Entrepreneurship acts as an amplifier of the ideas and also a catalyst to startups in synthetic biology. During the whole iGEM cycle, we carried out a series of market research and business analysis in order to better understand the prospect of the ERYbiotic. In-depth knowledge of the market is the first step to commercialization. Furthermore, not only about written materials, we also reach out to the factory and learn from the frontline of the industry, study the first-hand information and also the newest changes of technology. Lastly, we also communicate with the members from the iGEM Entrepreneurship Program Innovation Community(EPIC) and get a lot of useful suggestions through the conversations. We look forward to keep modifying our product and bring it to the market.
Mission
Our goal is to make probiotics more widely used, to continuously generate beneficial metabolites in the intestinal tract by relying on a stable carbon supply, to not only achieve the effect of lowering blood pressure and blood sugar level but also treat metabolic diseases, and to serve as a steady source of supply of multiple nutrients in daily life.
Vision
The application of probiotics has become increasingly popular and gradually occupies an important position. The abuse of antibiotics leads to the production of many drug-resistant bacteria. At the same time, the disorder of normal intestinal flora after using antibiotics makes applying probiotics more and more critical. The biological antagonism of probiotics and the promotion of vitamin absorption and other functions make them play a massive role in maintaining daily health. Our engineered probiotic can further release the potential of probiotics as nutritional supplements, endow probiotics with controllability, extend the existing time, and enable probiotics to produce probiotics and downstream products persistently. In addition, the combined use of erythritol can allow probiotics to gain acquired advantages. Then, the probiotics can change the ingested erythritol into beneficial products. Erythritol itself is a symbol product of a "low sugar diet." For people who need to limit their sugar intake strictly, it can help them reduce their sugar intake. At the same time, the products of probiotic metabolism can also continuously provide the nutrition the human body needs. With the combination of erythritol and modified probiotics, we are committed to introducing a product that is more in line with the lifestyle of modern people.
We constructed various kinds of simulations using the methods in computational structural biology to enrich the characterisation of the structural properties of the enzymes.
Innovation
1. Regulated probiotics: Probiotics mainly locate in intestinal. After ingestion, they produce healthy metabolites in the intestinal tract to promote intestinal health. However, these intestinal probiotics are usually not resistant to high temperatures and have a low resistance to acid, so they can easily be killed by enzymes and temperature in the oral environment during consumption. Only a tiny part of probiotics reaches the intestinal tract through the stomach and can play a role. However, intestinal probiotics must get a particular order of magnitude to play a role. The more active probiotics are, the better the effect will be. The small number of intestinal probiotics remaining is not dominant in quantity, and it isn't easy to compete for adequate nutrition. Therefore, the living condition is not ideal and can only produce a temporary effect. Our products can provide modified probiotics with a specific carbon source as nutrition by drinking a beverage containing erythritol after meals. And they can build a unique niche, avoid competition with other bacterial groups, and will not affect the growth of other bacterial groups, to achieve the long-term effect and regulate the metabolism of probiotics through erythritol.
2. Erythritol as an inducer of the probiotic: the regulation of probiotics does not depend on complex induction technology. It only requires the exogenous intake of erythritol to induce probiotics to produce downstream metabolites. Erythritol, as an edible sugar substitute, has been widely used in food and beverages, is easy to obtain, and is suitable for a healthy lifestyle of low sugar diet and for controlling the rise of blood sugar. It has played an essential role in the prevention and treatment of dental caries and in promoting oral health. 1 Using erythritol as an inducer of metabolic loci in probiotics can not only combine the advantages of erythritol in caries prevention and hypoglycemia but also provide a separate carbon source for probiotics so that they can grow and metabolize better.
3. PQQ and L-erythrose are metabolites of probiotics: the main role of PQQ involves direct action on a key enzyme. This key enzyme can participate in energy production in mitochondria. Therefore, PQQ helps to optimize energy production. Coenzyme Q10 is an important compound, which is also extremely important for the function of mitochondria. PQQ is more important than coenzyme Q10. Cells can produce coenzyme Q10 but cannot produce PQQ. Therefore, the human body must rely on diet or supplements to meet the body's demand for PQQ. At the same time, PQQ can also be used as an antioxidant to eliminate free radicals in the body.2,3 L-erythrose is a kind of small therapeutic molecule with the functions of lowering blood pressure and blood glucose, which can maintain blood glucose stability. It is a beneficial substance for people who need to control blood glucose. The engineered probiotics can not only use erythritol to produce PQQ, as the external supply of PQQ, but also produce L-erythrose to supplement the substances needed for human nutrition.
