underwent minimally invasive direct coronary artery bypass grafting (MIDCAB) for single-vessel disease, 25 who had symptoms or clinical signs of myocardial ischemia and 33 without CTA data due to renal dysfunction. The total 346 patients were divided into two groups: the patent group was defined as all anastomoses were patent and the occluded group was defined as at least one occluded anastomoses  and an intra-coronary shunt  was used during off-pump CABGs. In on-pump CABGs, cardiopulmonary bypass was established after standard ascending aorta cannulation and 2-stage venous cannulation of the right atrium, all distal anastomoses were performed after cardiac arrest. All procedures were performed by surgeons who had expertise in both on-pump and off-pump CABG, defined as more than 5 years of experience after residency training and completion of more than 100 cases of the specific type of surgery. The parameters were measured by The VeriQ system transit time flow measurement (TTFM) device . The parameter yielded by TTFM system included the mean graft flow volume (MGF), the PI (pulse index) and the diastolic flow fraction (DF). Satisfactory blood flow parameters criteria: The shape of blood flow waveform is stable and repeatable; PI < 5; MGF > 15 ml/min. If sufficient graft flow was not obtained, graft revision was considered and performed until diastolic graft flow was confirmed. All patients were given heparin 2500U per 4 h postoperatively, and then received antiplatelet therapy with aspirin, clopidogrel or both when tracheal intubation removed.After the drainage tube was removed, patients without contraindications received cardiac CTA prior to discharge. They were evaluated by Dual Source CT scanner SOMATOM Force CCTA . CCTA images were analysed with GE AW 4.6 advance Workstation  and were evaluated by 2 independent experienced radiologists. The number of grafts was counted by distal anastomoses, and the lesions of grafts and anastomoses were evaluated according to the FitzGibbon classification system. An occlusion of a graft was diagnosed if the graft was not filled by contrast medium along its whole length.Early asymptomatic graft failure: Graft failure in patients who do not have symptoms or clinical signs of myocardial ischemia  before discharge. Composite grafts: Y-SVGs: Y composite graft with a saphenous veins (SVG); I, Y-IMAs and SVG: an arteriovenous I or Y conduit with internal mammary artery (IMAs) and SVG; Y-IMAs: Y composite graft with LIMA and RIMA.P value < 0.05 or near 0.05 and variables based on clinical judgment were tested in a multivariable model using Logistic regression and ORs to determine the independent predictors of the occurrence of graft occlusion at graft-level. Optimal cutoff values of MGF, PI and DF to predict early graft failure, were determined by means of the ROC curve analysis. P < 0.05 was considered statistically significant. All the analyses were performed with SPSS version 26.0 and RStudio.Continuous variables were expressed as median and interquartile range (IQR) because most of the data were not normally distributed. Nominal and categorical variables were given as absolute numbers and proportions (%). The Mann–Whitney U-test was used for analysis of numerical data. Binary data obtained with Pearson’s χ2 test or Fischer’s exact test as appropriate. Intra-group analysis, the Bonferroni correction was adopted when appropriate.. Preoperative baseline data and perioperative data were included in the univariate analysis. Measurements of different grafts within the same patient were considered statistically independent. The variables with a univariable P = 0.266). We also compared the patency rates of the arterial and venous conduits in different target territories, and found that there was no difference between the arterial and venous conduits in different target territories (P > 0.05)  included in our study, 17 (45 grafts) had graft occlusion in the CT scans at 4–34 days before discharge after CABG. Among the 45 occluded grafts, there were 11 IMAs and 34 SVG grafts. Among which, there were two cases with four, 9 cases with three, 4 cases with 2 and 2 cases with 1 occluded grafts. There was no significant difference in patency rates among the different target territories (P = 0.003), and had a higher level in triglycerides .Baseline procedural characteristics and postoperative data of the patent group and the occluded group at graft-level are demonstrated in Table P = 0.022) and the postoperative CK-MB max  of the occluded group were both significantly higher than the patent group. The occluded group has higher PI , but lower DF . There was significant difference between the groups in antiplatelet treatment strategy (P = 0.02). There was no significant difference between the two groups in other baseline procedure data (P > 0.05).Baseline characteristics of the patent group and the occluded group at graft-level are given in Table P < 0.001) and DF < 63.5% (P = 0.007), however MGF < 18.5 ml/min was not found to be a statistically significant indicator of graft failure (P = 0.056).The results of ROC analysis of TTFM parameters are presented in Fig. P < 0.0083) and AO-SVG group were both significant higher than the composite grafts group , composite grafting ,PI  and new POAF  were independent risk factors that affect graft failure, while early postoperative dual-antiplatelet treatment with aspirin and clopidogrel was a protective factor   and there was no difference between the arterial and venous conduits (P = 0.059).Interest in understanding the factors associated with graft failure after CABG has been longstanding, but little study about the immediate (before discharge), asymptomatic graft failure factors and its occlusion rates. Previous studies have reported 1-year vein graft failure rates of 10–20%, limited data suggest that a substantial proportion of such occlusions occur much earlier . One majP = 0.000) was confirmed to be a predictive parameter of graft failure, which was similar to the results  reported by Gabriele Di Giammarco et al. [P = 0.000) and the AO-SVG subgroup  were both significantly higher than the composite grafts subgroup. In our study, there were total 25 composite grafts, of which 6 grafts was occluded including 3 Y-SVGs, 2 I, Y-LIMA and SVG, and 1 Y-IMAs. Although arterial composite grafts have been demonstrated to be both safe and effective for revascularization, studies assessing the safety and efficacy of using saphenous veins as a composite graft have produced conflicting results [In this study, graft failure occurred predominantly in female gender, which was similar to that reported in previous literature . Transito et al. . Optimalo et al. , 14. The results , 16. One results . In thisAn interesting finding of our study was that the dual-antiplatelet treatment with aspirin and clopidogrel was a protective factor of graft failure. The optimal antiplatelet strategy following CABG remains controversial, aspirin is considered the preferred antiplatelet drug to prevent graft failure after CABG. However, there is emerging evidence on the potential benefits of dual antiplatelet therapy with aspirin and clopidogrel or ticagrelor after CABG, including to prevent graft occlusion and adverse cardiac events post CABG –20. GrafAlthough we used the TTFM to confirm the quality of anastomoses, and if sufficient graft flow was not obtained, graft revision was considered and performed until diastolic graft flow was confirmed. There were still 17 of 346 patient have at least one occluded graft. However only 7 patient received percutaneous coronary angiography examination to verify the CTA-detected occlusion and were successfully re-intervened, considering the myocardial area of them remain unsupplied and might cause adverse cardiac event because of early graft failure.Several limitations of our study should be recognized. The first and most important limitation of this study was its descriptive nature, using a relatively small cohort of patients at a single institution. Second, the degree of the native coronary stenosis and collateralization of chronically occluded coronaries might affect the graft patency were not included in our study.Early asymptomatic graft failure is associated with both patient and surgical factors including female gender, high PI value, composite graft strategy and the new POAF. However, the early postoperative dual-antiplatelet therapy with aspirin and clopidogrel may be useful for preventing graft failure. Postoperative CTA scan can help to identify the early asymptomatic graft failure and to make further reinterventions.Additional file 1. Tables about the patency rates and grafting strategy.


