2. The questionnaire

Our team released an online survey and interview participants offline that collected 264 results in total, including 150 females and 114 males. The survey contains 17 questions and aims to explore several questions. First, are people aware of aging and its possible effects? Second, will people actively control their diet, exercise or choose anti-aging products to resist the effects of aging? Third, will an anthocyanin food additive suit their using habit and if not, what other forms can anthocyanin be in? Based on these questions, we analyzed the survey results

In the reported data, 75% of the participants are concerned about aging (Fig 2-A). However, only 50% of the participants have paid attention to food additives (Fig 2-B) and about 40% of participants have never heard of anthocyanin (Fig 2-C) only 21% of participants have intentionally consumed extra anthocyanin before (Fig 2-D). Among the methods provided to prevent aging, a healthy diet and improvements in nutrition are the most popular ones (Fig 3).

3. Experts interview

In addition to the perspectives reported by the public, we also collaborated with a variety of stakeholders including hospitals, nutrition companies, and local people to lay the foundation of our products.

We communicate and consult with many scholars in the field of synthetic biology and related people in the industry:

Yuanbai Liu (Fig 4), CEO of Dr. Laser Industry, Shanghai.

Jie Zhang (Fig 5), College of Food Science and Technology, Huazhong Agricultural University, MSc in Food Science; Henderson (Shanghai) Daily Health Products Company

Yueling Liu (Fig 6), Royal Beauty and Slimming Consultant

Yuqing Qin (Fig 7), Administrative Service Centre, Shanghai Municipal Administration of Market Supervision, Master of Food Science, Ocean University of China

Hongxia Wang (Fig 8), Ph.D., Professor of Shanghai Chenshan Botanical Garden (Mainly conducted research on the function and modification of sweet potato secondary metabolites such as anthocyanins and resveratrol.

Yan Zhong (Fig 9), Ph.D., Nutrition and Food Hygiene, West China University of Medical Sciences

We gained valuable information about different aspects of anthocyanin and other antioxidants with the aid of all professionals mentioned above.

(1) Consumer demand and types for anti-aging products

(2) Anthocyanin's ability of anti-aging

(3) dosage forms of anthocyanin products

(4) Synthesis and production method of anthocyanins

(5) Safety Concerns

3.1 Consumer demand and types for anti-aging products

For the consumer demand, Mr. Yuanbai Liu, CEO of Dr. Laser Industry, Shanghai told us that the demand for anti-aging products is very high and is increasing rapidly. The primary type of products that attract the audience’s attention is products that have anti-aging effects on the skin.,

In addition, Mr. Liu explained to us about the main types of anti-ageing products available in the market today. Through our discussion and further investigation, we discovered that the mainstream anti-aging products were:

(1) serums made from ingredients extracted from plants

(2) laser scintillation, i.e., the use of lasers to stimulate the skin cells to repair;

(3) fillers, such as skincare injections;

(4) rejuvenation materials, which are used to activate cell rejuvenation and are more effective than diuretic acid.

For anti-aging products taken internally:

(1) the first major category is anti-native/antioxidant;

(2) the second major category is NMN (β- nicotinamide mononucleotide), which can supplement NAD+ (nicotinamide adenine dinucleotide), a coenzyme that is in great demand by human beings.

3.2 Anthocyanin's function of anti-aging

Ms. Jie Zhang also told us that of chemicals with anti-aging properties. Anthocyanins were antioxidants, and the effects of most antioxidants were compared to those of the primary, most common antioxidants, such as vitamin C and vitamin E. In comparison, anthocyanins have a medium to high antioxidant effect, thus demonstrating a good level of anti-ageing. As for the verification of their anti-ageing power, experts said that it varied from person to person, and that the effect of anthocyanins was more pronounced in people with higher absorption capacity.

After interviewing Ms Yueling Liu, we were informed of the advantages of anthocyanins over other substances with anti-ageing properties, such as vitamin E and vitamin C, as described in the table below.

In terms of marketing, we learned that many of our customers also take anthocyanins. Anthocyanins were particularly effective for people who stayed up late at night, had dark circles under their eyes and had wrinkles. Some of our customers noticed a reduction in dark circles and a certain degree of refinement and brightening of the skin after taking anthocyanin products. This showed its excellent antioxidant properties.

3.3 dosage forms of anthocyanin products

From Ms. Yunqing Qin, we got information about the current dosage forms of anthocyanin products. Anthocyanin preparations were available in liquid, tablet, and capsule formulations. Depending on the formulation, they could be divided into single-formula products and compound-formula products. Although the content of anthocyanins in complex formulations was slightly lower, the targeted addition of other nutrients allowed the product to achieve its stated efficacy. The liquid form was characterized by fast absorption and short shelf life, making it suitable for people with fast-paced work life and the elderly but not easy to carry. Tablets were generally characterized by the addition of a disintegrant and include swallowed tablets and chewable tablets, which are often flavored and easy to take. Capsules were easy to take, but they may cause some stomach discomfort. This was due to the coloring of the product, which was more common in capsule products, where water and other oils were added to the capsule, reducing the effectiveness of the product's ingredients.