At the early stage of the project, we conducted a questionnaire survey to understand people's acceptance of probiotics and their willingness to purchase and use them. A total of 302 volunteers from different countries, regions, industries, and age groups completed the questionnaire. Among the interviewees of the questionnaire, the practitioners and students in biology, medicine, and related fields accounted for about 40%. Most of the other parts were not practitioners in life science-related lots, so it can help us understand the acceptance of probiotics and the willingness to buy probiotics from the professional and consumer perspectives. First of all, the table below illustrates that about 67% of the respondents have a positive attitude toward the safety and functionality of probiotics and are willing to buy and use probiotic products. The main types of probiotic products for people who have purchased probiotic products are probiotic food and drugs. Less than 2% of the respondents have never purchased probiotic-related products, indicating that probiotics are very popular daily and highly accepted. Among them, most purchased are yogurt containing probiotics for daily consumption or drugs made of probiotics to treat diarrhea, constipation, and other intestinal discomforts. In addition, there are probiotic health products for providing daily nutrition. Interestingly, few people take probiotics to lose weight and control their weight.
Fig 1. Consumer attitudes towards probiotic products
Fig 2. Purchase predisposition of the customers
Fig 3. The aim of buying probiotic products
For the use of sugar substitutes, we used a four-item five-level scale to investigate the public's attitude toward sugar substitutes. Questions 1, 3, and 4 are forward questions, and question 2 is reversed. 1. Questions 3 and 4 focus on the subjects' willingness to purchase sugar substitutes. In contrast, question 2 focuses on the subject's feelings about the taste of sugar substitutes.
Data analysis revealed that the public has a polarized attitude toward using sugar substitutes. Some people hold a conservative attitude towards sugar substitutes and seldom buy sugar substitutes, while others often buy sugar substitute drinks or products. Given this, we believe it depends on the public's understanding of sugar substitutes. Therefore, it is necessary to strengthen the publicity of sugar substitutes so that the public can understand the multiple benefits of sugar substitutes, the food grade application approach, the excellent taste, and the characteristics of almost no side effects. Only by eliminating the public's doubts can we solve this polarization of distribution characteristics.
Fig 4. Results of the survey
Based on the results of previous market research, we modified our products and improved the design of the subject. We have chosen probiotics with high acceptance and wide application as the chassis organism. Given the public's preference for probiotic food, our products can be packaged into yogurt or food for daily consumption. At the same time, probiotics can colonize the intestine by getting unique niches and sugar substitutes such as erythritol. Then, they can play an antagonistic biological role and resist intestinal flora harmful to human health. It can also regulate the intestinal microenvironment and treat intestinal discomfort and other symptoms. At the same time, the modified probiotics can also metabolize and produce daily nutrients such as L-erythrose and PQQ, which not only meet the needs of health products but also can be used as a drug to control blood sugar and antioxidants. Finally, we used erythritol as an inducer. As one of the low-calorie sugar substitutes, eating erythritol can control sugar intake without raising the blood glucose level. 4 It is one of the markers of a low-sugar diet. It can replace sugar, lactose, etc., which is usually eaten. The public's acceptance of sugar substitutes is gradually increasing. Based on this, we have jointly created a healthy low-sugar diet product to meet the needs of various consumers.
Field Research
In addition to the questionnaire survey on the market situation of probiotics, we also conducted field research on upstream manufacturers of sugar substitutes and downstream applications of sugar substitutes. We went to the front line to learn the scope of application of sugar substitutes and the views from industry professionals on sugar substitutes which helped us further understand the applications of sugar substitutes.
We were lucky enough to contact the famous domestic sugar alcohol-based sugar substitute manufacturer, Yuxin Sugar Alcohol Co., Ltd., and interviewed and communicated with Ning Jun, an expert from Henan Yuxin Sugar Alcohol Co.
