International Journal for Educational Integrity (IJEI). Tracey co-founded the journal with Helen Titchener, who worked alongside Tracey as Co-Editor for a number of issues before stepping away. In late 2019, Tracey invited me to join her as Co-Editor-in-Chief, starting in January, 2020. She knew she was ill when she invited me and made it clear that working alongside her during her illness was meant carrying on with the editorship after she was no longer able to do so. Although Tracey book-ended her connection to the journal with Co-Editors, throughout she remained its constant and exemplary leader, steering the journal to successively higher acclaim throughout the past 15 years.In this editorial we share our individual experiences of meeting and working with Dr. Tracey Bretag, co-founder and Editor-in-Chief of the IJEI. One of my tasks was to receive and compile the contributions. I made an editorial decision about how to order the contributions. I have book-ended the editorial with the contributions of Tracey’s Australian compatriots, starting with Helen Titchener’s, as one of the original co-founders of the journal. Next come the contributions of Tracey’s contemporaries in Australia, those who are actively engaged in professional life. Then, I have grouped the contributions by country: United States, United Kingdom, Czechia, Kosovo, UAE, Indonesia, and Mexico. There ordering of the countries is not in any way meant to represent a hierarchy or a preference, and nor is it meant to signal any kind of political statement. We, the educational integrity community the world over, are a global network of colleagues, due in no small part to Tracey’s work. The ordering of the contributions is almost certainly imperfect, and I am grateful for my fellow contributors for their graciousness and generosity. Every individual who has shared the reflections in this collaborative editorial has done so during the middle of the COVID-19 global pandemic, and at a time when we face unrelenting professional demands. The voice of every contributor matters and the ordering of the contributions was no easy task. Having said that, I intentionally ensured that the Australians have the last word, so to speak, as we conclude with the voices of two academic elders, professors emeriti, who have supported the journal as members of the editorial board, and Tracey’s work for many years.As contributors to this editorial, we share their personal and professional reflections about what working with Tracey has meant to us. Each contributor had a unique and special relationship with Tracey and Although many individuals have contributed to this editorial, we recognize that we are not alone in being inspired by Tracey. For every person who has shared their reflections in this collective editorial, there are likely at least a hundred others with similar sentiments and have their own personal connections to Tracey. We recognize that our words cannot begin to represent the worldwide impact Tracey had through her work and her vision.And now on to my own reflection…IJEI. The manuscript I had submitted for consideration was one of the first pieces I’d taken the lead on after finally securing a full-time tenure track position, after 22 years of being a sessional lecturer (otherwise known as a precariously-employed academic). The piece was a literature review about the research conducted in Canada on academic integrity. Through the revision process Tracey encouraged me to be bold, to go beyond a literature review, and set the stage for a research agenda in Canada on educational integrity, and to conclude with an evidence-informed call to action. I was inspired by her energy, her clarity of thought, and her passion for the work. I made the revisions and the piece was published Editor-in-Chief, International Journal for Educational Integrity.University of Calgary, Canada.I remember the day I met Tracey Bretag. It was 18 years ago in a seminar room at UniSA and the occasion was a presentation to introduce a brand new product to university staff. That product was Turnitin®. Tracey had recently begun her Doctor of Education studies and was passionate about pedagogical approaches to addressing plagiarism. I was at the beginning of my own PhD journey, and was passionate to lay bare the depth and breadth of the cheating behaviours of students.We happened to sit next to each other that day and, just as was the case with so many others that Tracey met at conferences and meetings over the following years, we became confederates and friends. Life-long friends.Within a fortnight, we had started what was later described by a professor of mine as a ‘cottage industry’. We were young academics, but not young in years and both of us were stubborn and tenacious. So, we did things that all young people should be roundly encouraged to do - we went and knocked on the highest doors and sold our dreams to people who held the ability to make them come true.We owed much to Professor Alan Bundy, the University Librarian. Alan was acting Pro Vice-Chancellor (Academic) in 2002. We told him that we wanted to grow an international community of academic integrity scholarship and described for him the emerging work in the rest of the world. He listened!International Journal for Educational Integrity in the closing ceremony of the conference and 2 years later the first edition was published.Alan lent us money (seed funding) and introduced us to the people who quickly became the inaugural committee of the Asia Pacific Forum on Educational Integrity. Within a year, the first conference, ‘Plagiarism and Other Perplexities’, was organised and held: We welcomed keynotes from the UK and the US and 200 participants from 12 countries. I was buoyed along by Tracey’s unending enthusiasm and energy and so, doctoral studies, jobs, and family responsibilities notwithstanding, we researched ways to establish a journal. We announced the founding of the When I had to walk away from my involvement in 2006, Tracey did what she always did, and stepped up to the challenge of doing it herself. Over the following decade and a half, Tracey sat next to countless academics, scholars, and students and never failed to give her time and energy to work with them.In so doing, she turned our cottage industry into the rigorous, internationally acclaimed, academic endeavour that it is today.Educational’ rather than ‘Academic’ when referring to integrity, and she was right. The integrity of our students does not begin when they step into university; it is established much earlier and we, as scholars, should extend our research and efforts into those areas.On a final note, it was Tracey who argued for the term ‘I am proud to have worked beside Tracey in those earliest of days. We became irrepressible accomplices and life-long friends. Life-long friends in a life that was too short.Helen Titchener.James Cook University, Australia.IJEI, where I was later honoured to join the Editorial Board. Tracey saw somethings in me that I had not seen in myself – an interest in academic and educational integrity, and the confidence to join the growing chorus of voices wanting to bring the issue out in the open, instead of pretending that the cheating and breaches of integrity did not exist. The IJEI is critical to this work, and I am proud to be so closely associated with it.A colleague encouraged me to write a conference paper in 2009 to document some work and results related to the use of Turnitin®. Little did I know that my first conference paper  would lead to me meeting Tracey Bretag for the first time, and that this fortuitous meeting would result in a change to my research and career trajectory. This meeting also led to my first publication in the IJEI, Tracey could nurture the community while providing an outlet for the talents of those she drew into the academic integrity interest sphere. Through the IJEI, she always encouraged individual insights and observations knowing that a range of perspectives was essential to developing a broader and better informed viewpoint on the breadth and extent of cheating behaviours, and how to pursue educative approaches to encourage personal integrity and learning.Many similar stories have been repeated by those fortunate enough to have known Tracey. She recognised in people the sparks of interest in academic integrity and encouraged us to connect, share learnings (appropriately acknowledged of course!) and pursue investigations into issues to encourage learning and quality teaching. Founding the IJEI that information and research about educational and academic integrity would be available to a wider audience regardless of location. The community of practice saw her bringing together like-minded individuals to work on editorial boards such as IJEI, scientific panels, conferences, books, research articles, training programs and presentations in Australia and around the world.Tracey’s passion and efforts highlighted that open discussions and research into the issues around student academic misconduct and contract cheating were essential to drive change in practices, policy and understanding. She also knew that through the open access approach of the As a role model she demonstrated how academic and educational integrity was worth exploring and researching, and modelled how our responses could contribute to student learning, societies and academic careers. The recognition of her guidance and work around the world is astonishing – but a testimony to her vision, vigour, passion, enthusiasm, drive, approachability, warmth, mentoring, encouragement and support. We are all richer for having known and being influenced by her, but saddened that she was lost to her family and the world too soon. Tracey created a momentum in the area influencing thought, process, policy and governance related to academic and educational integrity that we commit to continuing as an ongoing legacy to her leadership, collegiality and friendship.Ann Rogerson.Assoc. Professor Ann Rogerson SFHEA.Associate Dean (Education).Faculty of Business and Law, University of Wollongong , Australia.Chair UOW Academic Integrity Advisory Group.Friends and colleagues, Tracey and I shared a lot of very memorable moments in our journey together as researchers. I first met Tracey in 2006, and we worked on the research on the very contentious issue of self-plagiarism. I remember both of us going back and forth on how to define self-plagiarism. In one of the last pieces she wrote, Tracey mentioned us coming up with the definition of self-plagiarism. From there, through the years, whether it was how to use Turnitin® as an educative tool or how to have a more educative focus on academic integrity policy, or a more humane approach to breaches of