Aspergillus niger was engineered. In all fungal GH13 α-amylases that carry a carbohydrate binding domain (CBM), these modules are of the CBM20 family and are located at the C-terminus of the α-amylase domain. To explore the role of the domain order, a new GH13 gene encoding an N-terminal CBM20 domain was designed and found to be fully functional. The starch binding capacity and enzymatic activity of N-terminal CBM20 α-amylase was found to be superior to that of native GH13 without CBM20. Based on the kinetic parameters, the engineered N-terminal CBM20 variant displayed surpassing activity rates compared to the C-terminal CBM20 version for the degradation on a wide range of starches, including the more resistant raw potato starch for which it exhibits a two-fold higher Vmax underscoring the potential of domain engineering for these carbohydrate active enzymes.In the starch processing industry including the food and pharmaceutical industries, α-amylase is an important enzyme that hydrolyses the α-1,4 glycosidic bonds in starch, producing shorter maltooligosaccharides. In plants, starch molecules are organised in granules that are very compact and rigid. The level of starch granule rigidity affects resistance towards enzymatic hydrolysis, resulting in inefficient starch degradation by industrially available α-amylases. In an approach to enhance starch hydrolysis, the domain architecture of a Glycoside Hydrolase (GH) family 13 α-amylase from  A. niger is a member of the glycoside hydrolase family 13 (GH13) as classified by the Carbohydrate-active enzymes (CAZymes) database [see CAZy database at www.cazy.org (accessed on 25 February 2023)] [A. niger GH13 amylases have an ancillary starch binding domain (SBD) called carbohydrate-binding module family 20 (CBM20), always attached at the C-terminus [α-Amylases are enzymes that catalyse the endo-amylolytic cleavage at 1,4-glucosidic bonds of starch and find application in a broad range of industrial processes using starch as substrate . The α-aAspergillus niger glucoamylase [A. niger glucoamylase [Cryptococcus sp α-amylase [Neurospora crassa [Currently there are 15 CBM families listed in the CAZy database that have a starch binding activity and are considered to support enzymatic activity by increasing proper attachment on the polysaccharide [oamylase ,7. The Coamylase ,9. This oamylase ,11, Cryp-amylase , and AA1a crassa .Starch is a glucose polymer that occurs in nature in semi-crystalline granules consisting of linear glucose polymers with 1–4 linkages (amylose) and branched polymers containing both 1–4 and 1–6 bonds (amylopectin). Variation in the amylose:amylopectin ratio plays an important role in controlling the size and shape of the starch granules. A higher amylose content and a denser structure of the granule lead to an increased resistant starch fraction ,15. The Bacillus sp. AAH-31 [Enzyme engineering has led to the discovery of improved enzymes for a more efficient hydrolysis of substrates, in particular recalcitrant polysaccharides. Several enzyme modification strategies through molecular engineering have been applied to create improved enzymes, including mutagenesis, linker modification, and truncation, as well as N or C-terminal fusions ,22,23,24. AAH-31  and revi. AAH-31 . This en. AAH-31 . However. AAH-31 ,28. Alth. AAH-31 ,30.T. maritima xylanase to either the C- or N-terminus of A. niger GH11 xylanase resulted in an increase in thermostability as well as activity [A. niger glucoamylase [Cryptococcus sp increased the catalytic efficiency toward raw rice starch [Neurospora crassa on to the C-terminus of AA13 LPMOs from Myceliophthora thermophila resulted in a more than 50% increase in amylose binding [In xylanase engineering experiments it has been shown that the fusion of the C-terminal CBM9 from activity . CBM20 foamylase . Moreovee starch . For AA1 binding . HoweverA. niger, the simplest α-amylase domain organisation consists of a GH13 catalytic domain followed by a Domain of Unknown Function (DUF1966). Some of the A. niger amylases also have a CBM20 domain at the C-terminus, however an N-terminal CBM20 is not present in any α-amylases from this organism. Furthermore, according to the current database of protein families and domain organisation available in InterPro [A. niger, by assembling the GH13 catalytic domain with an N-terminal CBM20 domain. The opportunity that this approach could offer was supported by recent findings that the N-terminal fusion of CBM20 onto 4-α-glucanotransferases from the GH77 family was able to enhance the enzyme’s affinity toward granular starch [In InterPro , approxir starch .A. niger α-amylase carrying an N-terminal CBM20. The effects of this new domain architecture on substrate binding properties and catalytic activity as well as kinetic parameters are reported. This research suggests that the domain engineering of α-amylase is a valuable approach to obtain chimeric amylase with improved binding as well as hydrolysis especially with raw starch granules as substrate.Obviously in engineering the enzyme domain architecture, not only the order of domains but also the linker peptides connecting various domains can influence enzyme activity and substrate binding, as is demonstrated in studies showing that variation in linkers affects the kinetic behavior of modular α-amylase  and cellBaudoinia panamericana, which also contained a CBM20 at the N-terminus of the catalytic domain (XP_007679364.1). Furthermore, the actual signal sequence as well as the CBM20 and GH13 domain of our chimeric design were derived from A. niger.In designing a novel configuration of a GH13 α-amylase-containing modular enzyme, not only should the secretion signal as well as various fungal domains such as the activity domain and the substrate binding domain be considered but also the linkers connecting these different domains. In the research presented in this paper, a completely new design of α-amylase was explored. To avoid unwanted effects of de novo designed linkers, we took special care in using native linker configurations. This meant that we had to select configurations derived from different annotated genome sources where these enzyme configurations existed, since no N-terminal CBM20_GH13 amylase has been described. The linker connecting GH13 with CBM20 at the N-terminal position was derived from an uncharacterised algal GH13 family protein with an N-terminal CBM20 (JAC81539.1). However, as the annotated protein encoded by this gene was devoid of a signal sequence, the linker used in joining a signal sequence with the N-terminal CBM20 was derived from the hinge connecting a signal sequence from a fungal laccase from A. niger glucoamylase (glaA), as this was shown to be a versatile secretion signal [A. niger glucoamylase, representing a well and extensively studied enzyme containing a CBM20 [A. niger CBS 513.88, of which the accurately annotated genome sequence was available [A. niger CBS 513.88 has been widely studied [A. niger AB4.1 was used. In this case we could not use A. niger CBS 513.88 as a source as this strain does not have a gene encoding an α-amylase with a C-terminal CBM20 [A. niger AB4.1. The domain architectures of these three α-amylase variants are shown in The signal sequence was retrieved from n signal . Additio a CBM20 ,45,46,47vailable . Moreove studied ,50,51. TA. niger, but we have not determined if the α-amylases analysed in our research were glycosylated. However, this has no effect on the major conclusion of our research, i.e., that an N-terminal CBM20 domain results in a full active amylase.In modular CAZymes with CBMs, the modules are expected to act cooperatively during catalysis, and that degree of linker flexibility is essential for the movement of the domains. It was reported that the amino acid composition of the linker plays a role in determining the linker flexibility which further influences the interaction between domains ,53. SeveIn the chimeric CBM20_GH13, the natural linker used for connecting the two domains contains a large number of proline (Pro) residues, while the Pro residues are absent in the linker of the native GH13_CBM20 . Pro is For selecting the best performing transformants, 25 transformants from each construct were screened for activity on AZCL-amylose plates  showed a single protein band with amylase activity . All strFurther analysis of the enzymatic activity was conducted for the two CBM20 containing α-amylases obtained after the starch-binding purification, CBM20_GH13 and GH13_CBM20. The protein concentration of both samples was normalised to 0.5 µg, and similar band intensities were confirmed with SDS-PAGE . As showThe enzymatic activities of both α-amylases with N- and C-terminal CBM20 were also examined using various substrates, such as CNPG3 and several starches from potato, rice, corn, wheat, and soluble starch. Both enzymes were shown to hydrolyse a broad range of starches and CNPG3 substrates . SimilarFurthermore, to allow accurate comparison, the kinetic parameters of CBM20_GH13 and GH13_CBM20 were determined through measuring enzymatic specific reaction rates with various substrate concentrations and calculated in Unit per 1 mg of protein . The valIn all cases, the Vmax values of chimeric amylase with N-terminal CBM20 was higher than the Vmax of GH13_CBM20, especially for the CNPG3 substrate. As expected, the hydrolysis rate of the “simple” artificial chromogenic CNPG3 substrate was more rapid than the other “complex” starch substrates with a Vmax of 90.9 and 71.4 μmol/min for CBM20_GH13 and GH13_CBM20, respectively. CNPG3 is regarded as an easier substrate than the natural starch polymers, which have a much more complex structure, making them less susceptible to degradation by amylase.Among starches, both α-amylases with CBM20 acting on soluble starch and rice starch showed a higher Vmax compared to the Vmax for other types of starches. Moreover, especially for raw potato starch a very low Vmax was observed. It was reported that rice starch has smaller granule size distribution and is more porous compared to other common cereal starches such as maize and wheat, of which this small particle size and higher porosity provides more available surface for enzyme and water adsorption, affecting the high rate of hydrolysis by α-amylase . MoreoveFurthermore, in terms of enzyme affinity represented by the Km values, in general the CBM20_GH13 showed a lower Km than GH13_CBM20 . A low KA. niger used in this study exhibited an open structure in the substrate binding cleft  and complete medium (CM) were used [E. coli DH5α. For the elution of bound protein, the corn-based malto-dextrin from Merck (419672) was used. For enzyme activity assays, several substrates were used: AZCL-amylose (Megazyme), 2-Chloro-4-nitrophenyl α-D-maltotrioside (CNPG3) , and various starch granules purchased from Sigma–Aldrich, including soluble starch (S9765), rice starch (S7260), corn starch (S4126), wheat starch (S5127), and raw potato starch . All starch substrates used are insoluble. The 3.5-Dinitrosalicylic acid (DNS) solution was also purchased from Sigma–Aldrich (D0550) to conduct enzyme activity analysis based on the DNS assay with starch substrates.N52-1Not , was useormation . For culere used . Luria–BA. niger (database accession ANI_1_460094), the signal sequence and CBM20 encoding sequence from A. niger Glucoamylase (CAA25219.1), the linker region joining a CBM20 and GH13 domain from algae Neopullulanase tetraselmis (JAC81539.1), and a linker joining the signal sequence and CBM20 from a Fungal Laccase (XP_007679364.1). The signal sequences were predicted using SignalP 6.0 ). Protein domain analysis was performed using the HMMER program (Version 3.3.2) based on the conserved domain database available in Pfam [https://web.expasy.org/protparam/ (accessed on 20 December 2022)).In order to construct the new α-amylase with an N-terminal CBM20 (referred to as CBM20_GH13), we made the following amino acid sequence selections based on naturally occurring and nature-inspired configurations: the GH13 α-amylase encoding gene from  in Pfam . Based oFor the cloning of the chimeric amylase gene variant, BspLu11I and BamHI restriction sites were added at the 5′terminus and 3′terminus, allowing the chimeric genes to be cloned in pAN52-1Not plasmid , where BA. niger AB4.1 using primer pairs of OriAamAf and OriAamA_CBM20r. All final constructs with α-amylase variants were verified by sequencing using primer MBL852 and MBL858 (Macrogen Europe B.V). All the primers details are listed in http://www.pymol.org (accessed on 20 April 2023)).For generating the expression vector for the GH13 α-amylase without CBM20, assigned as GH13, the vector containing a chimeric CBM20_GH13 gene was used as a template for PCR amplification using primer pairs of AamA_2 and GH13AamARev. Meanwhile, for generating the expression vector of the native α-amylase with a C-terminal CBM20 assigned as GH13_CBM20, the complete gene encoding protein GH13_CBM20 (KAI3001921.1) was amplified from the genomic DNA of E. coli DH5α and each of the vectors used for the transformation of A. niger MGG029-ΔaamA pyrE−. Fungal transformation was carried out according to the protocol described in [A. niger pyrE selection marker vector in 10:1 ratio. PyrE+ transformants were selected on minimal media containing sucrose as an osmotic stabiliser [The three different expression vectors were amplified in ribed in , using tabiliser . Further8A. niger spores. These flasks were incubated in a shaker incubator at 180 rpm and 30 °C for 96 h. At the end of the cultivation process, the spent medium containing the secreted enzymatic activities was collected by filtration.For cultivation, submerged fermentation was carried out in 300 mL Erlenmeyer flasks with a 100 mL working volume of liquid complete medium that was inoculated with 1 × 10w/v) corn starch was pre-washed three times using 50 mM acetate buffer  in a 50 mL tube to remove traces of soluble saccharides. The spent medium was adjusted to pH5 with one third volume 200 mM acetate buffer pH 5. For enzyme binding, the pH 5-adjusted spent medium (45 mL) was mixed in a 50 mL tube which already contained prewashed 2% (w/v) corn starch and incubated overnight on ice while shaking gently using a rocking shaker (VWR®) in a cold room. Eventually, the corn starch with the bound protein was collected by centrifugation  separating it from the supernatant with any unbound proteins. Subsequently, the collected corn starch–enzyme complex was washed once with 50 mM NaAc buffer pH 5 by centrifugation  to remove any unbound protein or nonspecific protein binding. The supernatant and wash solution containing unbound protein were collected for determining the total unbound activity. The percentage of bound protein was determined by assaying enzyme activity in the spent medium and unbound fraction. Binding potential (%) was determined using the following equation:The starch binding purification was carried out based on corn-starch binding and the elution of protein as described in  with som® 44). To obtain the eluted protein, the corn starch was removed by centrifugation , and the supernatant containing purified protein was collected. The malto-dextrin molecules were removed through ultrafiltration with spin columns . The malto-dextrin was contained in the flowthrough, and the purified enzyme remained in the retentate. The purified α-amylase in the elution buffer  was then stored at −20 °C until being used for further experiments. The evaluation of α-amylase purification was performed by measuring the activity of the spent medium and all the fractions obtained from purification steps using CNPG3 and various starches as substrate  containing 2% malto-dextrin . The eluubstrate .A. niger transformants were spotted onto solid minimal medium containing 0.1% AZCL-Amylose (chromogenic substrate) and incubated at 30 °C for 24 h, essentially as described by the manufacturer protocol (www.megazyme.com (accessed on 20 December 2022)) and also as reported in [For the screening of the transformants, the spores of orted in . The amy2CO3. Subsequently, the 100 µL of each reaction sample was transferred to a 96-well plate , and the absorbance was measured at 405 nm using a plate reader . A standard curve was prepared using 2-chloro-4 nitrophenol (CNP) with a concentration ranging from 0.2 to 1.5 mM. One unit (U) of enzyme activity is defined as the amount of amylase that catalysed the formation of 1 μmol CNP per hour under assay conditions. This activity assay was performed in triplicate.The activity of α-amylase on CNPG3 was measured by mixing 50 μL of either spent medium or purification fractions with 50 μL of 10 mM CNPG3 dissolved in 50 mM NaAc pH 5 and incubation at 37 °C for 30 min as essentially described in . After i® 10 M). The soluble reducing sugar released was quantified based on a standards curve prepared with 0–10 mM glucose. A blanc without enzyme was included and its OD540 subtracted for all measurements. All experiments were carried out in triplicate. One unit of enzyme was defined as the amount of enzyme that produced 1 μmol of reducing sugar or equivalent to 1 μmol of glucose per hour.The enzymatic activity of the α-amylase variants was also investigated using several types of starches as substrates, including starch from rice, wheat, and corn, soluble starch, and raw potato starch in a DNS assay . These s® TGX™, #4561033, Hercules, CA, USA) as essentially described in [v/v) MeOH, 7% (v/v) Acetic Acid) and incubated at room temperature for 60 min while shaking gently using a rocking shaker (VWR®) and changed with fresh fixation buffer solution every 30 min. Subsequently, 35 mL of Sypro Ruby staining solution  was poured over the gel in a dark container and incubated overnight at room temperature with gentle shaking on a rocking shaker. The gel was destained with 40 mL of destaining buffer (10% (v/v) MeOH, 5% (v/v) Acetic Acid). Gel imaging was performed with the BioRad GelDocTM EZ Imager. Automatic image exposure and band intensity were selected. For the background image the colour gray was selected, while white was chosen for the Sypro Ruby stained protein bands (as shown in Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in 10% precast polyacrylamide gels (Bio-Rad, Mini-PROTEANribed in . The speFor the detection of α-amylase activity through zymography as essentially described in , samplesFor the kinetic parameters of the purified enzymes as described in ,83, the


In this study, we conducted research on the preparation of aerogels using cellulose and starch as the primary materials, with the addition of N,N′-methylenebisacrylamide (MBA) as a cross-linking agent. The chemical, morphological and textural characteristics of the aerogels were found to be influenced by the proportions of cellulose, starch, and cross-linking agent that were utilized. An increase in the proportion of cellulose led to stronger adsorption forces within the aerogel structure. The aerogel showed a fine mesh internal structure, but the pores gradually increased with the further increase in cellulose. Notably, when the mass fractions of starch and cellulose were 5 wt% and 1 wt% respectively, the aerogels exhibited the smallest pore size and largest porosity. With an increase in the crosslinking agent, the internal structure of the aerogel first became dense and then loose, and the best internal structure was displayed at the addition of 3 wt%. Through texture analysis and the swelling test, the impact of the proportion of cellulose and MBA on the aerogel structure was significant. Dye adsorption experiments indicated that MBA affected the water absorption and expansion characteristics of the aerogel by improving the pore structure. Lastly, in tests involving the loading of vitamin E, the aerogels exhibited a higher capacity for incorporating vitamin E compared to native starch. Aerogel is a unique material known for its special properties, including a large number of internal voids, low density, and various potential applications such as dye adsorption, heavy metal adsorption, and drug delivery systems . First dStarch is a biomolecule with abundant reserves on earth, offering numerous characteristics that make it highly attractive: readily available raw materials, high biodegradability, low cost, and a simple preparation process. Its molecular structure is characterized by a multitude of reactive hydroxyl groups, particularly the reactive ones that serve as the foundation for starch modification. Leveraging this feature, the hydroxyl groups on the surface of starch aerogels can be utilized as sites for various reactions, thereby expanding the potential applications of starch aerogels ,16,17. GDespite its advantages, starch-based aerogel still has some limitations. One of the main drawbacks is its relatively low strength, especially under wet conditions because the exposed hydroxyl groups in the starch molecular chain are easy to combine with water molecules to form hydrogen bonds. When in contact with water, the physical properties of the material are compromised, leading to a decrease in volume . To addrCellulose, a polymeric material formed by glycosidic bonds, offers a solution to these challenges. Unlike starch, cellulose possesses a strong molecular composition that is minimally affected by water, maintaining its strength even when immersed in water at temperatures as high as 100 °C ,20. MoreTherefore, this study focuses on the utilization of cellulose and starch, which possess strong biological functionalities, to develop aerogel materials. The objective is to create a composite aerogel by adjusting the ratio of these two raw materials in order to retain their inherent characteristics, namely, ease of preparation, biodegradability, and structural stability. The investigation systematically explores the role of cellulose, starch, and the cross-linking agent methylenebisacrylamide in the