The production range of Henan Yuxin Sugar Alcohol Co., Ltd. covers sugar alcohol products such as xylose, xylitol, erythritol, maltitol, L-arabinose and compound sugar. Through communication with the industry, we understand the steps required for industrial production, usually using yeast for fermentation. The fermentation method mainly uses glucose as raw material. Manufacturers with glucose production lines usually use corn starch for fermentation to produce glucose. To biosynthesize erythritol, we need to integrate upstream resources, select more suitable raw material sources, and reduce the initial price in order to control the cost.
In addition, through communication, we learned that erythritol has a wide range of downstream applications. Many products can add erythritols, such as candy, baking, jam, and even toothpaste, mouthwash, toothpicks, and other products, and currently, there is no strict limit on the amount of addition, so it can be seen that erythritol has a wide range of applications and strong demand and a good market prospect. When used in combination with probiotics, it can achieve a better effect.
The primary downstream purchasers of erythritol are food and beverage companies, and we interviewed Yuanqi Forest. Yuanqi Forest is a well-known domestic innovative food and beverage company in China with independent research and development, independent design, and the first company to use erythritol as the main raw material for beverages fully. It has multiple lines of products, such as Yuanqi Forest sparkling water. At present, low-sugar sparkling water containing erythritol accounts for most of the market share. It has set off the trend of sugar substitutes in China, making sugar substitutes widely known to the public and being the leading promoter and propagator of erythritol. The human body has a high tolerance to erythritol. 90% of ingested erythritol is absorbed by the small intestine and excreted through the urinary system, and about 10% is involved in the physiological effects in the body, which has a higher safety level compared to other sugar substitutes. Therefore, there is almost no quantitative requirement for erythritol in all countries regarding food safety. In addition, erythritol has the health effects of anti-caries and anti-oxidation by scavenging hydroxyl radicals in the body, so erythritol is widely accepted by consumers, and it is the only sugar alcohol sweetener that can be labeled as a zero-calorie, which has good publicity effect in the market. As the results of our questionnaire survey, the polarization of the attitude to the use of sugar substitutes needs to be improved by the publicity of downstream companies and the leading companies' leading effect. Still, it can be observed that the current downstream manufacturers are very active in the promotion, and the market of sugar substitutes is still expanding at high speed. The field survey results and our questionnaire respond to the market's attitude towards probiotics and sugar substitutes, so we can design products and genetic lines to meet the market's needs and make them more in line with popular tastes.
To understand sugar substitutes better, we also tried different kinds of sugar substitutes ourselves, mainly from the taste, sweetness, price and other aspects, erythritol compared to cane sugar sweetness to be a little lower, relatively less sweet, but better taste, with a cool taste in the mouth, suitable for summer drinking, and its sweetness is pure, close to cane sugar, without the bitter aftertaste, chemical taste or metal taste. Regarding price, beverages containing erythritol are currently a bit more expensive than beverages such as cane sugar, but their prices are still competitive due to their zero-calorie health concept.
Scope of Application
Erythritol does not raise blood sugar levels and, at the same time, does not cause insulin secretion, but can provide 70% of the sweetness of sucrose, so it is widely used as a sweetener in all kinds of food and beverage, which can adjust the taste of food and beverage and at the same time can control sugar intake, suitable for people who need strictly control sugar.
PQQ demonstrates a better antioxidant capacity than vitamins E and C, eliminating free radicals produced in the body.PQQ converts food and oxygen into energy needed by the body, and healthy mitochondria play an important role in supporting cognition and physical performance and delaying aging. PQQ can increase the number and quality of healthy mitochondria in aging cells, slowing the body's aging process and reducing metabolic disorders such as diabetes, cardiovascular diseases, etc. PQQ can also support immune function, as deficiency of PQQ may lead to immune dysfunction. L-erythritol can lower blood sugar levels, so modified probiotics combined with erythritol can be used as an extra daily nutritional supplement.
Erythritol has a good inhibitory effect on cariogenic bacteria in the mouth and can reduce the pH drop after sugar intake, thus reducing the acidic corrosion of teeth by acids in the mouth and reducing the degree of enamel demineralisation, thus preventing caries.
