1. Introduction

Soybean isoflavones (SI) are a bioactive substance with many effects, such as estrogen-like effects, anti-oxidation, and cell cycle regulation. It protects against osteoporosis, hardening of the arteries, and breast and prostate cancer, among other diseases. We found that equol (Eq), a metabolite of SI, has higher biological activity. However, only a tiny percentage of the population can metabolize SI into Eq, influenced by the intestinal environment, host genetics, and diet. Some bacteria in the gut microbiota can metabolize SI into Eq, but only a few people can synthesize Eq through gut bacteria. Therefore, it is of great significance to develop Eq into health products through synthetic biology technology to promote the health of non-EQ-producing populations and prevent diseases. 

Bacteria capable of degrading SI and its metabolites have been isolated, but achieving large-scale industrial production remains challenging. It is of great significance to design efficient multistage enzymatic chain reactions to improve the efficiency of Eq biosynthesis and to produce Eq as a dietary supplement nutrient in large quantities. This will help prevent diseases after menopause or old age and treat conditions such as premature ovarian failure and polycystic ovarian syndrome. Therefore, we have tried to build an efficient and safe system for Eq production, and for this purpose we conducted several kinds of human practice activities, as shown below, to answer these questions and better design our project.

2. Ground research

Prior to commencing the HP&IHP design, we conducted essential preliminary research, which involved reviewing relevant literature on synthetic biology, examining academic papers, studying Chinese health product industry standards, as well as familiarizing ourselves with applicable laws and regulations.

We obtained information on Chinese laws and regulations regarding health products through thorough research on government websites. Furthermore, during the course of our project, we had the opportunity to interview experts who provided us with valuable insights and relevant legal documents. These documents not only helped us gain a comprehensive understanding of the current state of health product development requirements but also served as a source of inspiration by highlighting the societal needs that our project aims to address.

Health food regulations: Eq health products belong to the category of health food, and are subject to the supervision of relevant regulations such as the Food Safety Law of the People's Republic of China and the Management Measures for Health Food. These regulations provide for the definition, classification, production, marketing, and labeling requirements of health foods.

Registration and filing: According to the Measures for the Administration of Health Food, the production and sale of health food requires registration or filing. Specific requirements include product safety assessment, production process, and quality control requirements.

Labeling and advertising: The labeling and advertising of health care products must comply with the provisions of the "Measures for the Administration of Health Food Labeling and Advertising". Labels and advertising should truthfully and accurately describe the efficacy of the product and the people for whom it is intended, and should not involve false claims or misleading content.

Nutritional supplements: Eq supplements may also be classified as nutritional supplements. According to the Measures for the Administration of Nutritional Supplements, nutritional supplements need to be documented and clearly declared as nutritional supplements on labels and advertisements. Detailed information is shown below.

In addition, through ground research, we collected Eq and SI-based health products currently available in the Chinese and world markets and analyzed the prices, application values, and usage of the corresponding products.

3. Survey

We wanted to understand our country's current health problems and comprehensive loss of estrogen abnormalities, including menopause-related problems and other estrogen disorders. By collecting relevant data and information, we can assess the impact of these issues on individuals and society, and inform the formulation of relevant policies and the provision of appropriate health services. So, we brainstormed and discussed in groups to form an initial 20 survey questions about female menopause and the symptoms associated with it. These included questions about whether the women themselves and other women in their families were going through menopause, the clinical symptoms they or their families experienced during menopause, and the impact of menopause on their lives. We expect that these questions will provide a preliminary understanding of the current rate of menopause and the severity of its symptoms among women in China.

In addition, we also want to understand the public awareness of estrogen analog health care products and market demand. These supplements may include products that supplement estrogen or other products with similar effects. By understanding the public's perception and demand for such products, we can better understand market demand trends and provide references for relevant companies and institutions to meet the public's health needs. Using the same methodology as above, we also initially designed about 20 questions to find out about currently known menopausal health products and their purchasing desires.

After a preliminary discussion, we agreed that an excessive number of questions had a non-negligible impact on the authenticity and effective completion rate of the survey. Therefore, we decided to reduce the number of questions and improve their quality. By combining these two purposes into one, we can collect relevant data and information in the same questionnaire to get a comprehensive picture of the abnormal estrogen health problem and the estrogen-analogue health supplement market in China. This helped further research and improved the provision of health services and products in related fields.