formation of aerogels. Additionally, several experiments are conducted to assess various aerogel properties, including morphology, swelling behavior, dye adsorption capacity, and mechanical strength. Lastly, the study explores the potential application of the developed aerogel for vitamin E adsorption.Potato starch was obtained from Hua’ou Starch Co., Ltd. ; Cellulose powder (particle size of 65 μm), N,N′-methylenebisacrylamide (MBA), and methylene blue were purchased from Aladin Biochemical Technology Co., Ltd. ; and Vitamin E was purchased from Macklin Biochemical Technology Co., Ltd. . All other chemical reagents used in this experiment were analytic reagents and purchased from Aladin Biochemical Technology Co., Ltd. .4)2S2O8 and NaHSO3 .Firstly, the water content of potato starch (PS) was measured by an IR-35 infrared rapid moisture analyzer . Then, a specific amount of PS and microcrystalline cellulose was taken in a beaker with 100 mL deionized water, resulting in a combined mass fraction of 6 wt% (dry basis) for the two materials. Then, a certain amount of the crosslinking agent MBA was added to the above solution  followed by the composite initiator  to form a network structure. See the In the crosslinking process, the starch particles were gelatinized in the aqueous solution to fully release molecular chains and expose active hydroxyl groups. Then, the initiators were added to the starch paste to initiate the free radical compolymerzation. They were decomposed to generate initiating radicals, such as SO3 container, and the evaporation of water was prevented by covering it with cling film. After allowing the mixture to cool to room temperature, it was placed in a 4 °C environment for 24 h to form a hydrogel. Subsequently, the material was placed in a −18 °C environment for another 24 h. Finally, the frozen gel was removed from the mold and subjected to a 24-h freeze-drying process. The resulting composite aerogel was labeled based on the proportion of its components, denoted as S(starch)*C(cellulose) or S*C*B (cross-linker MBA). For example, S5C1B3 indicates that the composite aerogel consists of 5 wt% starch, 1 wt% cellulose, and 3 wt% cross-linker.The solution was subsequently placed in a water bath at 90 °C for 0.5 h while being magnetically stirred at a rate of 1.67 r/s. Once the treatment was complete, the mixture was directly transferred into a 20 × 20 × 15 mmThe material was positioned next to a measuring tape and captured by camera to acquire an image depicting its external structure. The composite aerogel was segmented into multiple thin layers, approximately 0.1 cm thick, enabling microscopic examination using a Merlin scanning electron microscope  at an accelerating voltage of 5 kV. The magnification was 100×. One of the layers was carefully positioned on the microscope stage, securing its upper section to the carrier table using a conductive adhesive, followed by a 5-min gold coating using an MCM-100 Ion Sputter Coater . Subsequently, the material and the carrier table were positioned within the electron microscope chamber, where the air was evacuated prior to observation.The average droplet size and size distribution was determined through statistical analysis from optical microscope images using Image-pro Plus 6.0.−1 with 64 scans at a spectral resolution of 4 cm−1.The aerogel was ground into a powder, 3 mg composite aerogel powder and 200 mg potassium bromide powder were mixed well in a drying oven, and the dried mixture was pressed into tablets under a pressure of 20 Mpa at room temperature for 30 s. The obtained lamellar material was analyzed under an infrared spectrometer  between 4000 and 400 cmThe NMR spectra were recorded using an AVANCE digital 400 spectrometer  operating at 400 MHz for 1 H NMR spectroscopy and 100 MHz for 13C NMR spectroscopy, respectively. The 1 H spectra were recorded in 128 individual scans with a sweep width of 16 ppm and a delay time of 1 s. Prior to NMR analysis, 5 mg composite aerogel powders were dissolved in deuterium dimethyl sulfoxide (DMSO-d6) at 40 °C to obtain clear solutions.2 and placed facing downwards. TPA  testing was performed using a P/36R probe. The pre-test speed, test speed, and post-test speed were set at 3.0 mm/s, 1.0 mm/s, and 3.0 mm/s, respectively. The compression ratio was set at 50%, and the trigger force was 5 g. The compression was performed at intervals of 5 s, and the test was repeated at least 4 times under the same conditions. The data were fitted using the MACRO program in the Exponent system to obtain the hardness, viscosity, and elasticity indices of the samples.The determination of textural characteristics was conducted using a TA-XT2 type physical property tester . The prepared starch aerogel cubes were used as the samples, with one side measuring 20 × 20 mmM0 (g) represents the mass of the initial unsoaked sample; and Mt (g) is the total mass of sample after soaking in water at corresponding temperature.First, the mass of the composite aerogel was determined using an electronic balance. The prepared sample was placed in a beaker containing deionized water at room temperature (25 °C). After soaking for 0.5 h, the total mass of the material was measured, and the swelling ratio was obtained by Equation (1). The unit is expressed as g/g. This process was repeated at 50, 70 and 90 °C. Finally, the swelling ratio of the composite aerogel at four different temperatures was summarized and recorded.Firstly, 50 mL of an aqueous solution containing methylene blue at a concentration of 10 g/L was added to a 100 mL centrifuge tube along with a magnetic stirrer. Then, a specific mass of cross-linked starch aerogel was accurately weighed. The aerogel was introduced into the centrifuge tube, which was subsequently placed on the magnetic stirrer inside a water bath maintained at 25 °C to initiate the experiment. Once the experiment commenced, aliquots of the solution were extracted at the specific time intervals of 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h. Each of the seven extracts was then diluted to one-tenth of the original concentration, and the corresponding absorbance was measured and recorded. Based on the obtained data, absorbance curves for the extracts were plotted. Similarly, a standard curve for methylene blue was constructed using solutions of 0.01 g/L, 0.005 g/L, 0.0025 g/L, 0.001 g/L, and 0.0005 g/L. The instrument wavelength was adjusted to 660 nm, and a cuvette filled with deionized water served as the reference solution in the first compartment of the cuvette holder. The measurement of the adsorption of methylene blue refers to the amount of methylene blue material adsorbed per unit weight of the aerogel material. The unit is expressed as mg/g.0 and Ct (mg/mL) represents the concentration of Vitamin E before and after the adsorption process; V (mL) is the volume of the solution; and M (g) is the mass of sample.Five composite aerogel samples were chosen to assess the adsorption capacity of fat-soluble Vitamin E. A standard curve for Vitamin E was constructed using solution with concentrations of 0.05, 0.1, 0.2, 0.4 and 0.5 mg/mL. Initially, 25 mg Vitamin E was dissolved in 50 mL anhydrous ethanol to achieve the concentration of 0.5 mg/mL. 0.1 g of the samples were then immersed in the prepared solution and