Fig 5. Scope of Application
Market pain points and solutions
Probiotics have multiple effects on the human body, helping to improve the immune system and intestinal health, relieve constipation and prevent colds. However, the effect of probiotics is single-use, so it is necessary to replenish a sufficient amount of probiotics at regular intervals, which makes it challenging to maintain intestinal health in the long term. In this regard, modifying probiotics and giving them an independent carbon supply can effectively prolong the colonization time of probiotics to maintain intestinal health in the long term.
In addition, the ingested probiotics lacked modifiability, and it was difficult to control the content and type of their products. In contrast, the modified probiotics could be regulated by erythritol to modify their metabolic pathways and produce exogenous nutrients such as L-erythrose and PQQ, which changed the role of probiotics and made them more nutritious.
Although China's probiotic industry is a little behind the developed international, currently has been fully supported by our government, the industry is in a stage of rapid development.
Since 2005, China has launched comprehensive research on probiotics. China's National Natural Science Foundation, China's National Science and Technology Support Program, and China's "863" program have greatly supported China's probiotics academic institutions. Some of the research results of probiotics have been successfully transformed in the food industry, which are the basis for the rapid growth of probiotics industry.
In early 2015, with the help and funding of the "863" program of the Ministry of Science and Technology of China, the Functional Dairy Laboratory of the Ministry of Education of China Agricultural University established a probiotic resource bank, isolated and screened more than 10,000 strains of lactic acid bacteria from the intestinal tract of hundreds of thousands of healthy people and traditional Chinese fermented foods, and successfully transformed the key technology of probiotic production and obtain strains with excellent health utility, such as Bifidobacterium longum BBMN68, Bifidobacterium youth BBMN23, Lactobacillus casei L9, etc. Meanwhile, with the support of the Ministry of Science and Technology, technologies such as high-density fermenters for strains, freezing of strains, microcapsules for strain packaging, and strain activity retention were developed, realizing the independent intellectual property rights technology of probiotics in China.
In April 2018, China's Ministry of Health issued a notice on further standardizing the management of health food ingredients, which regulates the declaration of probiotic foods, etc. (or raw materials), and standardizes the management of health food ingredients involving fungi. China's Market Supervision Administration will further play a good supervision and management mechanism to escort the benign development of China's probiotic industry.
On the whole, China is paying more and more attention to the safety of healthy food, with high standardization. The state has made heavy efforts in many health food industry associations, formulated increasingly strict industry standards, and carried out a "fight against four non-safety" actions, aiming to promote China's health food industry development. The aim is to promote the healthy development of China's health food industry, strengthen the industry's self-discipline and integrity construction, and effectively let all the merchants in the market understand the harm of the health food banned by the state to the ordinary people.
At the same time, regarding the requirements of probiotics for infants and young children, the Ministry of Health of the People's Republic of China is very strict in controlling the domestic market of probiotics for infants and young children, and has published the Announcement on the List of Strains that can be used for infant and young children's food (Ministry of Health Announcement No. 25 of 2011). In accordance with the relevant provisions of the Food Safety Law and its implementing regulations, the Ministry has organized a safety assessment of the approved strains that can be used for food and a list of strains that can be used for infant food.
The government commit to promoting the development of the entire health care industry, which is another major initiative to meet the urgent needs of the masses, improve the quality of the whole population health, protect and improve people's livelihood, and is also an important strategy to enhance the service industry, effectively expand employment, form new growth points, and promote economic transformation and upgrading.
In addition, given the impact of some infectious and epidemic viruses, such as the new crown pneumonia that emerged at the end of 2019, which is rapidly spreading from person to person, the severity of the symptoms of individual performance depends on the resistance of the individual. The country's nutrition experts and the Health Commission call for attention to improve people's resistance by exercise and supplementation of multivitamins and probiotics to care for the gastrointestinal system.
With the rise of domestic per capita income, people have more and more disposable income, and they start to pay more and more attention to the requirements of quality of life and concern for their own bodies, and their life needs are diversified, which directly pulls the demand for healthy food, especially probiotics, which is a very cost-effective health food.
