Team:ULaval/Poster

Poster: ULaval

aSAP: a solution against maple polymers

Presented by Team ULaval 2020

Alexander E.¹, Cisneros Caballero A. F.¹, Côté M.¹, Gravel C.¹, Malaika Mutombo J. M.¹, Ouellet B.¹ and Ruel L.<sup>1

1</sup>iGEM Student Team Member, Université Laval, Québec City, Canada

Abstract

Maple syrup has a great cultural and economic importance for Canada. However, several issues can lower the quality of the final product. We engaged in conversations with experts to identify potential solutions to these problems, ultimately targeting ropy maple syrup. The ropy syrup has a very high viscosity, is not marketable, and damages maple syrup production equipment, which results in important economic losses, particularly for smaller producers. It is caused by the presence of dextrans produced by specific types of bacteria in the sap. aSAP, by team iGEM ULaval 2020-2021, will aim to develop an enzymatic treatment to degrade dextran and turn ropy maple syrup into a marketable product. Since maple syrup is stored at room temperatures, we are planning to use a dextranase from a psychrophile (cold-adapted) bacteria. In 2020, we have validated our candidate enzyme in silico, planned experiments to characterize and optimize it, and designed our implementation proposal.

Sticky Problem

Nearing the end of winter, to get out of its dormant state, a sap rich in saccharose, coming from the degradation of starch stocked in the root, flows through the xylem of the maple tree. Maple sap is then transformed through a 3 steps process into maple syrup¹.

1. Maple trees are connected to a tank with a tubing network to collect sap.
2. Sap is concentrated via reversed osmosis, which brings the sugar concentration from 2 to 3°B to around 20°B
3. The intermediary product is evaporated, leading to the final concentration of 66°B. It is equivalent to a boiling point of 104 ° C.

At this temperature, the Maillard reactions are greatly favoured and give the characteristic odour, flavour, and colour of maple syrup. From now on, maple syrup is evaluated in terms of its quality and is stored or sold.

In 2019, roughly 50M litres of maple syrup were produced in Canada, which represents a gross value of about 400M $US². This is equivalent to nearly 70% of the world production. Also, 83% of all exported maple syrup comes from Canada³.

According to the Federation in Quebec, the maple producers of Quebec (PPAQ), maple syrup is described as 100% authentic, natural, GMO-free, and preservative-free⁴. To ensure quality, it is sorted in terms of its sugar content, authenticity, clarity, colour, and taste⁵. Any differences concerning those quality criteria are subscribed to monetary penalties towards the producer. For our project, we focused on the class of flavour defects of maple syrup, which includes a variety of problems of different origins such as microbial, chemical, physical, etc⁶. Among the six subclasses of flavour defects, we considered the fifth and sixth; bud flavour syrup and ropy syrup. Bud flavour syrup occurs with the progression of spring and ropy syrup is caused in most cases by microbial fermentation.

References

1. Producteurs et productrices acéricoles du Québec, (2020). Production et technologie. Retrieved on June 14th, 2020 from http://ppaq.ca/lorganisation/production/production-et-technologie/
2. Statistique Canada, (2020). Tableau 32-10-0354-01 Production et valeur des produits de l'érable (x 1 000). Retrieved on November 8th, 2020 from https://www150.statcan.gc.ca/t1/tbl1/fr/tv.action?pid=3210035401
3. International Trade Centre, (2019). Trade Map. Retrieved on November 8th, 2020 from https://www.trademap.org/
4. Producteurs et productrices acéricoles du Québec, (2020). Le sirop d’érable du Québec. Retrieved on November 8th, 2020 from https://ppaq.ca/fr/sirop-erable/seve-au-sirop-erable/
5. Producteurs et productrices acéricoles du Québec, (2020). Québec Maple Syrup is Graded for Quality. Retrieved on November 8th, 2020 from https://ppaq.ca/en/maple-production/quebec-maple-syrup-graded-quality/
6. Producteurs et productrices acéricoles du Québec, (2020). Classement. Retrieved on May 14th, 2020 from http://ppaq.ca/producteurs/informations-pratiques/classement/
7. Lagacé, L., Camara, M., Leclerc, S., Charron, C., & Sadiki, M., (2018). Chemical and microbial characterization of ropy maple sap and syrup, Maple Syrup Digest, 9-19.

