In recent years, the concept of healthy diet has attracted public attention. An increasing number of people have begun to reduce the intake of added sugar and replace it with sugar substitutes to minimize the harm of added sugar to the body while tasting "sweet". In the past two years, a sugar substitute named erythritol has entered our vision. This sugar substitute with natural sweetness, zero calories and high safety has quickly swept the domestic beverage market.
During the early stage of our project, a study on the erythritol metabolic pathway specific to Brucella caught our attention. Considering the current problem in colonizing therapeutic engineered probiotics, we can transfer a metabolic pathway of erythritol into probiotic chassis EcN to establish an independent colonization system based on the independent metabolic niche of erythritol. By simply intaking a beverage containing erythritol, the probiotic product can be colonized. This is our "ERYbiotic".
The Integrated Human Practice of the project is carried out from 4 perspectives: "sugar and sugar substitutes", "erythritol", "intestinal flora and probiotics" and "engineered probiotic therapy". Improvements and feedback to the topic are carried out throughout the iGEM cycle.
Since ancient times, Chinese people have been fond of eating sugar. China is not only one of the earliest sugarcane-planting countries but also one of the earliest countries to refine sugar in the world. There were records of malt processing as far back as the Western Zhou Dynasty (about 1,000 BC). Today, sugar also plays a vital role in Chinese cooking. In Chengdu, Sichuan Province, the gastronomic capital, sugar can be found everywhere, from elegant restaurants to street food stalls.
Up to now, the artists of sugar painting are still active in Chengdu's markets and parks and have become a unique marketplace memory in the minds of local people. In August, SCU-China members visited Jinli Ancient Street and interviewed a sugar painter. Under her guidance, we learned how to make sugar paintings and experienced the process of sugar painting by ourselves. At the same time, we also know that the art of sugar painting, as intangible cultural heritage, has been highly valued by society and the government and is still passed down from generation to generation in her family.
Please check out our Jinli tour via video!
We also considered the possibility of incorporating sugar substitutes into the production of sugar paintings. However, when we asked in the interview, "Is it possible to make sugar paintings using sugar substitutes?" "It's hard to replace the malt syrup used to make sugar paintings," she answered. "Not only is the viscosity and setting process of maltose syrup not guaranteed by adding sugar substitutes, it also affects the flavor."
China's Food and Drug Industry Development and Regulation Research Center put forward: "Reducing sugar is becoming the trend of global food production and consumption development. In the Chinese market, reducing sugar, oil and salt is an important way to implement 'healthy China' in the field of food consumption." People began to pay attention to how to satisfy cravings without affecting their health. Thus, the concept of "sugar substitute" gradually came into our view.
Saccharin, a synthetic sugar substitute, was first discovered in 1879 by Constantine Falberg while studying coal tar derivatives and was put into commercial use. Since then, artificial sugar substitutes, because of their superior quality and low price, quickly became popular during the world war when sugar prices soared. However, with the development of related research, the safety of synthetic sugar substitutes has been doubted, and people begin to attach importance to the natural sugar substitutes from plant extraction or microbial fermentation. Examples include stevioside, ginsenoside, and erythritol, which has exploded in recent years due to the popularity of unsweetened Genki Forest sparkling water.2021 China Diet Beverage Market Trend Insight Report shows that: in 2020, the domestic diet beverage market size reached 11.78 billion yuan, compared with 2014 increased by seven times. The market will grow to 22.74 billion yuan in 2025, doubling in size within five years. The market will continue to climb to 27.6 billion yuan in 2027. (Fig.1)
Fig. 1 Diet Beverages Market Size and forecast in China (2021 China Diet Beverage Market Trends Insight Report)
To get a sense of the public's attitude towards sugar substitutes, we conducted a survey on the attitudes of people of all ages around us. The questionnaire was a five-level scale with four items, and a total of 336 copies were collected.