The annual GDP reached 1,143,670 billion yuan in 2021, an increase of 8.1% over the previous year at constant prices, and the per capita disposable income was 35,100 yuan, with a solid growth trend between the two. In the same year, China's per capita expenditure on healthcare was RMB 2,115, up 14.8%, with a growth rate of 1.2 percentage points higher than the national per capita consumption expenditure, and the residents' consumption structure further improved. The Questionnaire Survey Report of Urban Savers in the Second Quarter of 2022 released by the Central Bank also shows that when asked about the items they are prepared to increase their spending in the next three months, the residents chose the highest proportion for education, followed by healthcare, accounting for 27.4%, with people more willing to pay for health.
In addition, the burden of disease caused by chronic diseases in China accounts for about 70% of the total disease burden, and the proportion of chronic disease prevalence remains high. Under the effect of high-fat and high-heat dietary structure and irritating and irregular eating habits, obesity, cardiovascular diseases, and chronic diseases such as gastritis and chronic enteritis have seriously burdened residents. However, the unreasonable dietary habits of residents are difficult to change in the short term, and the development of probiotics offers the possibility to solve these problems.
The influence of many contemporary social factors has led to extreme demand for probiotics in the body.
The rapid development of the probiotic industry has also brought about rapid changes in the technology. The key indicators of probiotics are mainly the strain of bacteria, the number and activity of probiotics. Probiotics are extremely afraid of heat, and if the temperature exceeds 60 degrees Celsius, probiotics will lose activity, so manufacturers are required to always maintain a lower temperature in the production. In addition to low temperatures, the production of probiotics also requires drying technology. Drying low water activity protection technology is actually to reduce the free water in probiotics to a minimum so that they can only maintain the most basic life activities or even dormant state to ensure the basic activity of probiotics.
At the same time, while we take probiotics orally, probiotics will go through layers of our stomach acid, bile, and digestive enzymes to reach the intestines. When taken with the mouth, probiotics will be digested, and few probiotics will reach the intestines, not achieving the real sense of regulating the intestines and maintaining the intestinal flora. Therefore, this further gives rise to probiotic embedding technology. Currently, the common embedding technology includes double embedding technology, multi-layer embedding technology and composite embedding technology. Although the names of embedding technology are different, all of them are wrapped by one or more suitable materials to protect the activity of probiotics and facilitate the smooth arrival of probiotics in our intestines.
The realization and the standardization of the technology further enhance the quality of probiotics, promote the healthy development of probiotics, and provide consumers with correct, safe and effective healthcare probiotics.
Market Size
China's probiotics market reaches 101 billion in 2022, with a compound annual growth rate of 15%, and per capita consumption still has great room for growth. Taobao Tmall probiotics market overall performance has improved. Probiotics market annual total merchandise transaction (GMV) reached 11.94 billion yuan in 2021, with an increase of 23% year-on-year; probiotics category health products as the TOP1 category, GMV reached 6.32 billion yuan in 2021, with an increase of 28% year-on-year in a fluctuating upward trend. In the general diet segmentation track, probiotics grow the fastest. In 2020, probiotic GMV increased by 58%, vitamin increased by 50%, and glucosamine chondroitin increased by 35% year on year, making it the three fastest growing subcategories in TOP10. And in the overseas dietary segmentation track, probiotics are growing fast and in large volume. In the probiotics market, the combination of "probiotics + X" is more popular among consumers. For example, in the female probiotics market, mood-regulating probiotics are often used in combination with cranberries; and the unique innovation of this product is the use of "probiotics + erythritol". The combination of probiotics and sugar substitute is the first product in the market that can regulate the population density of probiotics, help the flora to achieve effective occupancy in the intestinal tract, ensure the diversity of the flora, enhance the body's own immunity and resist the invasion of foreign bacteria.
Fig 6. Global market distribution of probiotic products
Fig 7. Probiotics market size and growth rate
Fig 8. Erythritol consumption per capita in China
Fig 9. Total erythritol sonsumption in China
Potential customers
For the potential customers of this product, we specifically analyzed the values that each type of customer focuses on.
1) Individual consumers: For individual consumers, the economic effect and nutritional value are what most of them value. An economical price can attract consumers to buy the products and make the products competitive in the market. Therefore, we need to control the cost at the early stage of the product entering the market and make the price in marketing as much as possible to attract the customers to open the market and actively promote the transformation of Probiotics for the body benefits. It can also be combined with the use of erythritol to jointly create the image of a low-sugar healthy life in order to attract this type of consumers.