3.4 Synthesis and production method of anthocyanins

Before, we learned from Ms. Jie Zhang that the techniques used to create anti-aging products includes of organic solutions, membrane ultrafiltration, supercritical carbon dioxide extraction (more common), chemical synthesis, fermentation. And then, what techniques would be chosen for anthocyanin production. For this purpose, we visited Dr. Hongxia Wang and Ms. Yan Zhong.

The experts said that in contrast to extracting anthocyanins from various plants, biosynthesized anthocyanins were more stable and consistent. Anthocyanins were a family of flavonoids and consist of many variations in different species. For example, there were more than 20 anthocyanins in purple potatoes, but only five or six in purple tomatoes. In our design, the type of anthocyanins synthesized were determined by the genes that introduced the modified plant. Though the extraction techniques remain the same as the traditional ones, the synthesized single type of anthocyanin was easier to be isolated than in the traditional production, which had several types of anthocyanins extracted at the same time.

3.5 Safety Concerns

A series of interviews were conducted with professionals in order to ensure the biosafety of the biosynthetic anthocyanin process, to understand the possible negative effects of excessive intake and its advantages over other anti-ageing substances, with the following results.

(1) Biosafety

In terms of biosafety, our main concerns were the biosafety at the time of production and the safety of the final product. After interviewing Professor Hongxia Wang, we were informed that as the production uses plant cells, not viruses, the risk of leakage of the sump cells used in the extraction process to the outside environment was low and the probability of survival in the outside environment was low. This ensured to a certain extent the safety of the anthocyanins themselves when we synthesise them. The main concern about food safety in general was the perception that GM poses a risk to humans, but as we only used treated cells in the production process, the impact was not significant.

(2) Possible contamination

In terms of possible contamination from extraction, Professor Wang replied that there was a risk of contamination of the outside environment if organic solutions (such as methanol) are used, but this could be mitigated or even eliminated by recycling or using other solutions.

(3) Safety of the final product

In terms of the safety of our final product, experts believed that the risk was low due to the extremely low probability of environmental contamination caused by plant cells and the ease of degradation of the anthocyanins themselves. And although all other water-soluble substances were raised with the anthocyanins during the purification process in the crude extraction and may be harmful to humans when used directly, subsequent further purification could solve this problem.

(3) Intake of anthocyanins

Regarding the intake of anthocyanins, although consumption of anthocyanins did not cause skin discolouration, they were natural food colours and are not completely non-toxic, so excessive intake was not recommended. Experts advised that they needed to be used with proper intake and reasonable consumption to avoid discomfort. To sum up, we needed to test the anthocyanin content when we finally produced the food colouring and labelled it to remind our customers not to consume too much.

4. Project Implementation 

4.1 Selection of plant chassis cells

At the beginning, we plant to use tobacco cells as the chassis cells for anthocyanin biosynthesis, but we still had the question whether it was suitable as tobacco was not a plant that could be eaten directly.

For the design and refinement, we sought advice from experts on the selection of plant chassis cells, anthocyanin extraction, and the form of the product. For the selection of plant chassis cells, Dr. Hongxa Wang felt that our instructors were more familiar and experienced with such a system and had done similar experiments before. Compared to traditional tobacco cells, carrot cells do not contain alkaloids, so there would be no risk in the consumption of the product.

Therefore, based on the suggestion of Dr. Wang, our team decided to use carrot cells as plant ground cells.

4.2 Dosage form Selection of Anthocyanin Anti-aging Products

At first, we had planned to make our anthocyanin products into capsules, so that we could encapsulate the anthocyanin stock solution directly in the capsules. When we took the capsules, the anthocyanin stock solution could be quickly and directly absorbed by the intestinal tract.

After the interview with Ms. Yunqing Qin, we changed our initial mind. According to Ms. Qin, Capsules are easy to take, but they may cause some stomach discomfort. For the other dosage forms, they could also course problems: The liquid form was characterized by fast absorption and short shelf life, making it suitable for people with fast-paced work life and the elderly but not easy to carry. Tablets were generally characterized by the addition of a disintegrant and included swallowed tablets and chewable tablets, which were often flavored and easy to take. As for the complex formulations, though the content of anthocyanins was slightly lower, the targeted addition of other nutrients allowed the product to achieve its stated efficacy.

For this reason, we had adjusted from the initial choice, the capsule type, to a powder form as a type of food colourings. This was because, based on the public questionnaire, we found that the public preferred a harmless food additive and that its anti-aging effect made it more competitive, and that food colouring was more acceptable to the public than health supplements, plus the powdered form was a concentrated process that could be diluted to the target concentration more easily.