shaken for 30 min to ensure the complete adsorption of Vitamin E. The adsorption capacity of vitamin E in various types of samples was calculated based on Equation (2).Cellulose, being insoluble in water, primarily served a supportive and stabilizing role within the aerogel structure. When starch was added, the rigid cellulose molecule chains built more connection points between the free starch molecules (within the gelatinized starch), forming a denser internal structure. The SEM images of S4C2 and S3C3 revealed residual cellulose (irregular lumps as indicated by the arrows) within the aerogel pores. In this case, the cellulose primarily strengthened the starch structure and did not act as a connection site. The reason for this occurrence was that as starch content decreases, some cellulose remains unwrapped by starch molecules, thereby exacerbating pore enlargement. Additionally, increased cellulose content hampers the formation of separate pores. Consequently, we concluded that the starch-to-cellulose ratio in S5C1 is the most suitable. It could be seen that S5C1 was a more appropriate ratio, and the aerogels formed at this ratio were the densest and the cellulose was highly utilized.When the MBA proportion reached 5%, the aerogel began to exhibit laxity, resulting in an increase in pore diameter. On one hand, the addition of a small quantity of hydrophilic MBA led to more water content, causing the formation of larger ice crystals during the freezing process ,22. On t−1, which corresponded to the characteristic absorption peak of starch and cellulose (the stretching vibration of -OH groups). The stretching vibration generated by the saturated hydrocarbon C-H in glucose gave rise to a distinct sharp peak at 2900 cm−1, which was also a characteristic absorption peak of starch and cellulose.−1 that was related to the bending vibrations of the N-H bonds on the MBA chains [In the absence of the cross-linker MBA, no significant change in the fingerprint region was found in the IR spectra of S5C1, S6C0, and S0C6. There were subtle differences between the peak intensities and widths in the spectrum, which could be explained by differences in the hydrogen bond formation and water content between the components. In contrast, S5C1B1 containing the cross-linker displayed a new small peak at 1547 cmA chains . This ph1H NMR spectra of the samples are shown in The 2 (S3C3) < 3909 N/m2 (S4C2) < 6768 N/m2 (S5C1) < 9345 N/m2 (S6C0). Therefore, it could be inferred that the hardness of aerogels could vary significantly depending on the amount of starch present in them. It was observed that cellulose and starch served as the main precursor substances in aerogels. During the aging and pasting of the starch, the molecular structure changed from a dispersed state to a reconnected state, which played a crucial role in the overall network structure. Notably, cellulose was distributed discretely within the aerogel and lacked a continuous three-dimensional structure. As a result, it was challenging for cellulose to effectively support the aerogel, despite its more stable internal structure. This limitation also hindered further improvement in the hardness of the aerogel.In this study, the hardness, elasticity, and cohesion of the aerogels formed at different starch/cellulose ratios were systematically analyzed using a mass spectrometer (TPA), and the data are detailed in Likewise, S3C3 exhibited the lowest cohesion (0.18) and elasticity (0.22) due to its lower starch content. Starch played a vital role in providing support throughout the entire aerogel structure, and a higher starch content significantly affected the aerogel’s structure. Cohesion could be defined as the internal binding strength. The analysis revealed that the elasticity and cohesion of the samples remained stable (cohesion of 0.22–0.23 and elasticity of 0.27), except for S3C3, which experienced a significant decrease. This indicated that elasticity and cohesion were intrinsic properties of starch and exhibit less variation within certain ranges of starch composition. The decrease in elasticity and cohesion after reaching a certain threshold value was influenced by the decrease in starch concentration or the increased proportion of cellulose that hampered the rearrangement process of the starch molecules.2 (S5C1B0.5) < 6376 N/m2 (S5C1B1) < 8842 N/m2 (S5C1B3) < 9531 N/m2 (S5C1B5). Thus, the MBA content played a crucial role in the formation of aerogels, and its addition contributed to the enhancement of their structural properties.The TPA data obtained at various MBA contents are presented in detail in Regarding cohesion and elasticity, most samples (excluding S5C1B3) exhibited a noticeable decreasing trend with an increasing amount of MBA. In the absence of MBA data in , cohesioMoreover, it was observed that the S5C1B3 sample displayed a distinct cohesion and elasticity profile. This could be attributed to the reduced pore size of the aerogel. The smaller pore size in the S5C1B3 sample resulted in a decreased distribution density of the cross-linker, which in turn reduced the structural constraints on the aerogel, leading to improved cohesion and elasticity.The swelling properties of different composite aerogels are detailed in The curves depicting the adsorption of methylene blue over time for different starch-to-cellulose ratios are illustrated in The relationship between methylene blue adsorption and time at different cross-linker contents is depicted in 1H NMR provided evidence that the cross-linker MBA had indeed interacted with starch and cellulose. The aerogel showed a fine mesh internal structure, but the pores gradually increased in size with further increases in cellulose. The aerogels formed with a 3% addition of MBA exhibited the densest pore structure, consequently providing the largest porosity. Excessive amounts of the cross-linking agent hindered the swelling property of the aerogel to some extent, resulting in an initial increase and subsequent decrease in the adsorption capacity of the aerogel as the dosage of the cross-linking agent increased. The aerogel S5C1B0.5 showed good dye adsorbability, but too much of the cross-linking agent would inhibit its ability. Finally, based on experiments involving vitamin E adsorption, it was concluded that cross-linked starch aerogel S5B1 exhibited a significantly higher ability to adsorb vitamin E compared to S6C0. It can be predicted that starch–cellulose aerogel will have important application value in special fields such as drug delivery and food material loading in vivo.In this study, cellulose and starch were utilized as raw materials to investigate the properties and correlation of aerogels formed under the influence of varying doses of MBA. FTIR and