2) Medical field-related customers: engineered probiotic not only can be used for daily diet and health care but also has certain medical value. As a result, some medical field-related customers and companies may use this product to adjust and improve individuals with intestinal flora disorder or secondary infection after using antibiotics through probiotics with the independent ecological niche. Such customers pay more attention to the biosafety of probiotics, such as whether probiotics will put stress on the metabolism of other normal flora or inhibit the growth rate of other normal flora when they colonize the intestine for a long time, and whether probiotics will cause adverse reactions and side effects in the intestine, how serious these side effects are, and whether they will cause serious clinical reactions. In response to this, our probiotics depend on an independent carbon supply and do not pose direct competition with normal flora. The metabolites produced by the modified genetic circuit are completely harmless to the human body. PQQ is a nutrient needed by the human body itself and can enter the human metabolic pathway and be excreted naturally. L-erythrose is a food grade sugar, and its benefits to the human body have been verified, which can be metabolized and discharged by the human body, so the metabolic substances will not accumulate or remain in the human body and have better biological safety.
3) Biomedical companies: The probiotics we used are relatively mature chassis organisms that can be used for exogenous gene expression and biomodification. Biotech companies attach importance to practicality and functionality, and they hope to use probiotics as a more mature engineered bacterial modification chassis with more diverse regulation methods to achieve more complex line design and functional realization. And our product is characterized erythritol-based promoter, which realizes erythritol-induced metabolic gene circuit in probiotic bacteria, making probiotic bacteria become adjustable and providing a basis for more precise metabolic routes, which makes biotech companies more willing to buy.
1) Lack of official agency certification: When probiotics are applied to food, they should conform to the standards and regulatory requirements for food products of corresponding categories, and when applied to special food (health food, infant formula, special medical use formula), they should conform to the relevant national requirements. For products with probiotics added, it is not appropriate to specify the total colony index. If there are existing national standards, they need to be implemented in accordance with the existing standards, and the health function claims of food containing probiotics should also comply with the relevant regulations. Currently, the probiotics we use have passed the FDA's review and been approved for marketing, but for the stricter requirements of the domestic market, we need to be careful to prevent the risk factors in this area. If we cannot pass the national standards and regulations for probiotic products, we will face the risk of being banned from the market, and we will face great pressure in financial and technical aspects.
Countermeasures: Understand the latest audit standards in China, supplement the evaluation of pathogenicity and drug resistance of probiotics, verify the biological characteristics on the existing basis, such as gastric acid tolerance test, adhesion test and human epithelial cell adhesion test and virulence gene analysis, etc., and prepare the audit materials that may be required in time to prevent time urgency and uncertainty in the experiment.
2) Patent was registered: For a biomanufacturing company, the availability of patents with key technologies is the most critical interest protection barrier for the company. The decision on how to protect intellectual property is an integral part of the company's overall strategy to create a competitive advantage in the market and sustainable development. Patents are one of the key leverages for biopharmaceutical companies to build core competitive advantage and monopolize the market and raise funds. Patents require full disclosure of the details of the invention in exchange for exclusive rights for a certain period of time with the government that represents the public interest. If a patent is registered by another company first, it will significantly limit the company's growth and reduce its autonomy, as well as putting great pressure on low costs and incurring unnecessary overhead.
Countermeasure: Adopt a defensive publishing strategy. The publication is one of the pieces of evidence that can destroy the novelty of patent applications. Therefore, the developed technology should be compiled into public data as soon as possible and published in preprint or relevant journals to prove its timeliness in order to prevent other companies from taking the lead.
3) Unable to manufacture on a large scale which leads to higher cost: Although many biomanufacturing companies are able to design modified strains with perfect functions, the complexity of the gene circuit and the tedious procedural process result in a lot of effort and time required to build a strain that can successfully express the product. And due to the differences in raw materials and fermentation methods, setting up a mature production line also requires optimizing production conditions to achieve a yield that meets the requirements of industrial production. Therefore, large-scale production can fully release the production capacity, improve the turnover rate of fermentation tanks and other equipment, reduce the marginal cost, and thus reduce the overall cost.
Countermeasures: Conduct preliminary research on similar companies in the market to understand the production model they use, analyze the greatest difficulties in industrialized production, and actively carry out technical research and model changes.