academic integrity, we approached the issue with a sense of the holistic and nuanced nature of the problem.IJEI published, her hunt for reviewers (I was one) and her excitement at receiving interesting research. I worked with her as a project officer on the Academic Integrity Standards project (2010–2012) and Exemplary Academic Integrity project (2013–2014). We identified the five core elements of an exemplary academic integrity policy based on a review of all the policies in the Australian Higher Education sector and the literature. The paper led by Tracey was published in the IJEI in 2011. One of my last roles with Tracey was at the Office for Academic Integrity and I recall some of the innovative things we did to reach students on the International Day against Contract Cheating – instant scratchies, snap chat filters and petting Tracey’s dog, Peanut. The fun part was, we got to interact with students, and that was really Tracey’s gift. She knew her topic and was very passionate about it, but at the same time she had great compassion both for students and educators – we struggle as well with how to approach this issue. I remember Tracey used to say that ‘Perfection is the enemy of the very very best’. She wanted the IJEI to have continuity and longevity into the future. I witnessed her excitement when Springer took the IJEI on as publishers and finally she had the support she needed to publish her beloved journal.We had some very interesting conversations around the issues. I recall Tracey’s absolute commitment to getting the I am grateful for all the years we worked together and for the opportunity to be part of the academic and research integrity story. The last project that Tracey did – the Contract Cheating and Assessment Design project – I had gone away from higher education for a couple of years, but was fortunate enough to come back and be part of that project analysing the largest data set on the topic. Tracey’s lifetime of work on integrity has set Australia on its own track from the rest of the world. I know there are researchers all over the world that are working on this issue, but Tracey’s leadership on this has been phenomenal. Tracey was a dedicated advocate for integrity and a visionary who shall be sorely missed and forever remembered.Dr. Saadia Mahmud (formerly Saadia Carapiet).Online Facilitator.Australian Institute of Business, Australia.NB: Some of the text is the wording of my video tribute to Tracey for the memorial held at the University of South Australia on 26 October 2020.angles had been misspelled as angels. The version of the essay spun through the paraphrasing tool rendered this as Blessed Messengers. Tracey immediately suggested that this anecdote was the basis for an article in the IJEI. For goodness sake, I had never written a peer reviewed article and my PhD candidature was not even confirmed!A chatty phone call with Tracey led to me revealing an amusing and illuminating story about some students who had used an online paraphrasing tool. In the original essay, in a statement regarding CAT Scans, Five months later the article was published. It was those 5 months that revealed to me the warmth, depth and extraordinary power that was Tracey. She challenged, encouraged and sustained me through the process. Each word, every sentence, the concepts and contradictions were constructed through her expert guidance and supported by her wisdom and enthusiasm.Tracey gave me the courage to join the academic community of authors and has illuminated the path forward.Felicity Prentice.Lecturer.Health Sciences.La Trobe College Australia.IJEI under the Editorship of Dr. Tracey Bretag, both of which were memorable for how positive they were. I submitted my first article to IJEI as a corresponding author in 2008. An abiding memory for me from this submission was that I found the feedback and direction from Tracey, as the Editor, very helpful and supportive. After acceptance of the paper, I recall writing to Tracey to say “thank you” for her help and encouragement in getting the paper in shape for publication, with a statement to the effect that she was the most helpful Editor I had dealt with on a journal article submission. This was among the first half-dozen journal articles in my career; more than 30 papers later, I have not been similarly moved to provide spontaneous positive feedback to an Editor.I have had two experiences of submitting papers to IJEI. On receiving the reviews, I remember Tracey, as Editor, apologizing for delays that resulted from the fact that she was being treated for, or recovering from, breast cancer. I recall replying immediately saying that ill health was nothing to apologize for and that the resulting delays were completely understandable. Still, this reply from Tracey showed how important the operation of the journal was to her. Unlike my first submission, my second paper required substantial revision, and I remember Tracey again being clear in her guidance regarding how to address reviewers’ feedback and improve the paper.In 2010/11, I submitted my second paper to IJEI, I found that Tracey was a superb communicator and her enthusiasm for the journal was evident even over email. She clearly knew reviewers’ relevant expertise and expressed her appreciation for our help.As an occasional reviewer for Last year I had the privilege of working with Tracey on her final large project, development and delivery of Australia-wide academic integrity workshops and an academic integrity toolkit for the Australian higher education sector. Through this project, I got to experience Tracey’s enthusiasm, support, high professional standards, and goodwill in person. Tracey was the authentically kind, helpful, and supportive person in real-life as I had experienced as a journal contributor many years before. Tracey’s passing is a terrible loss to her family, friends, colleagues, students, and the community of academic integrity scholars.Guy Curtis.Senior Lecturer in Applied Psychology.The University of Western Australia, Australia.IJEI gave her. I will also always remember her compassion for the female students in engineering who report more often giving their work to others, than receiving work from others, while male students report the opposite. Tracey had a rare gift for the big picture and the details, and for distinguishing the offender from the offence.There are some disciplines and tasks which have for a long time been in the integrity “too hard” basket, non-text disciplines, and in particular mathematics with its proofs and calculations. Tracey had a wide vision for educational integrity, and wanted no discipline sidelined or ignored. She encouraged me to break the silence around mathematics, being only too aware of it because of the overview of the whole educational integrity landscape that editorship of Katherine Seaton. Associate Professor Katherine Seaton FAustMS, SFHEADepartment of Mathematics and Statistics.LaTrobe University, Australia.IJEI and put me in touch with someone who could co-author with me. She was always very encouraging of new researchers in this field, and given my professional roles for some years were in this area, it seemed a logical step to make for me to move into some form of academia through publishing outcomes of my studies. Having my first article published in IJEI was great, and a confidence booster as well!I cannot remember when I first met Tracey, but would think that it was around 2012 at the Plagiarism Conference in Newcastle, possibly the year before or after. That was my introduction into the circuit of plagiarism and integrity conferences around the world. I often met Tracey at these events and got to discuss differences between country approaches (myself then being based in the UK), over a coffee or a glass of wine at the networking events. At the time I had completed my master’s thesis on the topic of plagiarism, with a focus in outsourcing. Tracey suggested that I try to turn one of my papers into a journal article, something I’d not yet thought of. She offered to put it in IJEI, Dr. Helen Marsden (now Helen Titchener), so another connection to the journal! Tracey always took the time to talk to me about my research and how I was progressing. I remember being sat in the sun talking at length over a cold drink in Adelaide one hot and sticky afternoon, about the best ways to get information from students to open up and talk about their cheating behaviours. It is thanks to Tracey’s encouragement over 10 years ago that I find myself still writing and researching now.Following the publication, I ended up moving to Australia, meaning that I would bump into Tracey at national as well as international events. In 2015 I decided to take my interest in this area further, and commenced my PhD on the topic. One of my supervisors at the time was the co-founder of the Tracey was an inspiring advocate for integrity, may she rest in peace.Rebecca Awdry.Deakin University, Australia.I’m not sure if there was a disagreement the very first time I met Tracey, but if not, it was shortly thereafter. Finding and exploring our differences was one of the great joys of our friendship. We disagreed about policies, procedures, and language. We once stayed up hours after a conference’s conclusion, drinking wine and arguing passionately about plagiarism detection/text-matching software. I would give the world to be able to have that argument again.Despite being ideological allies and self-identified trans-pacific sisters, we argued—in the scholarly sense—constantly. (That’s how it is with sisters.) What made all of our disagreements useful, generative, and often delightful was that regardless of how passionately Tracey believed in her position, she believed just as passionately in testing her assumptions and convictions, which is one of the many reasons she was the perfect person to edit a journal on the subject of educational integrity.Many of our arguments started the same way: One of us would say something she thought was uncontroversial, and the other would shake their head like a dog shakes off water and say, “What?” and the game was on. The original speaker would state her case and be instantly rebutted. We’d line up our evidence and the debate would rage. After much explaining, nodding, head-shaking and brow-furrowing, the conversation would nearly always turn toward the same place: Talking about how we could test the ideas against each other and find out the truth. The arguments ended in research design plans and hugs.her journal—and I did think of it that way, as her journal —both rigorous and ground-breaking. As was the case in our arguments, no idea was sacrosanct or immune from challenge, but also, no idea was too unconventional to be explored. Just like