Impact Objectives• Analysis of proteins in plant remains to simulate interactions
between plant and phytopathogens• Contribute to our understanding of the foods and cuisines of ancientcivilizations• Unravel the foods and cuisines of Japonesian through understanding
more about ancient Proteomics

Insights into
ancient civilizations
Associate Professor Takumi Nishiuchi leads a project attempting to reconstruct the eating habits ofcivilizations by analysing ancient proteins found at archaeological sites
You are a plantbiologist and plant
pathologist. Can youtalk a little about thisfield of research?

Part of your research involves extracting
and purifying proteins from plant remains
such as carbonised rice from the different
archaeological sites. What are the main goalsof this research?

I use transcriptomics
and proteomics to study plant and pathogen
interactions at the molecular level. I am
fortunate to be based at the Advanced
Science Research Center which forms
part of Kanazawa University in Japan; wehave all the equipment needed for suchomics studies. My team collaborates withresearchers to support their omics studies,but we also work with archaeologists.

Ancient food cultures are spread over manyplaces. In particular, cereals were a staple
food and an important step in reading thehabits of ancient people. Rice is a staple
food in east Asia and the archaeologicalsites along the Chang Jiang river are thought
to be the origin of rice cultivation (ricecultivation in Japan and Korea is thought tohave been assimilated from China). We are
therefore studying carbonised rice and foodcrusts from different archaeological sites ineast Asia. We have already identified somecandidates for rice marker protein in plantremains and food crusts, and from there
have tried to identify marker proteins fromother excavated cereals such as commonmillet. These analyses are useful for theelucidation of materials in the food crusts
and will contribute to our understandingof the foods and cuisines of ancientcivilizations.

We began using proteomics to identify theamino acid sequence of collagen proteinsof excavated animal bones to identify
the biological species. Then, we tried toperform proteomic studies using somedifferent types of excavations. Amongthem, we identified many proteins derived
from not only plant themselves but alsomicroorganisms such as phytopathogens in
the plant remains. From there, we became
interested in the analysis of proteins in theplant remains to simulate the interaction
between plant and phytopathogens.

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What methods or tools have you used inyour analysis?

In this study, we usually use shotgun
proteomics, which refers to bottom-up
proteomics techniques where we combine
high performance liquid chromatography
with mass spectrometry. The protein
preparation step is very important for thisparticular study. After that, ancient proteinsare digested by trypsin and digested
peptides are purified using stage chips.
Then, purified peptides are separated bynanoLC, ionised by electronspay ionisationand then resulting fragment ions areanalysed by Orbitrap QE plus.
How are you investigating the Sungitani
Chanobatake ruins?
Many students are interested in the lifestylesof ancient civilizations and this includes
the foods they ate. With that in mind, I will
introduce our research about proteomic
studies of plant remains and food crust into
my classes. In addition, I sometimes guidethe student experiments of identification ofcollagen proteins in the excavated animalbones. The oldest rice balls were excavatedfrom the Sungitani Chanobatake ruins near
our university. These collaborations are very
important for us and our university and theresults of our research are of practical use to
the local community and wider society. lProteins of foods from the past
A team based at Kanazawa University are attempting to shed some light on the lifestyles of ancientJapanese civilizations by understanding the food they ate. The findings will highlight the importance ofproteomics and may help to restore ancient foods of the JaponesianStudying ancient civilizations is an effectivemeans of understanding how people, societies
and cultures have changed over thousandsof years, thereby providing important context
to present-day civilizations. Then there is
the idea of determining similarities between
humanity today and humanity back thenwhich, if nothing else, can generate a raft ofideas concerning what is common to all homosapiens.
There are many ways of studying ancientcivilizations, from reviewing the literatureand other texts that remain from civilizations
such as the Ancient Greeks, to excavatingpatches of land to uncover pottery, building
materials, bones and plant remains. Indeed,
these remains (coupled with modern analyticaltechnologies), enable scientists to gain insight
into the foodstuffs that ancient civilizations
grew and ate, and shed further light onto whatlife was really like for people all those years
ago.

Japan. He leads a team which is analysing
the ancient proteins in animal bones, plantremains, dental calculus and food crusts
that have been found at archaeological sites.
‘By identifying proteins in the excavations,
we want to uncover important clues thatenable us to simulate ancient foods, such asmeat, fish and cereals,’ explains Nishiuchi.
‘Based on the results we attain, coupled witharcheological data, we will be able to envisage
the lifestyles of ancient civilizations.’
His team has been working at ruins in China
and South Korea, as well as two Japanese ruins
located at Fukuoka prefecture. It is believedthat rice cultivation and related food culturesthat surfaced in Japan thousands of years
ago were introduced from China via SouthKorea. Thus, by focusing their studies at theselocations, the team are intent on analysing
samples that can provide an understanding of
the propagation of rice food culture. Gaining
such an understanding will provide fascinating
context to a food culture that continues toexist across Asia to this day.
PROVING THE EFFICACY
The methods of protein identification in
animal bones and dental calculus have alreadybeen long established, but many people(including scientific researchers), believe thatplant remains do not have enough stable
proteins to perform effective proteomicstudies. However, this is not a belief that
Nishiuchi subscribes to and some of his
team’s research has already attested to this.
‘Plant remains, such as seeds, definitely dohave useful proteins which we can use toOne method that has gained traction in recentyears is proteomics, which is the large-scale
study of proteins. By identifying proteinsin materials that have been found during
excavations, researchers are hoping to uncover
more information on ancient civilizations.
FASCINATING CONTEXTAssociate Professor Takumi Nishiuchi is a
researcher based within the Advanced Science
Research Center at Kanazawa University in