Characteristics of participants

Finally, we collected 301 questionnaires through WJX (a Chinese online survey platform). Among them, more than 60% of participants are female and the majority are aged under 45 years. Although, more than 30% of the participants were in perimenopause.

Results of the questionnaires

1. Most people are affected by menopause but do not try to relieve it with menopausal products. And most of them have never heard of Eq products. And most of the people who know about equol products just know, do not understand.

2. Nearly half of women are willing to buy our products, and nearly half of them are willing to share our products, which is very beneficial to our products, indicating that our products are relatively acceptable to the new buyers.

3. Most people accept the annual consumption of less than 5000 yuan in health care products, so our products are mainly economical.

Reflections

Overall, the current symptoms of menopause in women are mainly mood swings and sleep disorders. Daily work and life are affected to varying degrees. However, the vast majority of women seldom link these symptoms directly with menopause, and most of them think that it is because of the pressure of life, so few women will choose menopause health products or drugs to alleviate the symptoms. According to the survey, most women understand or even do not accept the use of menopausal supplements, so it is very important to promote awareness of women's menopausal health, and there is also a potential broad market for our project goal.

4. Stakeholder interviews

(1) Interview with Dr. Kaming Xue

Dr Xue is an attending physician from the Department of Integrative Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Dr. Xue gave us a brief introduction to synthetic biology techniques to construct efficient translation promoters for more efficient production of equol by E. coli. Equol is a metabolite of soy isoflavone, which has a high estrogen-like effect and can be used to help the treatment of menopausal women or estrogen-dependent diseases.

Through this interview, we have gained some reflections:

1. Now there is hormone replacement therapy, it is best to use a combination of non-hormonal drugs and hormone replacement drugs, which is more effective for relieving menopause.

2. Some families spend about 300 to 1,000 yuan per month on menopause relief. The specific situation needs to be judged according to the economic conditions of the individual family, and cannot be directly concluded.

3. For example, the incidence of breast problems, excessive or too little milk secretion, breast cancer, uterine atrophy, secondary sexual characteristics, external reproductive organ dysplasia, osteoporosis, and other populations are all related to female hormones and the need to supplement estrogen.

(2) Interview with Dr. Qi Li

Dr. Li is an associate professor at Sichuan Normal University, and her main research interests include microbial genome editing, microbial resource mining for Sichuan wine brewing, and microbial synthetic biology. Through the interview with Dr.Li, we learned that the commonly used genetic engineering techniques in bioengineering and production include gene knockout, carrier introduction, metabolic engineering techniques, genomics, and transcriptomics. However, there are some challenges and limitations in this area. One challenge is the lack of tools to perform gene editing on certain microbes, which limits the modification and application of engineered strains. To overcome this, researchers are constantly exploring new methods of gene editing and considering alternative chassis cells for expression and synthesis. Another challenge is the cost of growing bacteria, which can be expensive. To solve this problem, optimizing the medium can help reduce the cost. Safety is also a concern when using engineered strains in the final application scenario. To ensure safety, the information in the edible strain FPA file can be utilized, and the strain can be engineered to include DNA that does not naturally occur. Finally, the modified strain should be handled safely and scientifically. Finally, bacteria that have been modified through genetic engineering techniques do not exist in nature, so what to do with them during and after the experiment to ensure their safety for the natural environment as well as the biosphere is very important, and a core safety issue for the partners in the wet lab group to consider.

(3) Interview with Dr. Wu

Dr.Wu is known as a nutritionist of the Hong Kong Nutrition Society, a Registered Dietitian of China, a Member of the Chinese Nutrition Society, Senior Public Nutritionist of China. Dr.Wu thought scientific research supports the potential benefits of Eq on human health, including its anti-inflammatory and antioxidant effects, as well as positive effects on bone health and cardiovascular protection. However, there are potential risks and side effects associated with Eq and SI products, such as high estrogen levels, elevated equol activity, and potential stress on liver and kidney function. In the field of nutrition, products aimed at relieving menopausal symptoms in women mainly include herbal remedies, including SI. However, due to regulatory restrictions, these products cannot directly claim to relieve menopause symptoms in their marketing claims. Some established products in this category include Madame Oral Liquid and Viterkinen menopause capsules. In recent years, more and more foreign products have appeared, often in the form of dietary supplements, but they still rely on Chinese herbal extracts. In the healthcare industry, pricing strategies for marphenol-based products or soy isoflavones take into account factors such as research and development costs, marketing expenses, comparison with domestic and international products, data, and user feedback. Research and development costs include safety, independence, and functional testing to ensure product reliability. The marketing cost of Marmol products is higher because of the need for market education in a relatively niche market. The price can be determined by comparison with similar products on the market. In addition, we utilize extensive testing and user feedback to evaluate the effectiveness of our products and adjust our pricing strategies accordingly.