Fig.2 The histogram of frequency distribution of the total score of the scale
1.Then general public has a negative attitude towards the taste and purchase intention of sugar-substituted products, which may relate to the higher price and taste difference compared with sugar-sweetened beverages.
2."I will be attracted by the" zero sugar and zero calories "advertised by sugar substitute products." The average score of one question is much higher than that of "neutral", indicating that the advertising effect of sugar substitute products is relatively positive.
3.Consumers' taste perception and purchase intention for sugar substitutes increase with age and education level, suggesting that we should promote the health concepts related to dietary sugars and sugar substitutes to more young people, including children.
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Obviously, in the short term, sugar substitutes cannot replace the status of sugar in traditional cooking and diet. However, with the proposal of the concept of healthy sugar intake and the expansion of the advertising effect of sugar substitute products, the scale of China's sugar substitute market will further expand, and the health problems related to sugar substitutes will also attract more attention from us.
The 2021 China Diet Beverage Market Trends Insight Report shows that during the decade of 2010-2020, the ratio of natural sweeteners increased year by year, while the ratio of synthetic sweeteners showed reduced. In recent years, erythritol has gained momentum to replace aspartame and other artificial sweeteners, as shown below: from 2015-2019, China's erythritol consumption showed an obvious growth trend, with a growth rate of 89.7% in 2018 and 100% in 2019. This suggests that erythritol is a sugar substitute with high market impact and application potential.
Fig.3 Trend of sugar substitute use and erythritol consumption (2021 China Diet Beverage Market Trends Insight Report)
To understand the production and utilization of erythritol, related health issues and to explore the reasonable way to provide erythritol as a carbon source for engineered probiotic drugs, we talked to the manufacturer of erythritol—— Yuxin Sugar Alcohol Ltd. through an online interview; we also visited the factory of famous beverage brand Genki Forest in Dujiangyan and had a pleasant exchange with experts from Genki Forest Research Institute.
At the same time, as the stakeholders in the erythritol industry chain, we also collected their suggestions for our subject during the visit, which will be specifically mentioned in the “3. Improve In Communication”
To summarize our achievements, the following are our findings.
- The industrial production of erythritol is by microbial fermentation with glucose as the carbon source.
- Erythritol is a filler sugar substitute, which is more expensive than the general high power sweetener but has the advantage of "zero calories" and a good market foundation.
- Judging from the domestic market, the main application area of erythritol is still in the beverage industry. But other categories can be added, and there is no limit on the addition amount.
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- Erythritol is very stable in food and beverage process, easy to control the amount of addition, and can enrich taste and add color to the beverage.
- Erythritol is the only sugar alcohol sweetener that can be labeled as zero-calorie. The human body has a high tolerance for erythritol, but excessive intake may lead to diarrhea.
- during beverage development, compounding other natural high sweeteners (stevioside, rosmarinic acid glycosides) or dietary fiber can enrich the taste of beverages.
Learn MoreIn conclusion, erythritol is indeed a widely used and popular natural sugar substitute. As a carbon source substance required by engineered probiotics to establish an independent metabolic niche, it is safe for human body and its high stability ensures that processed erythritol can be ingested by human in various ways, which increases the possibility of engineered probiotics drug development. Meanwhile, whether our products are presented in the form of direct mixing of erythritol in engineered probiotic tablets or requiring the intake of erythritol drinks along with engineered probiotic tablets, the better market foundation of erythritol will allow our products to be more widely accepted in the market.
Are drinks sweetened with erythritol good? Do the repetitions of sugar substitutes enhance the taste of a drink? At the end of July, we invited members of SCU-China to conduct a blind test on six bottles of drinks purchased from convenience stores with sugar and various sweeteners.
After drinking each bottle, we invited the team members to rate the taste and sweetness of the drinks (out of 10) and comment on the drinks' taste. Let's look at the results of our evaluation!
In conclusion, in terms of taste and price, erythritol drinks do not have a significant advantage over drinks using other sweeteners. And, no matter which sweetener is used, the key to the taste is whether the flavoring of the drink is acceptable to the public and added other ingredients. Even so, driven by the concept of healthy sugar reduction, several team members said they were willing to replace sugary drinks with erythritol drinks.