Schematic flow of ancient proteomics
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identify plant species,’ says Nishiuchi. ‘The
innovation of the Orbitrap mass spectrometry
has made significant contributions to ourancient proteomic studies and by using theappropriate protein preparation from various
types of excavations enables us to collectmany important clues into what life was reallylike back then.’
One of the team’s greatest achievements todate has been to perform protein analysis inplant remains and food crusts found at various
sites. This has not been done many times
before, which not only provides fascinating
insight, but helps to demonstrate the efficacyof the tools and methods they are using. Byhighlighting the usefulness of their methods,

identify plant species in plant remains andfood crusts.’ Highlighting these markers were
important steps for the future of the team’s
research, as it gave them a reliable starting
point from which other studies at other sites
could begin from.

Elucidating the ancient biome could havesignificant positive benefits to protect current
ecosystems. lSECURING FUTURE FOOD
As well as the obvious importance tounderstanding which foods and plantswere cultivated in ancient civilizations, it
is possible that the team’s findings could
be translated into important applications
within industry and healthcare. ‘By identifying
the microorganisms around excavatedplant seeds, the team are able to gain anunderstanding of their condition - whether

FUNDING
This study was supported by the Japan
Society for the Promotion of Science(JSPS), KAKENHI Grant number 19H05345,
20H05820 and partly by Grant number
18H04176

By identifying proteins in the excavations, we wantto uncover important clues that enable us to simulateancient foods, such as meat, fish and cereals
the team opens up the possibility of similar
studies at various sites in countries around theworld, which can provide further insight aboutother civilizations.
PROTEINS OF CARBONISED RICE
Among food materials, cereals are often astaple part of any diet and so it was felt thatby uncovering more about them, the teamwould be able to better understand the foodof ancient people. They therefore set about
trying to identify proteins of carbonised rice
at some of the archeological sites they werelocated at. ‘We tested some methods ofprotein preparation and then improved one
selected method enough to identify more than
100 proteins by shotgun proteomic studies,’
outlines Nishiuchi. ‘From there, we foundthat proteins are commonly observed in the
excavated carbonised rice from different sites.
In particular, some proteins are commonlydetected with their many trypsin-digested
peptides by nano LC-MS/MS analysis.’‘In addition, these particular proteins werethermostable (meaning they are generally
unaffected by heat and are therefore more
resilient than other proteins),’ confirmsNishiuchi. ‘So, they were candidates asmarker proteins which the team could use to
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it is diseased or healthy,’ notes Nishiuchi.
‘It is possible that the team will find usefulmicroorganisms that can help plants retain ahealthy condition by activating plant immunity
or suppressing phytopathogen growth.’ If
this can be achieved, the findings could leadto innovations such as the development of
microbial pesticides that can help to prevent
crop disease. It would present a situation
where the food that ancient civilizations grew
would secure the future of food for moderncivilizations.
The next steps for the study will build on thework that has already been done by the team.
‘The proteomic study does not contain theamplification step, such as polymerase chain
reaction of DNA,’ explains Nishiuchi. ‘We are
therefore hopeful that by using these methods
in the future, we can use ancient proteomics
to provide accurate information about specificbiological species, including the plant itself
and the microorganisms that existed in theexcavated samples.’There is also the exciting possibility that by
employing the team’s methods across manydifferent excavations in archaeological ruins,
we will be able to understand the specificecological systems that existed back then.

Project Insights

COLLABORATORSDr Shin’ichiro Fujio (National Institutes
for the Humanities), Dr Takashi Gakuhari(Institute for Frontier Science Initiative,
Kanazawa University), Dr Shinya Shoda(Nara National Research Institute forCultural Properties)
ACKNOWLEDGMENT
Fukuoka City Archaeological Center kindly
supplied materials and photographs
TEAM MEMBERS
Dr Takumi Nishiuchi, Kana Sohara andMaki Togashi (Advanced Science ResearchCenter, Kanazawa University) and DrShinya Shoda (Nara National ResearchInstitute for Cultural Properties)
CONTACT DETAILS
Associate Professor Takumi Nishiuchi
T: +81 76 265 2772
E: tnish9@staff.kanazawa-u.ac.jp
W: https://nishiuchitakumi.com/BIO
Associate Professor Takumi Nishiuchi isbased at the Advanced Science ResearchCenter, Kanazawa University. He previously
worked as a Domestic Research Fellow at
the Institute of Biotechnology, AIST, as a
Research Associate at RIKEN PSC and as
an Assistant Professor at the Institute ofGene Research, Kanazawa University.