with our arguments, even though she was a giant in the field of academic integrity, she was willing to consider the possibility that views she had long held might not always hold sway.Tracey possessed the ideal but nearly non-existent combination of passionately held beliefs and openness to conflicting positions that made Our last argument wasn’t much of an argument at all. We were at an international function dinner, sitting beside each other and chatting as if our life depended on it. We’d finished eating and Tracey reached over and rearranged the silverware on my plate. The look on my face was enough of a question for her to explain that she did it to indicate that I was finished. I said, “Oh, is that how they do it here?” and she said, “This (pointing to her arrangement of the cutlery) means you are finished.” I shared with her that I’m never sure when I’m travelling, because even in the US, we have two different sets of table etiquette rules; I assumed everywhere was different. Tracey told me that she’d always assumed that Americans just didn’t have that kind of table etiquette because (her words) we “aren’t as stuffy as the commonwealth countries.” Then we compared different customs and habits, and gasped at our own misconceptions and assumptions until we were exhausted and laughing about how, after all these years, there was still so much we had to learn.That was Tracey in a nutshell. She would move the silverware on your plate, and that would launch an exploration of cultural competency and customs. Her endless curiosity, openness and willingness to “go there” made her an ideal editor and an adored and irreplaceable friend.Teresa (Teddi) Fishman.Former Director, International Center for Academic Integrity.United States.It is with great sadness to have to use the past tense when I talk about Tracey Bretag. She had so much energy and was full of the joys of life. Her presence would illuminate any room, large or small. She was, and still remains, a great inspiration to many people, me included, both in a personal and professional capacity.It is almost impossible to read anything about academic integrity without coming across Tracey’s name, as a researcher, editor or author. She was, quite rightly, seen as a leading authority in this field, initiating some of the most innovative and influential research conducted anywhere in the world. Her research and publications, completed together with many prestigious colleagues, will continue to be of relevance for many years to come.I’m personally greatly indebted to Tracey for helping me to develop my skills as a researcher and writer and for encouraging and supporting the first research that I led, 10 years ago, into plagiarism policies. I set out the details of how I first came across Tracey in my article within the Festschrift we put together for the well-deserved ENAI Lifetime Achievement Award, which was presented to Tracey at the  conference in Dubai in April 2020 (European Network for Academic Integrity (ENAI) educational integrity rather than academic integrity. We should use this term more, because it encompasses all educational levels. Researchers in this field are increasingly becoming aware of the need for young people to understand about integrity before they go to university, as it applies both to their education and personal lives. Gaining such knowledge and skills throughout their education will prepare young people either for working life or transition to higher education. This is an area where more research and development are needed.Tracey preferred the term Through the efforts of Tracey, together with researchers she worked with in Australia and elsewhere, there is far more information available today about threats to integrity and ways to challenge and reduce those threats. Tracey’s research helped us all appreciate how commercial contract cheating services are undermining standards in education globally.The best tribute to Tracey would be evidence of success with the “arms race” against corruption and malpractice in education. The most serious issue facing us today is commercial contract cheating in all its forms, including “essay” mills, degree mills, fake universities, including associated extortion and bribery. This is a global problem therefore we must continue to work together as a community of researchers and activists to find a global solution. To achieve success in this difficult endeavour, we must involve a range of interested parties, especially students.It is now our responsibility, as an international community of researchers in this field, to build on what has already been achieved by Tracey and others. We must continue to make progress in rooting out cheating in education, in whatever form it occurs.Dr. Irene Glendinning.Coventry University, United Kingdom.International Journal for Educational Integrity in 2005. The journal punched beyond its weight from the very first issue, securing contributions from world leading academic integrity researchers of the day, including Don McCabe and Jude Carroll. The journal later became part of the Springer Nature portfolio.People often assume that, since Tracey and I both started working on academic integrity research in the early 2000s, we’d known each other for a long time. We actually existed in separate circles. While my focus was on educational research and improving pedagogical practice within the UK, Tracey was making an impact on the world stage. Along with Helen Marsden (now Helen Titchener), she launched the Handbook for Academic Integrity she was editing .awe’. I wondered if there was anything else to add to the body of knowledge in this area of research!While carrying out my doctoral studies, I was pointed to the comprehensive work on academic integrity carried out by Tracey and her team in Australia. My initial reaction was that of ‘Then in 2012, I attended the 5th International Plagiarism Conference, on 16–18 July 2012, in Newcastle upon Tyne as a session chair and panellist. At the opening ceremony I saw Tracey for the first time, and I was amazed at her passion for this research area as she presented a keynote. I then went on to find her during one of the breaks; introduced myself and talked to her about my research on academic integrity, focusing on student plagiarism and Institutional response. The aura around her was so friendly, welcoming and enthusiastic that I found it difficult to be around Tracey without being infected by her passion - indeed, there were no dull moments around her.International Journal for Educational Integrity (IJEI). Tracey was not one to compromise on quality but would rather provide the guidance to achieve a higher standard in the articles the IJEI published. This peer review process was rigorous with very detailed feedback and I was quite excited when my paper was published in the IJEI.This initial meeting lead to several others as her work contributed immensely to the shaping of my research. Following my meeting with Tracey in Newcastle upon Tyne and discussion of my research with her, I was invited to submit a paper to the Handbook of Academic Integrity .Course Director, MSc. Management of Information Systems and Technology (MIST).Faculty of Engineering, Environment & Computing.School of Computing, Electronics and Mathematics, Coventry University, United Kingdom.Tracey provided keynotes at the International Integrity & Plagiarism Conference which ran in the UK from 2004 to 2014, in both 2012 & 2014. She thrilled delegates with her passionate and research-led presentations and words of wisdom, winning her many great friends in the UK and beyond. I will never forget the iceberg image from her 2012 keynote, showing the unconscious cultural values of integrity which lie beneath the surface!International Journal for Educational Integrity.During this time, I worked with Tracey and Teddi Fishman, then Director of the International Center for Academic Integrity, to establish an alliance of our three integrity conferences. Namely the ICAI annual conference, the Asia Pacific Conference on Educational Integrity and the International Integrity & Plagiarism Conference, as part of a shared commitment to ensuring that conversations on integrity should resonate with, and empower a global community, a vision that clearly was also fundamental to the International Journal for Educational Integrity, then published via the UNiSA Open Journal System. The accepted submissions then became part of the final issue for 2012, a year Tracey describes as “very busy year for academic integrity” in her editorial, with “numerous conferences, symposiums and research projects taking place around the globe” . She’s also on Facebook if you want to send her a message via Messenger. Please make her welcome at her very first academic integrity conference. I know you’ll have lots to chat about!Enjoy the conference, and know that I’ll be thinking of you all.With warmest regards,Tracey.”The email that I and others received sums up everything you need to know about Tracey and the care and concern that she had for others:Professor Phil Newton.Director Learning and Teaching.Medical School.Swansea University, United Kingdom.Professor Michael Draper PFHEA RLAUKAT.Director Swansea Academy Inclusivity and Learner Success.Dean Regulations and Student Cases.Swansea University, United Kingdom.I met Tracey for the first time at the conference Plagiarism Across Europe and Beyond in 2013. At that conference 2 years later, the idea of the European Network for Academic Integrity (ENAI) emerged. Since 2017, the conferences have been annual and provide a platform for sharing research results and experience and good practice examples. Tracey was a keynote speaker at almost all of them. We always appreciated her encouraging speeches, valuable feedback to other contributions, and enthusiasm she spread during informal talks and meetings.Tracey helped us a lot with establishing ENAI. She gladly shared her experience and constructively criticized our ideas and our work. It was a pleasure and a huge benefit to have her on the Advisory board. Without her, ENAI wouldn’t achieve what it is.International Journal for Educational Integrity, which, thanks to Tracey, provided a venue for the ENAI team’s publications. It started with a special issue in 2013, continued with the publication of selected conference papers in the following years, and resulted in a strong partnership between ENAI and IJEI. The aim of this partnership is clear: To share results of the academic integrity research, which allows any higher education institution to adopt good practice from elsewhere and build a culture of academic integrity.It was also the champion”, “absolute legend of the academic integrity world”, “very effective critical