Team:CDHSU-CHINA

Nowadays, it is nearly impossible for the patients with diabetes mellitus to enjoy the sweat foods, and our project is designed to solve that problem. So far, there is one thing that could help us reach our purpose-- Synsepalum dulcificum. The key is that the Miraculin in the Synsepalum dulcificum Could turn the taste of sour foods to sweat briefly, allowing patients with diabetes mellitus to enjoy the sweat taste. However, the current technology couldn’t make the collection of Miraculin from the Synsepalum dulcificum easy and efficiently. Our goal is to compound the Synsepalum dulcificum protein by using genetically modified technology, and we believe that the new compound method could increase the quantity of the Miraculin and decrease the cost of production, with the intention to help diabetes mellitus patients.
In the whole world, people prefer eating dessert and other food contain sugar. Thus, the proportion of diabetics increase obviously and the chance to get diabetes also rise. Treating diabetes takes long time and complicate series of periods.

Why Lactobacillus?

Lactobacillus, a kind of probiotic bacteria that allows carbohydrate to ferment and produce lactic acid, is generally used to produce various food products such as yogurt, cheese, and beer. Lactobacillus could contribute to the digestion process of human and help to keep digestion system healthy, so it is viewed as health-improved bacteria. Compared to E.coli, lactic acid bacteria is edible, so it is more ideal to use lactic acid bacteria instead of E.coli to be host bacteria.

What are Miraculin?

Miraculin is a glycoprotein with taste-modifying activity and is isolated from miracle fruit (Synsepalum dulcificum), a shrub native to West Africa. The protein can modify human's taste and turn a sour taste to a sweet taste. The mature peptide of miraculin consists of 191 amino acids and has taste-modifying activity in the forms of dimmers or tetramers, but not in monomers. Two histidine residues, exposed on the outside of the protein, are the main functional groups. In addition to the taste-modifying activity, it can also improve sensitivity of diabetic animals to insulin.

Why use Miraculin?

Mystery fruit flesh contains rich vitamins, citric acid, potassium, iodine and other substances, and its seeds are rich in natural mineral elements such as calcium, magnesium and potassium [1]. The reason why the mysterious fruit attracts people's attention is mainly because it contains a special kind of sugar protein, miraculin. This mysterious element cannot change the taste of food, but it can change people's taste. After eating the mysterious fruit, eating the original sour food, such as lemon, will become aromatic and sweet. Therefore, the mysterious fruit can be used as an additive in the food and pharmaceutical industries. It can solve the dietary bottleneck of diabetics who cannot eat sugar and those who do not want to eat sugar.

References

PAN Liping, YU Sili, LI Haihang . College of Life Sciences, South China Normal University, Guangzhou 510631, China 1994-2015 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
[1] Chen Ping, Yang Tongshun, Wang Huan. Guangdong Agricultural Sciences, 2008(3): 26-28.
[2] Paladino A, Costantini S, Colonna G, et al. Molecular modeling of miraculin: Structure analysis and functional hypotheses [J]. Biochem Biophys Res Commun, 2008, 367(1): 26-32.
[3] Ito K, Asakura T, Morita Y, et al. Microbial production of sensory-active miraculin[J]. Biochem Biophys Res Commun, 2007, 360(2): 407-411.
[4] Nakajo S, Akabane T, Nakaya K, et al. An enzyme immunoassay and immunoblot analysis for curculin, a new type of taste-modifying protein: cross-reactivity of curculin and miraculin to both antibodies[J]. Biochim Biophys Acta, 1992, 1118(3): 293-297.
[5] Theerasilp S, Hitotsuya H, Nakajo S, et al. Complete amino acid sequence and structure characterization of the taste-modifying protein, miraculin[J]. J Biol Chem, 1989, 264(4): 6655-6659.
[6] Tsukuda S, Gomi K, Yamamoto H, et al. Characterization of cDNAs encoding two distinct miraculin -like protein and stress -related modulation of the corresponding mRNAs in Citrus jambhiri Lush[J]. Plant Molecular Biology, 2006, 60: 125-136.

Experiments

Current experiments

A recent study by Paladino et al. [2] showed that the monomers of miraculin are inactive at any pH, whereas the dimers and tetramers exhibit an activity to change their taste at acidic pH. Molecular dynamics simulations at different pHs show that dimers exhibit a high degree of open configuration at acidic pH, which is consistent with other investigators' hypotheses. They also conducted mutation experiments to show that out of all the amino acid residues that make up the mystery, two histidine residues that are located in prominent parts of the protein structure are the active sites for their function. Ito et al. [3] also demonstrated through mutation experiments that Histidine-30 is an important site of mystery activity. Although mysterious fruit is a sweet protein, research shows that its amino acid sequence and protein structure are different from other sweet proteins (thaumatin, marrow, mabinlin, etc.). Nakajo et al. [4] also demonstrated that anti-stodotin antibodies are almost non-resistant to myosin by ELISA. The amino acid sequence extracted by Theerasilp et al. [5] has high similarity with the amino acid sequence of soybean trypsin inhibitor. Tsukuda et al [6] cloned two types of myogenic proteins, whose amino acid sequence is similar to that of miraculin. They all have a soybean trypsin inhibitor family module, but the function of proteins is quite different from that of mystery protein.

OurExperiment

Production of Mystery Fruit by Genetically Modified Lactic Acid Bacteria

Our goal is use genetically modified lactic acid bacteria to compound miraculin. At present, the extraction and separation of the mystery fruit is still limited to a small amount of operation. In response to this, we’ve devised an approach to solve such problem. Mystery fruit contains mysterious fruit protein, which has high extraction cost and low yield. The use of genetically modified lactic acid bacteria to synthesize mystery fruit protein. Compared with E. coli, the extract is safer, can be used directly, and has economic value: Mystery fruit can be used as an additive. It can be used in food and pharmaceutical industries to solve the problem of not using sugar. Dietary bottlenecks for diabetics and dieters who are unwilling to enter the diet.