Intestinal flora has been prevalent in academic and commercial circles in recent years. More and more studies reveal the complex relationship and interactions between intestinal flora and multiple systems of the human body, including digestive system and nervous system. Meanwhile, the colonization of engineered probiotics is highly correlated with the intestinal flora and the physiology of the human digestive system.
Probiotics are live microorganisms that are intended to have health benefits when consumed or applied to the body. They can be found in yogurt, fermented foods, dietary supplements, and beauty products. In addition, there are medicines for animals and plants.
Probiotic products applied to the human body are favored by the market for their effect on treat constipation or diarrhea, Daily healthcare, and lose weight reasonably.
Forecasts show that probiotic market of China will reach 101 billion in 2022, with a compound annual growth rate of 15%, and there is still great room for growth in per capita consumption. As the TOP1 category of domestic e-commerce, probiotics have the fastest growth rate in the general diet segmentation track.
Fig.4 Probiotics market size and growth in China(2021 China Diet Beverage Market Trends Insight Report)
In our "Questionnaire on Public Perception of Sugar Substitutes and Engineered Probiotics", we investigated the consumption and attitude of probiotic products among the public around us. The percentages of respondents who have purchased probiotic food, medicine, and health products are 96.0%, 49.3%, and 15.6%, respectively, indicating that the popularity of probiotics is indeed widespread. The most common purpose for the public to purchase probiotic products is to eat and treat constipation or diarrhea and for daily healthcare. Meanwhile, 70% of respondents said they would purchase probiotic products independently, while many respondents would also follow the advice of doctors, pharmacists, family members, and friends to purchase probiotic products.
Fig.5 The consumption and attitude of probiotic products among the public
The above results show that although the mechanism of action with intestinal flora is not fully elucidated, probiotic products have been welcomed by the market for their superior taste and significant effects. Learn More
Engineered probiotics are novel microorganisms produced by genetic editing of pre-existing wild probiotics. With the continuous development of tools and technological advances, the engineering modification of probiotics is becoming more diverse and feasible. In the past few years, several advances in the development of engineered probiotics have been made that will benefit humans.
Some probiotics achieve their anticancer effects by enhancing the apoptosis of cancer cells and preventing oxidative stress. (Badgeley A. et al, 2021; Aggarwal N. et al, 2020; Sehrawat N. et al, 2021) With the development of modern gene editing techniques, it has become possible to design probiotics that can play an active role in the fight against cancer.
The levels of metabolism-related substances are generally in a relatively stable state in the body. When biochemical processes in the body are hindered, the accumulation or deficiency of certain metabolites can cause diseases. (Hillert A. et al, 2020; Zheng R. et al, 2020; Soria LR et al, 2018) These diseases are difficult to treat and often require long-term control of lifestyle and dietary habits, causing serious financial burdens and stress to patients. (Binda S. et al, 2020) Engineered probiotics can break down or convert these accumulated substances and help treat such diseases.
However, the applications of engineered probiotics still face several challenges.
For example, many microorganisms have few actionable genetic tools, and only a small fraction of strains are currently available as bacterial chassis for research design. There are still a few engineered probiotics moving from the laboratory to the clinic. Even when it enters clinical trials, there is no guarantee that it will achieve the desired results, which hinders the efforts of some researchers to construct engineered probiotics. At the same time, some argue that GMOs are harmful and hinder the effective deployment of engineered probiotic therapies.
To develop an engineered probiotic for living therapeutics, we must consider factors besides theoretical route design, including more precise drug delivery, suitable colonization location and platform, and less toxic side effects. In order to explore the possibility of engineered probiotic colonization location, platform, and mode, we consulted Professor Yuan-ping Han from Sichuan University, who has deep insights into human digestive physiology and intestinal flora. We received some valid observations.