friend”, “prominent figure”, “the most selfless, honest and passionate persons you get the honour to meet”, “fairy godmother”, “family friend”, “bundle of energy, bubbling over with great ideas and good stories”, “inspirational friend”, or “super-leader in the promotion of academic integrity.” There is no doubt that all these superlatives are true. And this is how we will always remember professor Tracey Bretag.This editorial is too short to enumerate all Tracey’s contributions to ENAI and its community. We all valued Tracey as a colleague, expert, and friend. That is why ENAI appreciated Tracey’s efforts by a Lifetime achievement award earlier this year. Together with the award, ENAI published a Festschrift for Tracey Bretag . In ten chapters, members of the European academic integrity community describe what Tracey meant for them. In their contributions, Tracey is called as “Tomáš Foltýnek, President of the ENAI Board.Czechia.The issue of academic integrity was the preoccupation and main field of research and expertise of Prof. Tracey Bretag. It was she, respectful and professional academician Prof. Tracey Bretag, who became our academic integrity icon. Academic integrity in higher education was and continues to be one of the most challenging aspects for the academic community. Prof. Tracey Bretag gives a lot to this field as a creator of knowledge and as author of many scientific papers in the field of academic integration in education, and in particular in higher education.Handbook of Academic Integrity  we consider appropriate to grant recognition to Dr. Tracey Bretag who was one of the great pillars in promoting a culture of academic integrity worldwide.We met her in 2014 as a speaker at our 2nd Conference for Academic Integrity. Back then she chaired the International Center for Academic Integrity (ICAI). Then we met her again at the ICAI Conference in Vancouver. Later we accepted her generous offer to spend 3 weeks at UDEM as a visiting professor, then she did the same at the Universidad Católica de Chile.For more than 10 years she focused her research on all aspects of academic integrity.She obtained funding from several organizations such as the “Australian Learning and Teaching Council” to carry out different research projects.She was former President of the Asia-Pacific Forum on Integrity for Education and President of the ICAI’s Board.Handbook of Academic Integrity   campaign that I have been running since 2012, namely AK.SA.RA. . The results of the discussion and comments provided were materialized in a book chapter in the y Bretag , which bIJEI, on May 19, 2015. I immediately received this honourable opportunity with great enthusiasm and gratitude. My contribution as an editorial board member is not yet worth the amount of trust entrusted to me. One of the assignments given was the publication of Call for papers - Academic integrity in Indonesia: Building on 20 years of progress.Our collaboration continued when I was personally invited to join the editorial board of Since 1999, Indonesian government has been aware of various cases of academic misconduct and proclaim the importance of misconduct prevention. In 2010, Indonesian government announced a Ministry of National Education Regulation (MNER) on plagiarism in higher education. Unfortunately, these regulations gave only limited effect on the prevention and eradication of misconduct. The rise of Indonesian’s scientific publication coincided with the increase of ethical violations or academic misconducts, such as contract cheating, manipulation of authorship and citations, multiple submission, or plagiarism.To perpetuate memories of Tracey and carry on her extraordinary endeavour in the field of academic integrity, I would like to propose an acronym that frames the six fundamental values of academic integrity into her name. I propose to use the word “TRACEY” as the acronym for Trust, Respect, fAirness, Courage, rEsponsibility, and honestY. Hopefully, from now on, every time we talk about academic integrity, we will remember her, because TRACEY is truly the fundamental values of academic integrity.Ide Bagus Siaputra.Universitas Surabaya.Indonesia.Tracey and I first made contact on 18 September 2006 when she rang me to talk about integrity matters. We had a lot to talk about. She was organising the Third International Conference on Educational Integrity, to be held in Adelaide. After our phone conversation, she invited me to give a keynote speech.In December 2007, meeting before the conference, we hit it off immediately. Tracey had an engaging personality; she was genuinely interested in ideas, other people — and educational integrity. Her enthusiasm was infectious. I guessed that she had the same sorts of engaged interactions with others.By inviting me, Tracey showed her open-mindedness. I was an outsider to the educational integrity area, as a scientist and then a social scientist whose main interests were elsewhere. Furthermore, contrary to the usual focus on student plagiarism, I had written on a range of abuses by academics and other professionals. That suited Tracey fine.In the years that followed, we kept in touch by phone and email. When contacted by journalists or others about plagiarism and related matters, I often referred them to Tracey as Australia’s leading authority. I think she referred a few people to me.We shared an interest in self-plagiarism. Using your own text without acknowledging its prior use is widespread in some fields. It’s one of those borderline practices, like hyping your achievements in your curriculum vitae. Despite being deceptive, it is not officially malpractice. Tracey, in collaboration with Saadia Carapiet (now Saadia Mahmud), had creatively used Turnitin to track self-plagiarism among cohorts of Australian academics. It was the most thorough treatment of this topic I knew about.International Journal for Educational Integrity is one of Tracey’s gifts. It provided a home for a couple of my unusual articles, and I learned a lot serving on the editorial board and reviewing submissions. It is not easy to appreciate the effort required to set up and maintain a journal. Tracey’s wide range of contacts helped make it a success.The I still remember a visit to her beautiful home, where we had stimulating discussions. I also remember her frustrations in her job, including her thwarted attempt at promotion, and her satisfaction at finally overcoming this hurdle. My advice was not to get too upset about her academic rank because research showed that promotion, while initially pleasing, did not make people happier in the long run. Little did I know that only a few years later, with Tracey’s aggressive cancer, academic rank would be the least of her concerns.Tracey played a crucial role in the educational integrity area that will be impossible for any individual to fill. We can, though, be inspired by her commitment to do what we can in our own ways.Brian Martin.Emeritus professor of social sciences.University of Wollongong, Australia.Most of my own professional life was spent at the University of South Australia.It was there that, at first sporadically, I met Tracey. I had gravitated to become Dean of Research Degrees in one of the Divisions and I knew of Tracey’s interest in ethics. We exchanged views and she became fascinated with some of the ethical dilemmas I had faced in managing research degrees on the ground. She could not believe some of the wickedness that passed as acceptable practice.I retired in 2001 as Dean and a couple of years later I was asked to apply for the position of Director of the Ethics Centre of South Australia (ECSA). I was appointed and Tracey and I spent more time together. She would sometimes drop into my home and have a coffee with my wife and myself. I came to know her personal life, which exhibited great sacrifice and commitment, while at the same time she was able to maintain her academic roles. She would also establish the International Journal for Educational Integrity and invited me to the Editorial Board. In so many ways the Journal exhibited the qualities of excellence that Tracey had displayed in her own professional life.She honoured me by giving several addresses on behalf of the Ethics Centre, always well prepared and often controversial. For example, her ideas on academic self-plagiarism raised eye-brows and caused consternation among some university leaders who wanted to see more production of academic output, without any restrictions. The idea that a staff member might not publish a conference paper, an article and a chapter in a book (or perhaps even a book) without reference to previous incarnations of the substance was something that Tracey deplored and some academic leaders promoted.Vale, Tracey. What a great woman and parent; what a great academic and ethicist; what a great professional friend.Robert Crotty.Emeritus Professor.University of South Australia, Australia.International Journal for Educational Integrity.Editorial Board Member,


This paper investigates the conformational stability of porcine pancreatic lipase (PPL) in three non-aqueous organic solvents, including dimethyl sulfoxide (DMSO), propylene glycol (PRG), and ethanol (EtOH) through molecular dynamic (MD) simulation. The root mean square deviations (RMSDs), radius of gyration (Rg), solution accessible surface area (SASA), radial distribution function (RDF), hydrogen bond (H-bond), Ramachandran plot analysis, secondary structure, and enzyme substrate affinity of the PPL in the various organic solvents were comparatively investigated. The results showed that the backbone and active pocket RMSD, and hydrophilic ASA of PPL in three solvents increase with the increase in the solvent LogP, while the Rg, hydrophobic ASA, and H-bond between the solvent and PPL decrease. Among the three organic solvents, DMSO acts as a better solvent, in which the PPL can be loose and extended, and retains its native backbone in DMSO compared to PRG and EtOH. Moreover, Ramachandran plot analysis indicated that the PPL structure quality in DMSO was higher than that in PRG and EtOH. Also, the molecular docking results showed that PPL in DMSO exhibited the highest enzyme-substrate affinity. Porcine pancreatic lipase (PPL) is widely used in many industrial fields because of its high enantioselectivity in biocatalysis . This enPenicillium expansum lipase and Novozyme 435  by using MD simulation, molecular mechanics Poisson–Boltzmann  surface area method, and molecular docking, and the result indicated that the higher binding affinity between the substrate and enzyme mutants was partially responsible for the improved catalytic enzyme activity. Moreover, combining the computational and experimental results, the authors elucidate a