The rheological properties of starch gels were consistent with changes in the swelling power of starch granules. SEM observations confirmed that the gel structure became more regular, and the holes grew larger with the addition of PA below the ratio of 100:6.0 . The short-range molecular order in the starch gels was enhanced by the addition of PA, confirmed by FT-IR analysis. Mechanical experiments demonstrated that the binding strength of the starch gels increased with higher PA concentrations and decreased significantly after the aging process. TGA results revealed that PA promoted the acid degradation of starch molecules. This study provides a detailed guide for the preparation of starch-based adhesive and its applications in paper conservation.Wheat starch (WS) is a common adhesive material used in mounting of calligraphy and paintings. Potassium alum (PA) has indeed been used for many centuries to modify the physicochemical properties of starch. Thermal analysis revealed that the presence of PA led to an increase in the gelatinization temperature and enthalpy of the starch gels. The leached amylose and the swelling power of the starch gels exhibited a maximum at the ratio of 100:6.0 (WS:PA,  During this process, a crucial element employed as an adhesive is a meticulously prepared starch gel derived from wheat starch . Wheat sadhesive ,9. By em+) and sulfate (SO42−), as salting-out ions, were found to lower the swelling power and solubility of starch. In contrast, salting-in ion could increase these properties [2+) and magnesium (Mg2+) had a greater impact in reducing starch recrystallization rate compared to monovalent cations [+ and SO42−) and one multivalent cation (Al3+). As a result, the influence of PA on starch mixtures encompasses the comprehensive effects of these three ions working together.PA has already proven its significant effects on the properties of starch-based gels ,11. For operties ,16,17,18 cations . PA consw/w) used in restoration practices depend on the size of the works being restored. In general, the ratios range from 100:2 to 100:20 . However, the impact of PA on the apparent viscosity of wheat starch gels and its relationship with the binding capacity of the gel have not been thoroughly investigated. Furthermore, PA is an acid salt that has the potential to cause acid degradation and color change in paper relics, which has raised concerns among restorers [The use of PA blended with wheat starch suspension to enhance the apparent viscosity and adhesive strength of starch gels has been a practice for many centuries. The ratios of WS:PA (estorers . Therefo3+ ions present in PA can combine with water to form positively charged hydrated forms such as Al(OH)2+, Al2(OH)24+, Al3(OH)45+, and Al13O4(OH)247+ [w/w) increases by 8.7% compared to the blank sample. This increase indicates that more energy is needed to disrupt the starch granules. These findings are consistent with results concluded by Chen et al., who reported that multivalent cations can enhance the stability of the granules [The gelatinization curves of the WS and WS-PA mixtures were analyzed by DSC, and the pasting parameters were calculated using specialized software. (OH)247+ . The gel(OH)247+ . The pregranules .w/w) and decreases with the addition of PA above the ratio of 100:6.0 . Based on w/w), which is consistent with the results of leached amylose measurements. Different types of ions can significantly affect the pasting behavior of starch, in accordance with the Hofmeister series [+, Al3+, and SO42− ions. Wang et al. discovered that SO42− and K+ ions, as salting-out agents, could improve the stability of starch granules [3+ ions, as multivalent cation, could bind with water molecules to form hydroxyl coordination compounds (Al(OH)x(H2O)yn) which may promote the disintegration of starch granules [w/w), the PA could promote the disintegration of starch granules, with Al3+ ions playing a dominant role. However, when the addition of PA is above the ratio of 100:6.0 , the stability of starch granules is improved. There are two reasons to account for this phenomenon. Firstly, the SO42− and K+ ions play a dominant role as salt-out ions which could inhibit the breakage of starch granules. Secondly, the positively charged hydroxyl coordination compounds of Al3+ could act as cross-linking points through electrostatic interactions in the amorphous structure or on the surface of starch granules which inhibit the breakage of granules [The leached amylose of samples is an important parameter that indicates the leaching of soluble amylose starch and segmental amylopectin from starch granules during gelatinization. Swelling power represents the water absorption capacity of starch granules . Figure r series . PA consgranules . Conversgranules . The resgranules .w/w), the dynamic modulus increases with the presence of PA, as shown in w/w), the dynamic modulus decreases as the PA concentration increases further. These findings are in agreement with the observed swelling power of the samples, suggesting that the WS–PA gel has the most compacted structure at the ratio of 100:6.0 . The results indicate that the breakage of granules reaches the maximum at this specific proportion and more-soluble starch leaching out from the granules participates in the network formation of WS–PA gels.The dynamic rheological properties of WS and WS–PA gels at different ratios of PA are analyzed and presented in −1. Firstly, this phenomenon can be attributed to the non-equilibration of the sample [w/w), but it then decreases as the ratios increase further from 100:6.0 to 100:120 . The consistency index (K) calculated by the Herschel–Bulkley model represents the viscosity of the gels. However, the behavior of K varied between ratios of 100:3.6, 100:6.0, and 100:12 . This decrease can be attributed to the destruction of the compact crosslinked structure of WS−PA gels at high shear rates, resulting in a significant decrease in viscosity and the lowest fitted value of K. The yield stress required to change the structure of the gels increases from 1.14 Pa to 7.74 Pa with the ratio from 100:0 to 100:6.0 . This indicates that more external force was required to alter the initial structure of starch gels at low addition concentrations of PA. However, the opposite trend is observed as the ratios increase from 100:6.0 to 100:120  with the yield stress decreasing from 7.74 Pa to 0.37 Pa. This trend is consistent with the interpretation of the change in swelling power discussed in w/w) could result in obtaining the most compact structure of starch gels.e sample . Secondlw/w) is shown, and the others are displayed in −1 are shown in the −1, corresponding to the stretching vibrations of O-H bonds in starch [2 and -CH groups are detected at 3000–2800 cm−1 [−1 indicates the bending vibration of water molecules within the sample. The fingerprint region of 1200–800 cm−1 is sensitive to conformation of the samples, particularly the absorption peaks at 1022 cm−1 and 1047 cm−1, which corresponds to the relative contents of random structures in the amorphous region and the ordered structures in the crystalline region, respectively. The ratio of the absorption peak intensity at 1047 cm−1 to 1022 cm−1 serves as a measure of starch crystallization degree. The results are presented in −1 to 1022 cm−1 increases with the addition of PA, further indicating that during the storage, PA within the system can effectively promote the ordered rearrangement of starch molecular chains.The infrared spectra of WS and WS−PA gels are displayed in n starch . The sym800 cm−1 . A minorw/w). However, the opposite tendency can be observed at the higher ratios from 100:6.0 to 100:120 (w/w). The addition of PA promotes the breakage of starch granules below the ratios of 100:6.0 , and more starch molecules participate in the network formation of WS−PA gels. However, when