Slick Solution

Since bud flavour syrup is already a researched subject, we specifically worked on ropy syrup. It is caused by the presence of polysaccharide producing microorganisms, such as the dextran producing fermentative bacteria Leuconostoc mesenteroides¹, which induces a viscous texture and altered flavours by fermentation products. In addition to being unprofitable for the industry, ropy syrup causes further damages to equipment and lost energy resources. Currently, the ropy syrup is defined as a maple syrup making a string of ≥10cm. If needed, further analyses could be carried out to classify the syrup.

This year, we focused on the step reducing the viscosity to an acceptable threshold (inferior to 10cm). Currently, it is known that dextran producing fermentative bacteria Leuconostoc mesenteroides is correlated to an increased viscosity maple syrup. The use of a dextranase could decrease the viscosity by degrading the dextran polymer. According to fixed constraints (low temperature, high viscosity, high sugar concentration), we selected at first a dextranase from the psychrophilic organism Gelidibacter algens. A truncate of N-terminal and C-terminal extremity of the dextranase was done to increase activity and reduce proteolytic sensibility according to reports done with Streptococcus mutans dextranase².

Goals:

• Produce a dextranase treatment to transform ropy syrup into a base, which could then be used for other purposes (agrifood, alcohol or vinegar).
• Propose the treatment to be implemented at the end of the production line for better acceptability and easier manipulation.
• Work at low temperature (10-15°C) or ambient temperature to limit further Maillard reaction and energy loss.

References:

1. Lagacé, L., Camara, M., Leclerc, S., Charron, C., & Sadiki, M., (2018). Chemical and microbial characterization of ropy maple sap and syrup, Maple Syrup Digest, 9-19.
2. Kim, Y.-M., Shimizu, R., Nakai, H., Mori, H., Okuyama, M., Kang, M.-S., Fujimoto, Z., Funane, K., Kim, D., & Kimura, A. (2011). Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity. Applied Microbiology and Biotechnology, 91(2), 329–339.

Human Practices

Our project was chosen according to an untreated issue. This one was linked to a local problem from a product that we truly value, which is maple syrup. The timeline shows the initial goals we had until April, which included using synthetic biology to control the composition of the biofilm of maple syrup, different flavour defects, or the contamination of the tubing. After speaking to experts, we decided that the most helpful and feasible idea would be to revalue the waste of maple syrup resulting in the different flavour defects. Buddy syrup was already taken upon, so we focused on ropy syrup, which has no current solution. Also, our targeted clients had a say in a survey we did, in which 52 producers from 6 regions of the province of Quebec answered. By then we had already arrived in August.


From there, we continuously evaluated the usefulness, the potential issues, the risks, the alternatives, the fairness, and the benefits of aSAP, because we had in mind to make a real impact in the industry. As shown in the timeline, the application and the relevance of our project were validated by contacting organizations in this area (MAPAQ, PPAQ, and ACER Center) and scientists in the Department of biochemistry, microbiology and bioinformatics of Université Laval. Those consultations helped us change our course of action according to the needs of the producers and the regulation and science aspects. Those were made from September to October and will continue in the upcoming months. At this very moment, aSAP is an alternative that was never approached before. Futur steps for 2021 include a survey for consumers, further market research, collaboration with the PPAQ, and work with the community.

List of people we got in contact with

• 7 scientists and teachers mostly from the Department of biochemistry, microbiology, and bioinformatics of Université Laval
  • Manon Couture, Patrick Lagüe, Stéphane Gagné, Rong Shi, Marie-Ève Picard, Marie-Ève Paquet and Simon Hardy
• 3 scientists and experts in the maple syrup industry
  • Marie Filteau, Researcher at Université Laval in the Food Science Department.
  • Luc Lagacé, a researcher, microbiologist and expert in maple syrup from the ACER Research Center (Centre de recherche, de développement et de transfert technologique acéricole Inc.; french for Maple Syrup Research, Development and Technology Transfer Center Inc.)
  • Vincent Poisson, a forest engineer and a consultant for the “Club acéricole du Sud du Québec”, one of many clubs to help producers with technical management.
• 1 Professor, Jean-Michel Lavoie, at the University of Sherbrooke in the Department of Chemical Engineering and Biotechnology Engineering
• 1 teacher, Jean-François Sénéchal, at Université Laval in the Philosophy Faculty
• The PPAQ (Producteurs et productrices acéricoles du Québec, french for Maple Producers of Quebec) a private maple syrup federation and regulator
• The MAPAQ (Quebec Ministry of Agriculture, Fisheries and Food) for the criteria to be respected in the food industry
• The 52 maple producers who answered our survey!