structure–function relationship of mutant CgKR1-F92C/F94W: the indole ring of Trp94 forms a stable π –π stacking interaction with the imidazole ring of His 93, resulting in the significant spatial movement of the substrate  formed between the activity pocket and the substrate, resulting in the decrease in Km and the activity  simulation is a useful pathway in investigating protein structure and obtaining insights into the behavior of the enzymes at the atomic and molecular level. To date, there are several successful examples using MD simulation to illustrate the structure–function relationship of enzymes . For exaubstrate . Xu et aactivity . Moreoveeviously , but fewIn this paper, the effect of the three organic solvents, DMSO, propylene glycol (PRG), and ethanol (EtOH), on the conformational change of PPL structure was studied by using MD simulation, in order to give a molecular insight of the PPL structure in the organic solvent.The structure of the PPL was obtained from Protein Data Bank (PDB code: 1ETH). The topology formats of DMSO, PRG, and EtOH were generated by the PRODRG for small molecules for the GROMOS96 43A1 force field .via the NVT ensemble , followed by the NPT ensemble . The PPLs in DMSO, PRG, and EtOH were marked as PPL-DMSO, PPL-PRG, and PPL-EtOH, respectively.The MD simulations were performed with the Gromacs 4.5.3 package  by using1 constructed by our team. The change of PPL in aqueous as control during molecular simulations. The data outputted from Gromacs were also arranged by using the Magical Platform.The active pocket of PPL is composed of Gly77, Phe78, Ile79, Asp80, Trp86, Tyr115, His152, Ser153, Leu154, Asp177, Pro181, His264, and Leu265. The active site includes Ser153, Asp177, and His264. The average density of PPL solvents at the last 1 ns was calculated. The average root mean square deviation (RMSD) of the PPL molecules at the last 1 ns was also calculated. The average radius of gyration (Rg) analysis of the PPL molecules backbone at the last 1 ns was also calculated. Secondary structure analysis, including the alpha helix, beta sheet, and the loop content of PPL, was also calculated by PyMol . The hyd2 constructed by our team. The molecular mechanics Poisson–Boltzmann surface area (MMPBSA) analysis was performed using the g_mmpbsa tool  after simulation and the original PPL structure from the PDB database were used as a receptor. Dicaprylin (DAG) was used as the model ligand. AutoDock Vina  was usedbsa tool .The partition coefficient of the solvent in between octanol and water can be defined as the oil–water partition coefficient  of the organic solvent models, which was calculated according to Wang’s method . The solThe RMSDs of the PPL molecules in different organic solvents during the simulation process were calculated. As shown in 2). It is worth noting that the hydrophilic surface area of the PPL in the organic solvent increased gradually as the polarity of the organic solvents decreased. Also, compared to PPL in the aqueous solution, the ratio of the hydrophobic area to the total surface area increases significantly, revealing the hydrophobic part of the protein molecules and thus becomes more accessible. Thus, combining the Rg analysis of the PPL in the presence of different organic solvents, these results demonstrate that the compression of the PPL molecules (the decreasing of the Rg) in the organic solvents was mainly due to the exposure of the hydrophobic part of the protein on their surface.According to the surface area analysis result of the PPL molecules, the hydrophobic surface area of the PPL in the three organic solvents was bigger than that in the aqueous solution  of solvent molecule to PPL. The first RDF peak at about 0.3 nm belongs to the H-bond between the amino acid residues of the PPL and the solvents .The total number of the H-bond between PPL and the solvent is shown in Based on the secondary structure analysis, the alpha helix structure of the PPL decreased with the increase in the polarity of the organic solvent. Moreover, the beta sheet structure content of the PPL in DMSO was similar with the PPL water and significantly higher than that in PRG and EtOH. What’s more, the loop content in the three organic solvents increased from 48.21 to 62.50%, with the increase in the solvent LogP.Ramachandran plot is an objective method of evaluating the quality of a protein structure, as shown in Moreover, for PPL in PRG and EtOH, the regions (Glu2-Ser12), (Lys137-Gly141), , (Lys240-Ile249), , and  exhibited a relatively high RMSF value than that of PPL in DMSO, indicating higher flexibility and lower rigidity of these regions. The region (Glu2-Ser12) of PPL-DMSO belongs to a beta sheet, while that of PPL-PRG and PPL-EtOH belongs to a loop structure . On the Molecular docking is a useful way of investigating enzyme substrate affinity. As shown in via the salt bridge, while none of the salt bridge can be observed between DAG and PPL-PRG/PPL-EtOH. These results illustrated that the PPL in the DMSO solvent exhibited the highest enzyme-substrate affinity compared to PRG and EtOH.Moreover, PLIP was used to investigate the PPL–substrate interaction. The greatest number of the substrate-binding sites can be observed in PPL-DMSO. The number of amino acid residues demonstrating hydrophobic interactions with the DAG substrate in PPL-DMSO was six, while that in PPL-PRG and PPL-EtOH were both five. Three residues in PPL-DMSO were detected to interact with DAG through hydrogen bonding, while in PPL-PRG and PPL-EtOH, there was only one. What’s more, two residues in PPL-DMSO can interact with DAG After simulating 20 ns under the same conditions, the free energy results of the three organic solvents were calculated by the MM/PBSA method . The totVia simulation, the RMSDs, Rg, SASA, RDF, H-bond, Ramachandran plot analysis, secondary structure, and the enzyme substrate affinity of the PPL in the various organic solvents were comparatively investigated. The results showed that the backbone, active pocket RMSD, and hydrophilic ASA of PPL in the three solvents increase with the increase in the solvent LogP, while the Rg, hydrophobic ASA, and H-bond between the solvent and PPL decrease. Among the three organic solvents, DMSO was the better solution compared to PRG and EtOH for the following reasons: (1) PPL can be loose and extended and retained its native backbone in DMSO, (2) higher PPL structure quality as indicated by Ramachandran plot analysis, and (3) PPL in the DMSO solvent exhibited the highest enzyme-substrate affinity according to molecular docking results and MMPBSA analysis.In this paper, the conformation, structure, and substrate affinity in the three organic solvents are discussed. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.Z-SC, Y-DW, and S-LC conceived and designed the experiments. Z-SC, Y-DW, and K-PH performed the experiments. Y-DW, Y-JL, W-HJ, M-JX, and Y-SL analyzed the data. Z-SC, Y-DW, JZ, and S-LC wrote the manuscript. All authors contributed to the article and approved the submitted version.S-LC was part-time employed by the company Foshan Wu-Yuan Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Starch, a plant-derived insoluble carbohydrate composed of glucose polymers, is the principal carbohydrate in our diet and a valuable raw material for industry. The properties of starch depend on the arrangement of glucose units within the constituent polymers. However, key aspects of starch structure and the underlying biosynthetic processes are not well understood, limiting progress towards targeted improvement of our starch crops. In particular, the major component of starch, amylopectin, has a complex three-dimensional, branched architecture. This architecture stems from the combined actions of a multitude of enzymes, each having broad specificities that are difficult to capture experimentally. In this review, we reflect on experimental approaches and limitations to decipher the enzymes’ specificities and explore possibilities for in silico simulations of these activities. We believe that the synergy between experimentation and simulation is needed for the correct interpretation of experimental data and holds the potential to greatly advance our understanding of the overall starch biosynthetic process. We furthermore propose that the formation of glucan secondary structures, concomitant with its synthesis, is a previously overlooked factor that directly affects amylopectin architecture through its impact on enzyme function.The online version of this article (10.1007/s11120-019-00704-y) contains supplementary material, which is available to authorized users. Starch, the primary energy storage of most plants, is the second most abundant glucose polymer on earth after cellulose and the main source of energy in human diet. Although starch exclusively consists of glucose units that are either α-1,4- or α-1,6-linked, its structure is surprisingly complex. It is composed of two distinct polymers—amylopectin and amylose, which together form massive semi-crystalline granules within the micrometer range Fig. . These pPlants produce starch in different tissues, but always in the plastidial compartment—the chloroplasts of leaves and amyloplasts of non-green tissues. In the chloroplast, a proportion of the carbon fixed during photosynthesis by the Calvin–Benson cycle is directly allocated to the formation of starch, while the remainder is exported to the cytosol and used primarily for sucrose production  of ca. 10 Manners . In cont Manners ; Fig. 1. Manners . Amylose Manners . Despite Manners .The tree-like architecture is one of the most remarkable and intriguing features of amylopectin Fig. . AlthougAn alternative to the cluster model is the building block backbone model by Bertoft, in which the long chains spanning several lamellae are oriented perpendicular to the shorter chains constituting the crystalline lamellae Bertoft . InsteadThere is evidence for further levels of organization on a larger scale Fig. . These lThe insoluble nature of starch allows the plant to store energy in an osmotically inert and compact form. The storage efficiency of starch in plants can be compared with that of glycogen, which is the storage carbohydrate of animals, fungi, and many bacteria . Because of the complex and largely unknown branched structure of glycogen, it is not feasible to establish a universal correlation between the molecular size and the weight of the molecule  with a typical length c = 4.2 nm , the introduction of branches by branching enzymes (BEs), and the selective removal of branches by debranching enzymes  plus the number of elongation steps (m) divided by the total number of chains (N):The resulting probability curve for the length of an individual chain , BEs