5. Business Plan Presentation

After gaining a preliminary understanding of equol health products and their effects through questionnaires and interviews, we conducted a preliminary business analysis of our products and wrote a business plan. We presented our business plan to professional business investors. We presented our business plan to Dr.Wu, which mainly included the results of the pestle analysis, SWOT analysis, and 4P analysis. After that, we also interviewed Dr.Wu about cost planning, merchandise sales, and promotional design.

Some important advice on our business plan (Please click here to check the Entrepreneurship):

(1) At present, the market has more products of the same type or the same efficacy, highlighting the advantages of the ingredients or efficacy is very important to the core competitiveness.

(2) It is necessary to consider the regulatory policies of domestic and foreign markets. At present, the policies on food and health products in mainland China are very different from those in other regions, and products targeting mainland China cannot claim additional efficacy, so it is worthwhile to think about how to demonstrate their competitiveness under the existing laws.

(3) Positioning of product price and assortment is very important, and it is necessary to design different products at different prices for different age groups and different income levels. Therefore, the concentration of ingredients and the efficacy of the corresponding products need to be further researched, designed, and examined.

6. Conclusion

(1) As the public's awareness of health increases, the issue of women's menopausal diseases and estrogen-related disorders has gradually come to the attention of the public, and how to efficiently and safely promote women's health is a serious health consideration.

(2) Eq is an effective food-derived estrogen analog that has been proven through scientific research and practical clinical application to help women relieve menopausal symptoms and slow down aging.

(3) Currently, part of the population is unable to synthesize Eq by autogenous metabolism, while the traditional chemical synthesis method of this substance is inefficient and relatively costly, so constructing an efficient and safe biosynthetic pathway of Eq through synthetic biology technology is an effective solution to the production and thus the promotion of women's health.

(4) The safety risks and potential natural damages of synthetic biology technologies are an issue worth thinking about, and how to further safeguard the environmental damages of the technologies is the focus of our experiments that need to be further designed and considered in the future.

Our project tried to build an efficient and safe system for Eq production, to achive industrial production. Next, we aim to produce Eq product disigned for middle-aged and elderly people mainly.

Moreover, our product fall into two main categories of capsules and granules, which is already known and accepted by the public.

As for implementation of our project, we plan to sell and promote on both online and offline locations. While, cooperation with large companies will be a great method as well.

6. Reference & Ground research List

Cameron, D. E., Bashor, C. J., & Collins, J. J. (2014). A brief history of synthetic biology. Nat Rev Microbiol, 12(5), 381-390. https://doi.org/10.1038/nrmicro3239 

Chen, K., Lang, H., Wang, L., Liu, K., Zhou, Y., & Mi, M. (2020). S-Equol ameliorates insulin secretion failure through Chrebp/Txnip signaling via modulating PKA/PP2A activities. Nutr Metab (Lond), 17, 7. https://doi.org/10.1186/s12986-020-0426-8

Cubillos-Ruiz, A., Guo, T., Sokolovska, A., Miller, P. F., Collins, J. J., Lu, T. K., & Lora, J. M. (2021). Engineering living therapeutics with synthetic biology. Nat Rev Drug Discov, 20(12), 941-960. https://doi.org/10.1038/s41573-021-00285-3

Deng, H., Gao, S., Zhang, W., Zhang, T., Li, N., & Zhou, J. (2022). High Titer of (S)-Equol Synthesis from Daidzein in Escherichia coli. ACS Synth Biol, 11(12), 4043-4053. https://doi.org/10.1021/acssynbio.2c00378

Sekikawa, A., Wharton, W., Butts, B., Veliky, C. V., Garfein, J., Li, J., Goon, S., Fort, A., Li, M., & Hughes, T. M. (2022). Potential Protective Mechanisms of S-equol, a Metabolite of Soy Isoflavone by the Gut Microbiome, on Cognitive Decline and Dementia. Int J Mol Sci, 23(19). https://doi.org/10.3390/ijms231911921