4) Inadequate exposure of probiotic products: The product model of "Probiotic + X" is gradually gaining attention in the market. For example, mood-regulating probiotics are usually used in combination with cranberry, but its audience is still limited, and its market share is still in the development stage. The market is still in the development stage, and the market is still in a wait-and-see attitude towards the combined use of the product model, and the publicity is insufficient, resulting in a limited audience.
Countermeasures: Produce relevant materials about popular science to educate the public about the advantages of "1+1>2" in this product combination. In addition, we can actively promote the healthy lifestyle of combined use of erythritol and probiotics and take it as a breakthrough to develop potential customers.
5) Low turnover: Turnover constitutes the main income of the company and also reflects the company's operating ability. Low turnover may be caused by several factors, and the decline in turnover will lead to the company facing greater operational pressure.
Countermeasures: Integrate upstream raw material resources, improve bargaining power, or directly increase product unit price.
6) Severe clinical symptoms: Although genotoxicity testing and various clinical tests have been done before marketing, individual differences may still lead to different clinical symptoms after taking the product. Although the possibility of severe clinical symptoms is very slight, once it happens, it will be devastating to the product's image.
Countermeasures: Expand the sample size of the clinical trial, extend the duration of the trial, and observe the differences between the experimental and control groups and their clinical performance for a long time.
Fig 10.
Competitor Analysis
Fig 11.
Partners
We are committed to creating "Probiotics + X" product portfolios. Erythritol, used in combination with probiotics, serves as both an inducer and a general sweetener. Its sales boundary is also the sales boundary of engineered probiotics. The combination of the two can achieve a win-win situation and jointly create a healthy life concept. For the system's long-term development, we actively communicate with other scientific research teams to promote academic and technical exchanges to develop more feasible and safer probiotic chassis organisms.
Driven by the Yuanqi Forest, China's sugar-free beverage market has exploded. The sugar-free beverage market is expected to reach 22.74 billion yuan in 2025. Due to the downstream demand growth, China's erythritol demand is expected to reach 285,000 tons in 2025, with a compound growth rate of 34.15% from 2021 to 2025. The vast sugar substitute market can give ample environment for engineered probiotics, while the market size of the global probiotic industry in 2016-2020 is growing year by year. The global probiotic market size was $42.8 billion in 2016, and by 2020, the global probiotic market size will grow to $56.6 billion, an increase of 7.6% over the previous year. Besides, The "Probiotics + X" product portfolio is still a new concept; coupled with the current probiotic market pattern is still not fixed, innovative product designs contribute to competing in the stock market.
In addition, unlike the functional probiotic products on the market, which use mixtures made of a variety of live bacteria, our products take a single strain. However, the single strain can biosynthesize a variety of useful downstream products through genetic route design, which makes itself function like various live bacteria, saving the cost of isolating and mixing different strains. It makes the price more competitive, the project's investment reporting rate predictable, and the cost payback period shorter and more commercially feasible.
Biotherapy techniques developed based on Nissle 1917 have high clinical application value5, and therapies for a variety of diseases, such as inflammatory bowel disease (IBD), have also been validated6. In 2016, Keimyung University Dongsan Medical Center conducted clinical trials using Nissle1917 for the treatment of intestinal discomfort like constipation, proving that Nissle1917 biosafety is high and meets clinical standards.
Our experiments proved the feasibility of erythritol as an independent carbon source. Bacteria can grow in an M9 medium, which uses erythritol as the sole carbon, demonstrating that engineered probiotics can metabolize erythritol and achieve superior colonization in the gut.
Fig 12. Growth curve in M9 culture medium
The financing stage of the project is initially expected to be the following four phases: initial insider finance, angel finance, venture capital, and public equity market. In the five-year from 2022 to 2027, it is expected to carry out the initial internal financing (seed round) and round angel stage, getting the financial support of the "angel person" at the end of 2027 and starting the venture capital A round financing (pre-A) stage in 2028. The duration of the A, B, and C rounds of financing and the amount of financing depends on the company's operating scale and capital turnover, which is expected to be completed in 2030. After 2030, the project will consider entering the private equity financing stage and make preparations for IPO, Trade Sale, M&A, Mergers and Acquisitions, and buybacks by company management.