1.The colonization of engineered bacteria is a complex process influenced by multiple factors, and an important aspect to consider is the location of bacterial colonization. While intestinal luminal bacteria are quickly eluted with defecation, mucosal bacteria can adhere stably to the intestinal wall.
2.The engineered probiotic bacteria colonize the wall of small intestine, the colonization density will not be high due to natural barriers such as small intestinal Paneth cell defensins. At the same time, the small intestine has a strong absorption effect on bacterial toxins, and the colonization of engineered bacteria may have negative effects on the human body.
3.Currently, engineered probiotic therapies for cancer are still in the developmental stage, and the effectiveness of probiotic therapy is currently controversial due to the complexity of the cancer development process and the complex relationship between therapeutic substances and the tumor microenvironment.
These contrary opinions remind us of the need to prove our theory with more reliable evidence. It includes exploring more reliable delivery methods and colonization strategies and proving that it is non-toxic to humans.
Learn MoreOf course, we should also listen to views of customers on an emerging concept as "engineered probiotics." After a brief introduction to engineered probiotics, we surveyed the subjects' intuitive impressions of the functions and safety of engineered probiotics and their willingness to try engineered probiotic therapy.
Learn MoreIn addition, the practical problems of developing engineered probiotics that encountered in health, foods, and drugs, especially safety and legal issues, cannot be ignored. We invited synthetic biology entrepreneurs from EPIC-China to discuss these issues in depth from their perspectives and to explore the most feasible solutions for us through extensive data search.
Learn MoreSCU-China attaches great importance to collecting opinions during the process of Human Practices and thinks about how to feedback on the project design appropriately. Since July, we have been getting valuable opinions on the improvement of the project from the interviews and exchange sessions. Some of the comments were decisive for the advancement of the project. We gradually improved the project, optimized the design, and developed a preliminary business plan during this period.
At the time of the interview with Yuxin Sugar Alcohol Co., we had only completed our initial project design and had given less thought to the extension of the project. Our initial positioning of the product was health food, and we were eager to know what other difficulties needed to be overcome as the subject moved toward practical application.
Learn MoreWe realized the difficulties before the products developed through synthetic biology went to market. We hoped that we could get a satisfactory answer by discussing with more stakeholders who know more about the commercialization of synthetic biology products.
We verified the utilization of independent carbon sources by EcN and simulated its co-culture under anaerobic conditions with glucose competition in subsequent modeling work. We concluded that erythritol could confer sufficient competitiveness to EcN in small intestinal flora for colonization, even in the presence of glucose.
We had already achieved milestones in our experimental and modeling work and welcomed the first inter-school meeting. We had a general understanding of other team's projects. The guests from CCIC, Song Lin and Jinrong Liu, and other participating members affirmed our preliminary work and raised some questions and suggestions.
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In China, the Regulations on the Safety Management of
Agricultural Genetically Modified Organisms and its
supporting regulations, sectoral laws of higher rank, and other
relevant regulations constitute the current basic management system
of GMOs. Detailed and strict rules have been made on the products’
research and testing, production, processing, and operation.
However, for the time being, there is no precedent of engineered
probiotic health food entering the market in China or even in the
world.
On the pharmaceutical side, Synlogic's SYNB1618 engineered probiotic
for the treatment of phenylketonuria has been approved by the FDA to
enter Phase II clinical trials, suggesting that we could consider
engineered probiotics designed as drug delivery platforms with
erythritol-independent ecological site colonization characteristics.
Mutaflor® Escherichia coli strain Nissle 1917 entered the Australian
Register of Therapeutic Goods in February 2012 and is still today
the only probiotic in Australia registered at the AUST R level which
is effective in treating constipation. The existence of these two
drugs indicates their safety and effectiveness, which shows the
possibility of our product being approved by the market.
Probiotic microencapsulation technology is based on freeze-drying technology, which encapsulates a protective film on each probiotic unit to make it resistant to the enzymes of the digestive system and cross the gastrointestinal tract. We considered that the final presentation of the product could be in the form of an encapsulated probiotic powder or tablet on top of an additional intake of erythritol.
Through the two previous exchanges, we saw more possibilities for the applying our engineered probiotic products. In response to the problems we've encountered in the commercialization of the subject, we contacted EPIC-China. We met with EPIC-China members and iGEM seniors: Wenxi Li (SZU-China captain 2020) and Rui Su (ShanghaiTech-China captain 2021, founder of SynMeta) had a communication. They raised several points that need to be emphasized in the startup process, which caused us to think about them.
We tried to contact the domestic probiotic companies by email and phone to make them our main stakeholders for cooperation and communication. But due to time and space constraints, unfortunately, we did not have good contact with them.
The next step in market research is first to understand what
products domestic probiotic companies generally make, how the
products are sold, and the current progress in terms of in vivo
colonization. We also need to understand our target consumers'
needs. Integrate the results of market research and think about the
market position of the product and our chance in the market.
We conducted detailed research in the entrepreneurship section.
In early September, we also released a questionnaire on public perceptions of sugar substitutes and engineered probiotics, and we got a general idea of public attitudes toward sugar substitutes.
The intestinal flora is a very complex system, and the addition of engineered probiotics adds a lot of uncertainty to it and complications related to human physiology that need to be considered in the design of the subject. To address these issues, we would like to seek expert advice. On August 20, 2022, we met with Prof. Yuanping Han at the School of Life Sciences, Sichuan University. He expressed a strong interest in our topic. We discussed several physiological and therapeutic-related issues on the topic in detail and obtained many meaningful hints.
Learn MoreAs a large food company utilizing erythritol, Genki Forest has rich experience and professional insights into the application of erythritol, the commercialization process of food, and the assurance of food safety. During the visit, they made many theoretical and application suggestions to SCU- China's subject.
Learn MoreFirst, we performed prokaryotic expression of EryD to investigate its inhibitory effect on bacterial growth and gene expression. Subsequently, we used bioinformatics to optimize the codon of EryD , improve its binding specificity to EryO and reduce its metabolic toxicity by altering specific amino acid residues.
Ultimately, we chose to consider the possibility of developing the project as both a colonization platform and a drug.
To synthesize erythritol from the front end by biological methods was a part of the original conception of our project. However, considering the project's purpose to solve the problem of colonization and the limitation of experimental time, we finally chose to give erythritol from an exogenous source. After the downstream colonization and functional verification experiments are completed, we will turn our attention back to the upstream to try to extract, ferment and produce erythritol from straw and other wastes.
During the exchange, SCU-China values input from experts, companies, and other teams, both inside and outside the industry. In the communication, we saw the concerns and attitudes from different regions, disciplinary backgrounds ,and ages about the subject and the social topics of design. Whether positive, constructive, or negative, we listened to them and then actively discussed and reviewed the information, hoping to optimize and improve the topic from more perspectives.
Again, we sincerely thank all parties for the attention, advice, and encouragement given to SCU-China!
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2. https://www.nccih.nih.gov/health/probiotics-what-you-need-to-know
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7. Zheng R, Li Y, Wang L, Fang X, Zhang J, He L, et al. CRISPR/Cas9-mediated metabolic pathway reprogramming in a novel humanized rat model ameliorates primary hyperoxaluria type 1. Kidney Int. 2020;98(4):947–957. doi: 10.1016/j.kint.2020.04.049.
8. Soria LR, Brunetti-Pierri N. Targeting autophagy for therapy of hyperammonemia. Autophagy. 2018;14(7):1273–1275. doi: 10.1080/15548627.2018.1444312.
9. Binda S, Hill C, Johansen E, Obis D, Pot B, Sanders ME, et al. Criteria to Qualify Microorganisms as "Probiotic" in Foods and Dietary Supplements. Front Microbiol. 2020;11:1662. doi: 10.3389/fmicb.2020.01662.