the ratios are above 100:6.0 , the addition of PA inhibits the disintegration of granules. Hence, the structure becomes irregular and the size of pores grows smaller. Additionally, the lyophilized samples with the ratios above 100:30  are brittle, which is perhaps related with the high crystallization degree of starch gels.3+, which generates various hydroxyl coordination compounds. These compounds can participate in the network formation of the starch gel. Additionally, a significant amount of hydrogen ions is generated during the hydrolysis process of Al3+, which facilitates the breakage of glycosidic bonds [ic bonds . As a cow/w) resulted in an increase in folding endurance from 1.96 ± 0.18 to 2.49 ± 0.13, tearing strength from 267.33 ± 15.81 (mN) to 417.66 ± 20.77 (mN), and tensile strength from 0.96 ± 0.08 (kN·m−1) to 1.18 ± 0.10 (kN·m−1). This indicates that the adhesive strength of starch gels was enhanced with the addition of PA. Interestingly, these results were not consistent with the changes observed in the apparent viscosity measured through steady rheology experiments. The reason for this phenomenon can be attributed to the positively charged hydroxyl coordination compounds of Al3+, which act as ion-bridges to connect the starch fragments and cellulose fibers of the paper [w/w), indicating that the higher crosslinked density between cellulose fibers and starch fragments improved the hardness of the paper samples [w/w) decreased by 19.9%, 37%, and 11.5%, respectively. The reason for this phenomenon can be related to the hydrolyzation of Al3+ ion, and a large number of H+ ions are generated during this process, which promotes the acid hydrolysis of glycosidic bonds in both starch chains and cellulose fibers. The decrease in mechanical properties of the testing samples after aging experiments highlights the significance of controlling the addition of PA to the starch gel in practical applications, as it can affect the durability and stability of the paper relics.he paper ,38. Thes samples . HoweverWheat starch  was provided by Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China. Potassium alum (PA) was provided by Sinopharm. Deionized water was used in this work. Xuan paper was obtained from China Xuan Paper Company Group, Jingxian, China.w/w), including 100:0, 100:1.2, 100:2.4, 100:3.0, 100:3.6, 100:6.0, 100:12, 100:30, 100:60, and 100:120, were labeled and used for further experiments. The starch gels were prepared by heating at 95 °C for 20 min under constant stirring. After heating, the resulting starch gels were stored at ambient temperature for 24 h.Different concentrations of PA solutions were prepared. Wheat starch was added into PA solutions to obtain 4% wheat starch solutions by oscillating. Samples with ten ratios of WS:PA  [0 represents the yield stress (Pa), τ represents shear stress (Pa), K is consistency index (Pa·sn), γ represents shear rate(s−1), and n is the flow behavior coefficient.The rheological measurements were carried out by a rheometer . The experiments were conducted at a temperature of 25 °C using a parallel plate . Before testing, the samples were transferred onto the rheometer plate and equilibrated for 5 min. In order to analyze the dynamic rheological properties, the liner viscoelastic region was determined by a strain sweep measurement, and the strain for the frequency sweep experiment was set at 1% . The fretion (1) .Τ = τ0 v/v) to adjust the pH of the solution to 5.0–6.0, and subsequently 0.01 N I2-KI (0.05 mL) was added. The mixture was reacted at room temperature for 30 min, and the absorbance of solution at 620 nm was measured using a spectrophotometer . Amylose was used as the standard to calculate the dissolution amount of amylose.The leached amylose and swelling power of WS and WS−PA gels were obtained according to the report of Chen et al. with a slight modification . The slu2 atmosphere [The thermal analysis of wheat starch with different concentrations of PA was conducted using a DSC  under a Nmosphere . To perf2 atmosphere, and the heating rate was 20 °C/min. The temperature range was set from 30 °C to 600 °C. The weight loss of the samples was recorded as a function of temperature, indicating the decomposition or volatilization of different components contained in the samples [The WS−PA gels were prepared according to the methods in  samples ,45.−1 to 500 cm−1. To collect the spectra, each sample was tested at a resolution of 4 cm−1, with an accumulation of 64 scans. One specific parameter of interest for evaluating the molecular order of starch gels is the ratio of absorbances at 1047 cm−1 and 1022 cm−1. This ratio provides an indication of the short-range molecular order present in the starch gels. By comparing the intensity of these two absorbance peaks, it is possible to evaluate the structural characteristics and organization of the starch gel [The lyophilized samples were obtained following the procedure described in arch gel .The lyophilized samples were obtained following the procedure described in The starch gels were obtained as per the methods described in The tensile strength was measured according to GB/T 12914-2008 at a constant elongation rate of 20 mm/min by a tensile strength tester , and the size of paper samples was 15 cm × 0.75 cm.The folding endurance was measured according to GB/T 475-2008 by an MIT folding endurance tester . The size of paper samples was 15 cm × 1.5 cm, and the force was set at 4.98 N.The tearing strength was performed according to GB/T 455-2002 by a tearing strength tester . The size of paper samples was 63 cm × 50 cm.The color change of paper samples before and after the aging process was determined by the color coordinate values (Equation (2)) using an NR10QC colorimeter .ΔE = [.All the measurements were conducted in triplicate to ensure accuracy and reliability of the results. The results were subjected to statistical analysis using SPSS statistical software . To assess the significance, analysis of variance (ANOVA) was performed on the data. In this case, Duncan’s test was used to further analyze the data, with a significance level set at w/w). The rheological properties and SEM observations support this phenomenon. In practical applications, the addition of PA enhanced the bonding strength of the starch gels. The adhesive ability of the starch gels was improved with addition of PA. However, the addition of PA at high concentrations could have a negative effect on the surface color of paper samples. Therefore, it is advisable to carefully control the concentration of PA when adding it to starch gels. Keeping the addition of PA at low concentrations, specifically below the ratio of 100:6.0 , could help to mitigate the negative effects of PA on the degradation and color change of paper relics while still achieving the desired enhancements in rheological and adhesive properties.The addition of potassium alum to wheat starch gels was found to be an effective method for modifying their physicochemical properties. The experimental results show that addition of PA increases the gelatinization temperature and enthalpy value of the starch gels. However, it was observed that the leached amylose and swelling power of the starch gels reached a maximum at a certain ratio of WS:PA, specifically at the ratio of 100:6.0 (