theoretically have many possibilities regarding the cleavage site of the donor chain and the site onto which the chain is attached. Data from in vitro studies suggest relatively loose constraints, such as ca. DP 12 as the minimal length of a chain to become donor for BE action and DP 6 as the shortest transferred chain segment Fig. , 2014. OΩC the number of positions where the BE can cleave an α-1,4 bond, assuming that (1) only chain segments with exactly DP 6 (counted from the non-reducing end) can be cut, (2) these segments must not carry any branches themselves, and (3) the remaining chain that is left behind is of DP ≥ 6. With these constraints, a chain can be cleaved only at one position at most, and ΩC simply equals the number of glucan chains with DP ≥ 12 whose outermost branch is at least 7 glucose units distant from the non-reducing end.Assuming only very basic constraints for both enzymatic steps of a single chain transfer by a BE, we can estimate the total number of possibly resulting glucan structures as follows. Here we assume that all branches are distinguishable, which tends to be true for complex biological polysaccharides like amylopectin. We denote by ΩP the number of possible paste positions within the glucan, for which we—as a strong simplification—only exclude the glucoses whose C6 is already engaged in forming a branch point. Finally, we denote by Ω the total number of resulting glucans for a single chain transfer. It corresponds to any combination for the hydrolysis of an α-1,4 bond, followed by the creation of a new α-1,6 branch point, and can be estimated as:In addition, we denote by Ω indeed has the potential to become even larger as the number of chains increases. The plethora of theoretical branching possibilities, however, needs to be corrected by the fact that BEs have additional constraints, as considered below. In reality, the distribution of branch points in amylopectin appears to be tightly governed and not homogenous; rather regions with relatively high branch frequency alternate with relatively unbranched regions, underlying the semi-crystalline nature of starch  and share their domain structure over the whole protein  is not affected, we only reconsider the possibilities for the position of the new branch point. In addition to the glucoses engaged in α-1,6 bonds, we now also exclude one spacing glucose above and below each branch point, all non-reducing ends and the reducing one. When performed on the example glucan from Fig. ΩP from 24 to 10.Taking this putative minimal distance between branch points into account, we can re-estimate the total number of resulting glucans for a single chain transfer. As the number of outcomes from the hydrolysis of the α-1,4 linkage  and the acceptor chain simultaneously—this would help prevent BEs from transferring the cut chain segment to water . Indeed, crystal structures of rice and cyanobacterial BEs suggest that glucans are not only bound to the active site, but also to additional starch-binding sites at the protein surface, although the actual intermediate with at least one covalently bound chain has not been captured  and one limit-dextrinase . ISA1 and ISA2 have been implicated in starch biosynthesis under normal conditions and are the focus here. ISA3 and limit-dextrinase are primarily engaged in the process of starch breakdown  may be inaccessible to ISA, whereas branches where chains are unpaired might be accessible. Accordingly, the association of glucan chains  would prevent excessive branch point hydrolysis. If trimming by ISA helps linear chain segments to associate, its action would be self-limiting and would prevent further debranching. This concept requires that the glucan is generally competent to crystallize, i.e., SSs and BEs have endowed it with linear chain segments sufficiently long to form double helices (most chains DP ≥ 10) and more-or-less adequately spaced branches to allow double helices to associate into crystalline lamellae  may rapidly convert malto-oligosaccharides into glucose and maltose molecules that could be exported and further metabolized in the cytosol, as observed during nocturnal starch degradation Supplementary material 2 (EPS 571 kb)Below is the link to the electronic supplementary material.


Rheological analysis displayed shear thinning behaviour of nano starch samples as evaluated using Herschel-bulkely model and Power law. The nano starch samples exhibited comparatively low thermal gelatinisation temperatures as compared to native counter particles. Moreover, the nano-encapsulated starch samples offered more resistance to in-vitro digestion and showed control release of folic acid at target sites.Ball milling offers green approach for size reduction of starch granules to nano scale size. In this research work, the starch from two underutilised cereal varieties viz. foxtail starch (FS) and sorghum starch (SS) were milled to achieve the desired nanometric range with mean particle diameter of 467.98 and 271.12 nm for nano foxtail (FSN) and nano sorghum starch (SSN), which were highly stable as revealed by zeta potential analysis. Functional attributes like solubility, swelling index, apparent amylose content, emulsifying and pasting properties were evaluated. Scanning electron microscopy (SEM) clearly revealed damaged starch granules produed by the process of milling. X-ray diffraction (XRD) displayed decrease in crystallinity upon milling to 16.08% (SSN) and 18.56% (FSN) and disappearance of some peaks. Attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR) also revealed reduced crystallinity as confirmed by the decreased absorbance ratio of 1047/1022 cm It has extensive applications as raw material in different food, pharmaceutical and textile industries due to its properties like biocompatibility, storage stability, less toxicity and excellent drug delivery vehicle3. This constantly mounting usage of starch is leading a way to modify this essential polymer in order to meet specific industrial requirements4. One such modification is nano-reduction of this macromolecule, which is trending now-a-days due to its customised small size, improved functional attributes, greater mechanical strength, better bioactive retention, improved and controlled release behaviour of encapsulated functional ingredients leading to significant improvement in various applications of this polysaccharide5. However, the method of size reduction of this polymer into nanometric range plays a significant role due its impact on the polymer functionality, yield and time consumption. In order to minimise changes in the basic characteristics of starch polymer simple physical nano-reduction method i.e., ball milling provide benefits over other reduction methods which include simple operating process, less contamination chances, high size reducing frequency and yield6. Moreover, nanotechnology is being used in various fields especially in drug and food industries in order to enhance the stability of various unstable nutrients or bioactives. Folic acid  methyl]amino]benzoyl]-l-glutamic acid), a vital water soluble natural vitamin present in various foods like green vegetables and fortified foods7 promotes the red blood cells formation and is also regarded as an anti-anaemia factor. Furthermore, it averts neural tube defects, Alzheimer’s disease, cardiovascular diseases, pregnancy-related complications etc9. In spite of these advantageous properties there are certain limitations associated with this bioactive like under acidic environment its easy degradation, hence low bioavailability8. Therefore, to increase the bioavailability and bio accessibility of folic acid it needs to be protected under harsh conditions. Nano-encapsulation could be an interesting approach to safeguard this essential nutrient from degradation leading to its high surface-to-volume ratio for improved intracellular penetration, higher bioavailability as well as its gradual release at target sites11. The daily requirement of folic acid in a normal adult is about 200 μg daily which needs a high dose to meet such prerequisite. The current approach can significantly decrease the dose requirement of this essential vitamin to meet the daily body demand. With these advantages of nano biopolymers like simple organisation, nano-molecular complexes formation and desirable functionality they are receiving prodigious attention compared to the complex structured polysaccharides. Therefore, the current work targets to study changes in functional properties, structural attributes, thermal properties, and rheological characteristics of starch extracted from two minor cereal varieties i.e., sorghum and foxtail millet when reduced to nano range. The study also aims to compare performance of starch from both varieties as wall materials for folic acid and its in vitro release behaviour in human GI tract conditions.Starch is a carbohydrate biopolymer, non-toxic in nature, highly biodegradable and an inexpensive polysaccharide abundantly present in plants, cereals, crops and fruits has grabbed much attention in recent years due to its wide applications. Structurally, it is semi-crystalline composed of an amorphous (amylose) and non-amorphous (amylopectin) region whose proportions depend on factors like the botanical source, degree of plant maturity and cultivar differences of the same species12. Moreover, millet starches exhibit A-type starch pattern13, which is easily susceptible to breakage due to the presence of unbranched amylopectin portion when subjected to shear forces during milling14. Changes in the zeta potential values was revealed by the DLS. The zeta potential values were found to be negative in all the samples under investigation. The surface charge of FS and SS was found to be − 44.64 mV and − 21 mV which increased to − 24.27 mV and − 12.16 mV for FSN and SSN, indicating a shift towards greater stability and less possibility of aggregation15. Overall, SS revealed the highest stability both in the macro and nano cases. The increased exposure of hydroxyl groups (–OH) on starch nanoparticles when subjected to ball milling process might have contributed to the increase in zeta potential16.The mean particle size and zeta potential of native and nano starch granules is shown in Table 17. The ball milling treatment bought significant changes in the granular structure of starch breaking it into very small fragments and disarraying the smooth and flat surfaces into irregular and rough surfaces. As can be seen, the fragmentation of SSN is more pronounced when compared to FSN which was also revealed earlier by the average particle size analysis. The small granules have grooves and fissures on the surface without any clear defined granular structure in both the cases. The milling process affected the granules inducing the irregularities to the surfaces and converting the granules into nano-range, thereby affecting the physico-chemical and functional properties of the starch particles. Furthermore, it can be clearly seen that the starch granules exposed to process of ball-milling tend to clump together forming cluster which might be attributed to the agglomeration of damaged granules due to the rupturing of hydrogen bonds present in the granules which possibly link with each other forming the lumps18. The results are in good agreement with the data obtained in DLS. Similar results were observed in maize starch by Liu et al19 after ball milling treatment.The granular surface topographic images of native and ball milled starch samples are displayed in Fig. 20  of SS and FS was found to be 59.07 °C and 76.13 °C which showed a shift to some lower degrees of temperature viz 47.61 °C and 58 °C when nano-sized. Similar trend was followed by the peak gelatinisation temperature (TP) which showed a shift from 88.8 °C (SS) to 75.37 °C (SSN) and 103.51°C (FS) to 92.47°C (FSN). The conclusion temperature (Tc) for SS and FS was detected at 100.65 °C and 118.90 °C which also decreased to 89.34 °C and 112.34 °C for SSN and FSN, as revealed from the figure. Moreover, the endothermic enthalpy which is an indicative of crystallinity and presence of double helical structures reduced for nano-starch samples when compared to native starch samples. The gelatinisation enthalpy value reduced from 16.83 J/g (native) to 11.31 J/g (nano) for sorghum starch and 15.83 J/g (native) to 112.76 J/g (nano) for foxtail starch. The possible reason for decrease in enthalpy on nano sizing might be the fact that native starch samples are semi-crystalline in nature and have double helical structure due to which it needs higher temperature for gelatinisation. In contrast, decreased ordered structure in nano-starch and conversion to single helical structure caused by the mechanical agitation during ball milling process leads to the decrease in gelatinisation properties27. Similar findings of reduction in gelatinisation temperatures has also been revealed by Ding et al.28 for RS4 nanoparticles.The thermographs of native and nano-reduced starch samples are depicted in Fig. 29 reported 28.7% of amylose content in native sorghum starch. The amylose content difference depends on the factors like botanical origin, granule morphology of the starch and starch extraction methods30. After milling process, the apparent amylose content of the nano-starch particles increased significantly (p > 0.5) to 42.53% and 45.05% for FSN and SSN. The physical shear forces during milling easily destroys the branched amylopectin converting it into the smaller linear amylose units31. Moreover, the increase in amylose content was found higher for SSN which might also be ascribed to its greater destruction and lowest average mean particle size as already revealed by the SEM images and DLS data. The increase in amylose content upon nano-reduction can directly affect the functionality of the starch which is considered beneficial for the diabetic patients, since this kind of starch shows low glycaemic index32. The increase in apparent amylose content of resistant starch upon nano-reduction was also reported by Ding et al.28.The amylose content present in native and nano-reduced starch samples is presented in Table 33 observed much lower lightness value (76.20) for native foxtail starch. Moreover, a significant increase in L* value from 85.14 (FS) and 90.23 (SS) to 90.98 (FSN) and 94.26 (SSN) was detected with substantial reduction of b* value in nano starch samples. This increase in whiteness may be explained on the basis of increased surface area upon size reduction resulting in the higher refractive index values in milled starch particles. Similar increase in lightness values was observed in acid modified pearl starch by Balasubramanian et al.34.Color is considered an important parameter in food industry with high lightness values usually desirable in terms of consumer acceptability. The calorimetric values of native and nano-reduced starch granules are presented in Table 32. Hence, the reason for the increase in swelling power on size reduction might be the shear forces during milling process which disrupts the crystalline part converting it into an amorphous structure with less amylose-amylopectin, amylose-amylose and amylopectin-amylopectin associations so that water easily gets entrapped. Similarly, the results revealed highest solubility index for native starch samples at 90 °C with SS showing high solubility (9.08%) than FS (8.04%). This corresponds to the high amylose content in sorghum starch , since solubility of starch mainly depends upon amylose leaching35. The nano- reduced starch samples showed increased solubility at all temperatures compared to native starch samples. The reason attributed to this might be that upon ball-milling both intra and inter hydrogen molecular bonds weaken and more hydroxyl groups gets exposed leading to more interaction with water molecules resulting in improved solubility. However, the native samples have strong internal structure resisting the leaching of amylose and are therefore, comparatively less soluble. Similar increase in swelling and solubility upon nano-reduction have also been earlier reported for waxy rice starch upon nano-reduction using ball milling (BM) and octenyl succinic anhydride (OSA) modification36.The swelling power and solubility index of native and nano-reduced starch samples are presented in Table 37 where he found bitter yam starch with lowest particle size showed higher emulsion forming property compared to other large sized starch particles. Moreover, among all the samples analysed the emulsifying property of SSN was found to be significantly higher which is consistent with the results obtained by DLS (explained earlier) showing its lowest mean particle size diameters. Thus, ball milling can significantly improve emulsion forming ability of starches.To understand the effect of particle size on the emulsion forming abilities, the native and nano-reduced starch samples were analysed and the values obtained are depicted in Table 38. This is attributed to the prominent structural disintegration of the nano-starch samples resulting in the formation of linear chain molecules which pertains to reduced pasting parameters and gelation ability. Hoover et al.39 reports that the structural changes like crystallinity transformation, molecular order loss, amylopectin and amylose dispersion can significantly contribute to the reduction of viscosities and increase in the pasting temperature. Similar decrease in the pasting parameters were observed by Tan et al.40 for G80 corn starch samples after planetary ball milling at different times.The pasting properties of native starch and changes upon nano-reduction was evaluated using RVA and values are presented in the Table 41 as shown in Fig. The frequency sweeps were measured to determine the changes in storage modulus (G´) and loss modulus (G´´) over the frequency ranging from 0.1–100 Hz. The G´ (elastic modulus) was dominant over the G´´ (viscous modulus) in both native and nano-reduced starches throughout the frequency range which is indicative of its viscoelastic behaviour0) (Table 2) value was found higher in Hershel bulkily model, thus can be considered best fitted for the evaluation of rheological parameters of millet starch gels. Overall the nano-reduced samples showed a proportional shear rate whereas native ones presented non-linear curves characteristic of pseudo-plastic flow behaviour fluid42. The yield stress values were found highest for native ones with maximum value for SS than FS. Similarly, FSN revealed higher yield stress than SSN. This indicates that the compact structure of native starches provides higher resistance to structure breaking at same shear rate. Nano-reduction resulted in a weak gel structure with higher ability to flow. The consistency index (k) indicative of strength of molecular association of the gels decreased in nano starch in the order of SS > FS > FSN > SSN indicating that native samples have more structural strength. Flow behaviour index (n) calculated from both the models were found to be less than one for all starch samples confirming shear thinning (Non-Newtonian) behaviour. Bhandari et al.43 attributed shear thinning behaviour to higher amount of breakage of the intra and inter molecular associative bonding system in starch network micelles due to shearing at high rates. The reduction in interchain association due to milling in nano-reduced samples might have resulted in lower gel network junction formation and less resistance to flow. Similar rheological behaviour was observed by Shah et al.44 in acetyl modified starch.Plots of shear stress (s) versus shear rate data for native and nano-reduced starch pastes are shown in Fig. 0) Table . Co-effi45 reported 50.20% folic acid encapsulation using horse chesnut starch matrix. The nano starch samples showed higher encapsulation efficiency than native starch samples. The efficiency for folic acid encapsulation increased to 78.76 and 73.43% for SSN and FSN. The encapsulating efficiencies are consistent with the particle size of the starch granules. Lower the size, better is the efficiency to form a film around the bioactive and retain the same for the further release at different target sites46.The encapsulating efficiency of native and nano reduced starch samples for the retention of folic acid is shown in the Fig. 47. Under the simulated gastric conditions, the native starch samples showed significantly higher release than nano encapsulated systems. The SS sample showed a release of 34 µg/mL during the first 30 min while as FS released 45 µg/mL of folic acid. However, the nano-starch samples retained the bioactive and released only 15 µg/mL (SSN) and 17 µg/mL (FSN). During the next 60 min it showed a non-significant increase with FSN retaining the higher level of bio-active. This can be attributed to the fact that nano-reduced starch samples show high swelling index (as discussed earlier) thereby increasing the path length to diffuse through the nano-starch core material, thus protecting and retaining the bioactive under harsh conditions48. Similar gradual release pattern of curcumin was also observed from sago starch nanoparticles by Chin49. Another study also showed the similar results where beta glucan nanoparticles resisted gastric phase digestion more than native beta glucan for the target release of alpha tocopherol50. Under simulated gastric conditions the free folic acid was fully released with 52 µg/mL and 59 µg/mL released at 30 and 60 