DBTL

To find a way to organize our project, we decided to follow the Design-Build-Test-Learn cycle (DBTL cycle), a work structure presented to us by Michel Guertin.

Design
Define the problem, propose multiple solutions, and identify the biological components to construct.
Goals :

• Define the problem: ropy maple syrup, which causes important economical losses for maple producers.
• Consider a solution: treat ropy maple syrup with enzymatic treatment to remove dextrans, which cause its ropy texture.
• Find where and how to deliver our enzyme in the maple syrup production system : introduce our dextranase directly in the ropy maple syrup after its production (see Proposed Implementation).
• Find dextranase based on ropy maple syrup conditions :
  • High viscosity (between 400 and 5000 cP at 15 °C¹)
  • pH between 5 and 6¹
  • Temperature below 20 °C (maple syrup storage temperature)
  • High sugar concentration (66 °Brix1)

Considering all the above ropy maple syrup conditions, the deciding factor is the temperature at which our dextranase would work. Most dextranases come from mesophilic or thermophilic organisms, but the storage temperature would not be optimal for such types of dextranases. To respond to this, we consider a dextranase from a psychrophilic organism, Gelidibacter algens (figure 1, (1) and (2)).

Build
Synthesise, assemble and/or edit the components.
Goals :

• Overexpress dextranase gene in E. coli
• Purify dextranase

Expression vectors of our candidate dextranase from Gelidibacter algens and control dextranase from Streptococcus mutans, both from dextranase family 66, are prepared by Twist Biosciences. Both organisms’ dextranase pET-28a plasmid constructions (figure 1, (5)) are designed using SnapGene to be expressed in E. coli BL21 DE3.

References :

1. Lagacé, L., Camara, M., Leclerc, S., Charron, C., & Sadiki, M. (2018). Chemical and microbial characterization of ropy maple sap and syrup. Maple Syrup Digest, 9–19.

DBTL

Test (planned for 2021)
Characterise all the components that we build to identify the ones that work best.

Goals:

Learn (planned for 2021)
Analyse all the data generated to start the next DBTL cycle.
Goals :

• Analyse results of the Test section and draw conclusions.
• Do we need to adapt our dextranase? Do we need to restart a DBTL cycle?

Proposed Implementation

How to implement our project in the real world?

The PPAQ, the federation regulating maple syrup production in Quebec, buys maple syrup from producers.
They evaluate the quality of each barrel produced and classify it along with different criteria. Good maple syrup barrels are stored in warehouses until they’re sold to the industry or to customers.

Ropy maple syrup is typically thrown away because there is no way to implement it into other maple products. To do so, producers have to pay fines to correctly get rid of this syrup. It is estimated that between 2008 and 2017, 4.23 million USD were lost due to ropy maple syrup¹.

With our project, we envision the PPAQ buying our dextranase treatment because they’re the ones in possession of ropy maple syrup after its classification. Since they can store their barrels for many years, this is the perfect place to implement our treatment.

The PPAQ will add our treatment to the defective barrels, wait for our treatment to degrade dextrans, and sell this new product to industries or consumers. For now, we have many candidate treatment methods such as a liquid or solid treatment or a filter that would contain the dextranases. With our treatment, producers will be able to make a profit from their ropy maple syrup barrels.

Is our project safe?

To ensure the safety of our product, we evaluated the laws and regulations put in place by different government agencies in Canada². All of these agencies will have to approve of our product. The most important safety aspect is that we ensure that our product and its creation are food safe.