Fig 13. Funding strategy
Relying on the IGEM Association of the Sichuan University's Academy of Life Sciences, this project has won 10,000 yuan of the 2021 "Good Doctor Double Creation Outstanding Team Scholarship" of the Academy of Life Sciences of Sichuan University, 2,000 yuan of the "Top 100 Student Collective" of Sichuan University in 2022, and 2,000 yuan of the "Top Ten Student Groups" of Sichuan University in the 2021-2022 academic year as the initial internal financing fund. Besides, the project is also supported by the Academy of Life Sciences of Sichuan University, collaborating for the follow-up experimental tasks. Additionally, we will actively participate in relative forums, exchange meetings, academic science and technology competitions, etc., to seek seed investors who favor the project, obtain their financial and resource support, and strive to raise 1 million yuan of project funds. In the initial internal financing stage, the project is in the laboratory research and development (R&D) validation stage, so the commercial team and marketing model is just beginning to shape but not yet developed practically.
After the initial internal financing is completed, we plan to register a joint stock limited company with a registered capital of RMB 100,000. The entrepreneurial team accounts for 90% of the shares, while 60% are owned by the founder, 30% by the co-founder, and employees of the enterprise own the remaining 10%.
Fig 14. Shareholder Structure
In the angel round of financing, the project product is verified to be feasible, as the erythritol and probiotics joint-use product can be produced, and the commercial team is beginning to take shape. But the marketing model and promotion are still our shortcomings. The project urgently needs to attract a lot of capital investment, and it lacks marketing promotion to attract industry downstream manufacturers and partners. At this stage, we continue to deepen our market network through our core users, expecting to broaden the market sales channels while striving for more funds to integrate. We also seek help from large and medium-sized investment institutions and angel investors by demonstrating the advantages of the project and the "Blue Ocean Market" of the product. Finally, we seek to raise a total of 5 million yuan in financing.
Five million yuan financed in the angel round is expected to be invested in planting equipment acquisition, technology introduction and R&D, product marketing promotion, personnel recruitment and training, etc. The specific distribution of funds is as follows:
Fig 15.
The corporate equity structure has been changed and adjusted to make the angel investors hold about 20% of the shares. At the same time, the shares held by previous shareholders should be adjusted accordingly: the entrepreneurial team holds 75% of the shares, of which 55% are owned by the founders, 20% by the co-founders, and 5% by the employees of the enterprise. The adjusted equity structure is shown in the following figure:
Fig 16. Shareholder structure after adjustment
Since venture capital, project products, business teams, and marketing models have matured. It has a relatively fixed market share in the market for probiotic health products. There are a large number of loyal downstream users and potential collaborators. The operation of the enterprise project has gradually stabilized, as the registered erythritol probiotics joint use related patent trademarks, and with a certain scale of production biopharma plants, some products are widely understood and recognized.
FINANCIAL STATEMENTS
1. Based on the project development in 2022-2025, combined with the financing of internal finance and angel round finance, and according to the development of the health products market scale, the corporate financial data for 2026-2030 are expected to be shown in the following table:
Fig 17.
The above figure predicts the operating revenue, operating costs, operating expenses, and operating profits of the enterprise in 2026-2030. The following figure compares operating income with operating costs separately. It shows that in the first two years, because of the company's inception, Low visibility, low market share, revenue costs, and expenses are at a low level, so make the profits. With the expansion of the company's market share, revenue, costs, and expenses are gradually rising, but the growth rate of operating income is much higher than that of costs and expenses.
Fig 18.
2. The cost projections for the period are shown in the following table:
Management costs consist of two parts, fixed and variable. Fixed parts include depreciation and amortization; variable parts include office expenses, salary benefits, and other expenses.
Fig 19.
Sales costs include labor, advertising, consulting, and other costs, as shown in the following table:
Fig 20.
Financial costs include, inter alia, labor costs, interest on loans, and other expenses, as shown in the following table:
Fig 21.
3. Calculate the current ratio and the debt-to-asset ratio of the enterprise in 2026-2030 by analyzing the financial data listed above. It can reflect the solvency, operating capacity, and profitability of the enterprise from the side, as shown in the table below:
Fig 22.
A detailed business plan is provided.