min. After this when the encapsulated systems are subjected to simulated intestinal conditions, with the increase in the time viz., 30, 60 and 120 min the bioactive carrier system degrades gradually and release of folic acid increased significantly. The nano starch carrier systems showed significantly high release than native carrier systems, with SSN displaying the highest release of 84 µg/mL after 120 min. The underlying reason might be that at higher pH and presence of bile salts significantly affected the nano-encapsulating systems due to their high surface area leading to rapid release of the folic acid. The results clearly reveal that nano-encapsulating systems are favourable for the delivery and target release of bioactives as compared to micro encapsulating system The similar increase in resveratrol release from zein/pectin core–shell nanoparticles under simulated gastric conditions was observed by Haung et al51.The nutraceutical activity of any bioactive is valid if it is released slowly and absorbed in the gastro-intestinal tract such that it gets absorbed in the blood stream and exhibit beneficial health benefits to the body. The in vitro release behaviour of folic acid encapsulated in native and nano-starch structures at different simulated conditions viz simulated mouth, gastric and intestinal conditions are depicted in Fig. Setaria italica L.) and Sorghum (Sorghum bicolor L.) were procured from local market of Srinagar, J&K, India. The samples were cleaned and foreign materials were removed manually and then stored at room temperature in air tight containers. The chemicals and reagents used for the study were of analytical grade and bought from Himedia Laboratories Pvt Ltd and Sigma Aldrich Pvt Ltd.Foxtail millet  was then prepared and pH 9 was maintained. The slurry was then kept for 1 h and mixed at regular intervals. The mixture was then filtered through clean muslin cloth, followed by centrifugation at 3000×g for 15 min. The upper layer containing impurities was scrapped off while as lower starch was collected and washed three times with distilled water and then kept in hot air oven at 40 °C for drying.Starch was extracted according to the method described by Shah et al.16 with slight modifications. The starch was nano-reduced by ball milling method . The starch (10 g) was kept in ceramic pot and ten 8 mm Zirconium balls were added and left for rotation at a speed of 600 rpm for 1 h.Starch nano-reduction was carried out by the method of Jhan et al.45. Briefly, 0.01% w/v of sample was dispersed in mili-q water and sonicated in a bath sonicator for half an hour, particle measurement was then taken using a Zeta-sizer . For measuring the zeta potential, 0.01% of the sample was dispersed overnight in 0.1 Mm KCI at pH 6 in order to equilibrate the sample solution.The particle size and zeta potential of starch samples was determined by following the method of Ahmad et al.The basic morphological structure of starch samples was examined under vacuum conditions using Zeiss EVO 50 scanning electron microscope (SEM). The samples were analysed at 20 kV acceleration potential.53.The XRD patterns of the starch samples were recorded using a X’Pert PRO PANalytical, Netherlands, operated at 40 kV voltage and 35 mA current and X-ray line of λ = 1.5418 Å. The measurement range of diffractograms was recorded from 10 °C to 45 °C for 2θ at a step size of 0.02/min. The relative percent crystallinity (RC) of samples were evaluated using the equation of Rabek−1 at a resolution of 4 cm−1.The infrared spectra of starch samples were obtained using ATR FTIR spectrophotometer  within the range of 40–4000 cm54. Starch sample  was weighed in a hermetic platinum pan and diluted with distilled water (8 μL) with the help of micropipette. Prior to the measurement, the pan was allowed to stand for 1 h at room temperature. The pans were then heated at the temperature range of 20–250 °C at a rate of 10 °C/min. An empty platinum pan was used as a reference.The gelatinization properties of starch samples was evaluated using a differential scanning calorimeter  by the method of Gani55. 20 mg of starch sample was mixed thoroughly with 10 mL of KOH (0.5 M). The dispersion was then transferred to a volumetric flask (100 mL) and volume make up was done using distilled water. 10 mL of test starch solution was pipetted into another volumetric flask (50 mL) and 5 mL of hydrochloric acid (0.1 M) was added followed by addition of 0.5 mL of iodine reagent. The volume was finally diluted to 50 mL and the absorbance was measured at 625 nm . The content of amylose present in the starch samples were determined using a standard curve which was developed using standard amylopectin and amylose blends from potato starch.Apparent amylose content of the samples was evaluated by following the method of AshwarThe color parameters of the starch samples was measured using Color Flex Spectrocolorimeter . Results were obtained in terms of L* (lightness), ranging from 0 (black) to 100 (white); a* (redness), ranging from + 60 (red) to − 60 (green) and b* (yellowness), ranging from + 60 (yellow) to − 60 (blue) values.32 with slight modifications. Specifically, starch 0.25 g (W0) was dispersed in 10 mL of double distilled water and heated for 30 min at 60, 70, 80 and 90 °C . After this, the mixture was cooled at room temperature and centrifuged at 1500×g for half an hour. The supernatant was carefully removed and left over swollen starch sediment was weighed (W1) . The collected supernatant was dried at 105 °C in an oven until a constant weight (W2) was attained. Swelling power and solubility index of the starch samples were calculated using the equations given below.The swelling and solubility index for the starch samples was evaluated following the method of Shah et al.41. Starch suspension (1%) was mixed with 5 mL refined oil and homogenised for 15 min followed by centrifugation  at 1100g for 5 min The emulsion capacity and stability was calculated by the following equationEmulsifying activity and stability was determined by the method described by Khan56.A rapid viscos analyzer (RVA)  was used to determine change in viscosity of starch samples during heating and cooling cycles as determined by Ashwar et al.44. Briefly, 6 g of starch was dispersed in 100 mL distilled water and was heated at 90 °C for about 20 min with constant stirring. The gel formed was then left to cool for about 1 h. The flow properties like yield stress, flow behaviour index, and consistency coefficient was evaluated from the flow curve of shear rate (r) versus shear stress (t) by fitting the data into two rheological models i.e., Herschel-Bulkley model and Power law whose equations are given below.0 (Pa) is the yield stress, K is the consistency index (Pa sn), and n is the flow behaviour index, σ is the shear rate.The rheological properties of starch suspension was determined using rotational rheometer  by the method of Shah et al.To evaluate the visco-elastic range, amplitude sweep test was carried out for each starch gel with constant frequency of 1 Hz and shear stress ranging from 0.01 to 100 pa. The appropriate shear stress was selected i.e., 1 Pa, after linear viscoelastic range analysis. Frequency sweep test was then carried out at the frequency range of 0.1 to 100 Hz.12 with slight modifications. Solution containing folic acid (10 mg) and acetone (50 mL) was prepared followed by the addition of starch samples (1 g) and kept for stirring at 600 rpm for 20 min. During this period, distilled water (100 mL) was added drop wise into the solution. The dispersion formed was kept on magnetic stirrer at room temperature until acetone gets fully evaporated. The resulting suspension was centrifuged at 4000 rpm for 40 min for the separation of nanoparticles with encapsulated folic acid. The obtained samples were washed three times with ethanol for the removal of excess folic acid. The resulting folic acid loaded starch samples were kept for drying in an oven at 30 °C for 2 days.The encapsulation of folic acid in starch samples was carried out by following the method of Guevaraa et al.47 using folic acid standard curve obtained with known concentrations having regression equationEncapsulation efficiency calculation was carried out by the method of Noor et al.g for 10 min. The supernatant was collected and absorbance was measured using UV-spectrophotometer at 272 nm. Pellet was then recovered carefully and subjected to simulated gastric conditions by suspending the pellet in simulated gastric juice (10 mL). The conditions were prepared by dissolving 3 g /L pepsin (3 g/L) in sterile NacL solution (9 g/L) with adjusted pH to 3.0. After the intervals of 30 and 60 min, the solution was centrifuged at 10,000g for 5 min and absorbance was taken at 272 nm to measure the release of folic acid from macro and nano carrier systems. The pellet was again recovered and resuspended in simulated intestinal juice (10 mL), the conditions were prepared by dissolving bile salts (3 g/L) and pancreatin (10 g/L) in PBS. The samples were centrifuged  at 30, 60 and 120 min interval and absorbance was again taken at 272 nm. Percentage folic acid released was calculated by the formulae:The folic acid release behaviour was studied under gastrointestinal conditions (GI) for macro amd nano starch samples as carrier systems. Folic acid loaded starch samples (10 mg) were suspended in simulated mouth conditions (10 mL). The conditions were prepared by dissolving a-amylase (0.2%) in phosphate buffered saline (PBS), pH 7.2. The sample and amylase solution was then vortexed for five minutes followed by centrifugation at 5000The experiments were carried out in triplicate and results were expressed in terms of mean ± standard deviation (n = 3). The data was analysed using commercial statistical package SPSS (IBM statistics 22) software and the significant difference was determined by analysis of variance (ANNOVA) at 95% confidence level.On nano-reduction, the starch granules showed enhanced functional attributes like high swelling and solubility index, better emulsion forming properties and low pasting parameters. The decreased retrogradation tendency and better thermal stability reflects its use in various food and non-food applications. Also, this physical technique effectively disrupted the supra molecular structure of starch granules creating nano structures with desired in-vitro release of folic acid in various gastric intestinal conditions which offer its potential application as nano-vehicle for target delivery and release of bioactives at the desired sites for the development of future functional foods and use in pharmaceutical industry. Thus, ball milling proved to be the powerful means for nano-sizing the starch granules.