Our treatment would have to be considered as a food processing aid, a product that doesn’t affect the intrinsic characteristics of maple syrup and the residues of our treatment should be negligible³. If our product can’t be considered as a food processing aid, we will have to add it to the food additive list of Canada Health.

Discussions with the PPAQ have led to a potential collaboration, which will help us with all of these implementation and safety considerations. They are open to using our product and would be a valuable partnership for the development of our project.

References :

1. Lagacé, L., Camara, M., Leclerc, S., Charron, C., & Sadiki, M. (2018). Chemical and microbial characterization of ropy maple sap and syrup. Maple Syrup Digest, 9–19.
2. Government of Canada, (2020b). Canadian Standards of Identity: Volume 6 – Maple Products.
3. Retrieved on October 25th, 2020 from https://www.inspection.gc.ca/about-cfia/acts-and-regulations/list-of-acts-and-regulations/documents-incorporated-by-reference/canadian-standards-of-identity-volume-6/eng/1521132920112/1521132920549
4. Government of Canada, (2020). Food Processing Aids. Retrieved on October 25th, 2020 from https://www.canada.ca/en/health-canada/services/food-nutrition/food-safety/food-additives/processing-aids.html

Science Communications

We were committed to communicating science to general audiences.

• Radio interview with Radio-Canada: Due to the cultural significance of the maple industry in Canada, we wanted to communicate our project to general audiences. As such, our team considered radio would be an appropriate platform to do so and reached out to Radio-Canada, a national news broadcaster. The interview was held in French to reach our French-speaking community and really interested the presenter, who offered to promote our project at Radio-Canada.
• Science communication online conferences: A second way, in which we communicate science to a wide audience, was by co-organizing two conferences together with Team iGEM Concordia. Because of the COVID-19 pandemic, we decided to organize them virtually, with the main focus being how synthetic biology can impact the food industry in terms of sustainability since this was the common ground of our projects. The format of our conferences was set to be a panel discussion, featuring panelists from academia, industry, and investors. The conferences were a success, reaching 160 total participants from 19 different countries.

Collaborations

We participated in several collaborations with iGEM teams around the world:

• Calgary: JulyGem was an event organized by Team Calgary around the theme of translating synbio to the real world. It included workshops on public engagement, science communication, and entrepreneurship, as well as presentations by 14 iGEM teams. This event was a nice opportunity for our team to have a first glance at the diversity of iGEM projects at this year's competition.
• Concordia: Team Concordia organized a Mini Jamboree with several teams. We only participated in the poster presentation part because of time restriction. It was a great opportunity to readapt our Giant Jamboree presentation thanks to the pros and cons given by the judges at this event. The event allowed our new members to get in touch with scientific communications and learn from their experience. Finally, we co-organized two science communication online conferences with Team Concordia (see science communication section).
• TU Delft: Team TU Delft organized a worldwide video collaboration for teams to get to know the diversity of projects from this year's competition. Briefly, teams submitted a 10-second video explaining who they are and what their project is about.
• AUC-Egypt: Following our online webinar on our computational tool for toehold riboswitch design, Team AUC-Egypt reached out to us. Together, we were able to debug our tool together to make it more helpful for new users, and team AUC-Egypt could produce the riboswitches they needed.
• UPCH-Peru: Team UPCH-Peru invited several teams to share information about their projects and discuss how they contribute to the United Nations Organization Sustainable Development Goals. As such, we identified that our project fits with the three following goals:
  • Industry, innovation, and infrastructure: Our project innovates by helping transform would-be wastes into a usable product and fosters further innovation by providing a source for new maple products.
  • Sustainable cities and communities: Our project aims to help maple producers reduce their waste output and increase the sustainability of their industry.
  • Responsible consumption and production: Our project helps reduce the energy used to deal with waste products and improve the productivity of the maple industry in a sustainable way.

Acknowledgements and Sponsors

Special thanks to:

Teachers	Students
Steve Charette Hélène Deveau Michel Guertin	Djebran Alkozai Carla Bautista José Campano William Chevarie Florian Echelard Olivier Lavoie Candice Lemaille Jean-Michel Proulx François Rouleau Danilo Tubic Martine Voisine

Our Sponsors: