2 The anthocyanins biosynthesis cell system (ABC System)

Due to the unstable source of raw plant materials, the occupation of limited farmland and the great quantity of production waste that coursing environmental pollution and waste of resources, the scale of the current anthocyanin industry is serious limited. The yields for this industry are far from meeting consumer demand. fortunately, our anthocyanins biosynthesis cell system could completely solve these problems.

Structure of ABC system

The ABC system mainly consists of two parts of work. The first part was the transgenic induction of high-yield anthocyanin carrot hairy roots, which could be supplied by professional biological laboratories. The second part was the in-vitro culture of hairy roots and the induction for anthocyanins synthesis. This part was the main object to expand for the mass production of anthocyanins, including the following basic equipment and conditions:

(1) Equipment and materials: a) Flask; b) Shaker; c) Liquid Ms medium and d) β-estradiol.

(2) conditions: a) 25oC; b) Dark and c) 90 rpm.

We could obtain high yield of anthocyanins following these basic steps:

a)Incubate the hairy-root tips in a shaker protected from light at 25oC and 90 rpm with weekly changes of medium.

b)After about 30 days of incubation, add β-estradiol solution and make sure it reaches a final concentration of 2 μM.

c)Continue to culture the hairy roots for 120 h in a shaker for anthocyanin synthesis.

Target Users

Anthocyanin manufacturers will be the largest target users of the system. For this system, the raw material for anthocyanin production were carrot hairy roots that not only had much higher contents of anthocyanins, but also could be rapid proliferated and continually grown in vitro and left much less waste during the anthocyanin producing progress. As the result, more and more anthocyanin production enterprises would choose this system to pursue for greater economic benefits.

Application

The carrot transgenic hairy roots in vitro kept proliferating stably and continually. Through subbreeding by Cutting into a lot of small segments, a short hairy root could grow into sufficient amount of qualified raw materials. Furthermore, the grow conditions of these hairy roots were easy to reach as the can survive well in a simple MS liquid medium at room temperature and in dark. Thus, the anthocyanins biosynthesis cell system could be easily amplified in factory floor.

Safety and possible challenges:

As the transgenic media, both E. Coli and Agrobacterium rhizogenes strains with containing target genes was used for Explant infection in the early stage of the system construction. Therefore, some people worried that hairy root disease will be caused to plant outside through the spread of these bacteria. In fact, it was unnecessary. Any waste from the project related to organism including E. Coli, Agrobacterium rhizogenes and hairy roots will be autoclaved before dumping and no hairy root disease will be caused outside.

People’s another concern is the environment pollution caused by chemicals used, because the chemical beta-estradiol was added into the liquid medium to induce anthocyanin biosynthesis in hairy roots. The fact was that beta-estradiol was low toxic and would irritate the skin, mucous membranes and eyes and make them itch. Meanwhile the quantity of beta-estradiol used was quite low and only 2 μM as the final concentration. The low concentration of beta-estradiol could be effectively removed with the conventional wastewater treatment.

Modeling

We had investigated and predicted the yield of anthocyanins after adding different amounts of β-estradiol to find out the optimum amount of β-estradiol to increase the yields by modeling based on the gradient experiment data.

With our fitted modeling, it indicated that we could convincingly conclude that a higher dosage of inducer will only further increase the production of anthocyanin in a small amount if not at all. And if to put this into real world scenario, such model will also help us figure out the best production-to-investment ratio within plausible range. From the point of view of increasing production, we found that the optimum amount of β-estradiol to increase the yield of anthocyanins was about 10 μM.

Future development

In the future, while promoting the expanded application of this system, we will further improved soem high yield, stability, durability and environmental adaptability cell lines of this carrot hairy root system by means of genetic modification and synthetic biology, and change and optimize category composition of anthocyanins. At the same time, it will be committed to developing a new production system with different plant cells as chassis to meet the different needs of anthocyanin production and consumption.

In addition, although the modeling result advised us to choose 10 μM. Of the final β-estradiol concentration to induce anthocyanin biosynthesis, there question we should answer with more future experiments - Whether the carrot hairy roots survival in this high β-estradiol concentration and whether it will cause much environment pollution?

2 A protective phytochemical quantifier (PPQ) for anthocyanins

The isolated anthocyanins are highly instable and very susceptible to degradation (Giusti & Wrolstad, 2003). Their stability is affected by several factors such as pH, storage temperature, chemical structure, concentration, light, oxygen, solvents, the presence of enzymes, flavonoids, proteins and metallic ions (Rein, 2005). Besides, of potential importance to human health is the relatively high concentration of anthocyanins in the diet. The daily intake of anthocyanins in the U.S. diet is estimated to be between 180 and 215 mg whereas, the intake of other dietary flavonoids such as genistein, quercetin and apigenin is only 20–25 mg/day (Hertog et al., 1993), and in China, it was currently defined a specific proposed level of 50 mg/d for anthocyanins (Chinese Nutrition Society, 2013). To help our users to use our anthocyanin products in a more manageable way, we designed a storage tank for anthocyanins, protective phytochemical quantifier (PPQ), which was convenient for people to weigh anthocyanins for every day.

Structure of PPQ

Our container provides a perfect environment to store our anthocyanin products, especially in Shanghai, the subtropical monsoon zone, where the air is humid and warm. Our device was mainly composed of the following parts:

(1) Tank: The tank was divided into three functional areas. a) Outermost layer, to insulate the heat. b) container, to storage anthocyanins. c)Silicone desiccant, to maintain the low humidity inside the container.

(2) Controling temperature and humidity: With the aid of a temperature and humidity sensor and a cooling plate, it was able to monitor temperature and humidity inside the tank.

(3) Pouring out the powder: A stepper motor was used to rotate the screw so that the powder can be rotated out bit by bit with a PID algorithm applied to control the speed.

(4) Quantifying: The measuring structure was divided into upper and lower layers, which is similar to a coffee machine with a gravity sensor under the structure.

Target Users

Anthocyanins are natural, non-toxic food colourings and also a good type of health care products and they could be used and intaken by everyone every day, especially for the people who prepare diet.

Application

Consumers could use our PPQ to storge anthocyanin powder products for quite a long time to protect them from light, high temperature and high humidity. They could easily measure out the precise quantity of anthocyanins they needed every time.

When people needed to measure the quantity, they could input the value of the powder quantity they wanted and the system would automatically measure precisely the amount of powder.

Safety and possible challenges:

All material used that had contact with anthocyanin wer good for food safety. The wires, DC power supplies, and step-down modules we used to build the circuit were all market currency versions that had been checked for proper use. Under the condition that the circuit input and output were well connected. We used the step-down module to step down the battery voltage. The frequency of the voltage was within our calculation range.

Future development

PPC is currently too big for children and patients. We would like to further our inclusivity consideration in the future by developing our home-based anthocyanin storage device into a smaller, more portable one.

Additionally, the software can be adjusted to control the inner environment of the tank to fit into the preferred ones of any powder products. Changing the innermost tank and delivery system, we are able to store and measure any form of products with such single device.