Cell-free protein expression technology and Brazil sweet protein co-plastic health new "sweet" point!

 

 

 

In recent years, the frequent occurrence of epidemics has shown a clear correlation with the increase in sugar intake. Problems such as obesity, diabetes, hyperglycemia and dental caries have emerged one after another. According to the World Obesity Map published by the World Obesity Alliance in March 2023, It is estimated that by 2035, more than 4 billion people in the world will be overweight or obese, accounting for more than half of the world's total population. Obesity can lead to endocrine and metabolic abnormalities, often accompanied by a variety of diseases. As a global public health problem, diabetes is particularly difficult in China. With the improvement of material living standards, diabetes has gradually become a common disease in China. The number of people aged 20-79 with diabetes in China has increased from more than 2000 million in 2000 to more than 0.14 billion in 2021.

 

As people pay more and more attention to healthy diet and the demand for reducing sugar intake increases, sugar-free food is favored by the majority of consumers because of its low sugar and low calorie characteristics. According to the report "analysis of market size and share of sugar-free food and beverage market-growth trend and forecast (2024-2029)" released by Mordor Intelligence, the market size of sugar-free food and beverage is expected to grow from US $19.17 billion in 2023 to US $23.3 billion in 2028, with a compound annual growth rate of 3.98 per cent in 2023-2028.

 

In the rapid development of the food industry, many innovative companies are exploring new ingredients to meet global nutrition challenges. In China, the development of sugar-free food industry is particularly eye-catching. With the improvement of residents' health awareness and the enhancement of sugar reduction demand, the market demand for sugar-free food is increasing year by year. Sugar-free beverages, sugar-free cakes, sugar-free snacks and other products are favored by consumers. At the same time, Chinese sugar-free food companies are constantly innovating to improve product quality and taste to meet the diverse needs of consumers. As a new type of sweet compound, sweet protein stands out for its natural and low-calorie characteristics. Unlike sucrose, sweet-tasting proteins do not trigger fluctuations in insulin in the body. Therefore, they are expected to be an ideal substitute for artificial sweeteners and sugars in the food and beverage industry.

 

At present, there are 7 kinds of sweet proteins that have been studied more: Thaumatin (Somatin), Monellin (Monellin), Miraculin (kiwi fruit), Curculin (thaumatin), Mabinlin (caper), Brazzein (Brazil Thaumatin) and Pentadin (perittin). Compared to chemically synthesized sweeteners, they are safe and non-toxic; compared to other natural sweeteners, they are low in energy. As a substitute for sucrose, they do not cause hypertension, hyperlipidemia, diabetes and other diseases, and have potential applications in the food and pharmaceutical industries. Among them, Brazilian sweet protein is a kind of natural source of high-power sweetener with great application potential, which has attracted much attention because of its unique advantages such as good solubility and thermal stability.

 

 

 

Illustration: Amino acids, functional properties, and biological hosts of sweet proteins

 

Brazilian sweet protein is a kind of sweet protein isolated and purified from the fruit of the wild plant Pentadiplandra Brazzeana Baillon in western Africa, with a molecular weight of 6.5 KDa and a sweetness 2000 times that of the equivalent sucrose. Its advantages over several other sweet proteins are clear, including:

 

1. Has a very strong sweet taste, and its sweetness lasting, pure taste, no metallic taste or other bad taste;

2. High thermal stability and pH stability, and can maintain its sweetness characteristics under different temperature and pH conditions;

3. The molecular weight is small, the water solubility is good, and the aqueous solution still maintains the sweetness after 4h heat treatment at 80 ℃;

4. After digestion, it has little effect on blood sugar, does not cause dental caries, and shows good safety;

5. Can work synergistically with other sweeteners to improve the overall taste and flavor of the food.

 

This makes it potentially useful in the food industry, especially when developing low-sugar or sugar-free foods. In addition, Brazilian sweet protein can also work synergistically with other sweeteners to improve the overall taste and flavor of food. It can be used to make various beverages, candies, cakes and other foods, providing consumers with more healthy and delicious choices.

Therefore, it is particularly important to realize the commercial production of Brazilian sweet protein so that this sweetener can be used on a large scale in the food and beverage industry.

 

 

Figure: Backbone functional area of wild-type Brazzein

 

However, Brazilian sweet protein is isolated from tropical plants, but this tropical plant is difficult to bear fruit in other environments, which limits its development and application. The cost of using chemical synthesis methods is a key issue. The second is the technical difficulties faced in the expression of Brazilian sweet protein. So far, Brazzein have been expressed in microorganisms, fungi, plants and mammals, expression products or low yield, or no sweet taste, still can not achieve the desired effect. In addition, the stability and production process of Brazilian sweet protein also need to be further studied and optimized. Therefore, there is an urgent need for a process that can produce sweet protein on a large scale.

 

Cell-free protein expression technology stands out because of its unique advantages. It is a method of protein synthesis in vitro using the essential components of protein synthesis in cell lysates (such as ribosomes, transfer RNA, aminoacyl synthetases, etc.). Cell-free protein expression technology offers several significant advantages over traditional live cell-based protein expression systems:

 

1. High expression level: The cell-free system is not restricted by cell growth and replication, so all added substrates can be fully utilized to achieve a high degree of protein expression. This high efficiency enables cell-free protein expression technology to produce large amounts of target proteins in a short period of time;

2. The purification process is simple: Since there are no other cell components in the cell-free system, the complicated steps of cell lysis and separation of intracellular components are avoided, thereby simplifying the entire workflow;

3. Flexibility: The CFPS system has good adaptability to conditions and can react stably in a variety of temperature and pH environments. In addition, the protein expression process can be optimized by adjusting the concentrations of added substrates and cofactors to meet different experimental needs;

4. Short expression cycle: The cell-free system saves time for cell growth and protein expression cycle and is therefore more rapid. This rapidity makes cell-free protein expression technology suitable for research and application scenarios that require rapid access to experimental results;

5. Suitable for the expression of special proteins: Cell-free protein expression technology is particularly suitable for the preparation of various types of proteins, including difficult-to-express proteins, toxic proteins, complex proteins, etc.

 

In recent years, cell-free protein expression (CFPS) technology has been gradually applied to the food industry. This makes the large-scale production of Brazilian sweet protein possible, making it more widely used in food, beverage and other industries. Perotin Bio has been committed to the research and development and commercial application of cell-free protein expression technology. It has been deeply cultivated in the industry for many years. Relying on the independent research and development and unique cell-free protein expression technology platform, Perotin has successfully developed the preparation process of Brazilian sweet protein, which can realize the large-scale production of Brazilian sweet protein.

 

References:

Novik TS, Koveshnikova EI, Kotlobay AA, et al. Sweet-Tasting Natural Proteins Brazzein and Monellin: Safe Sugar Substitutes for the Food Industry. Foods. 2023;12(22):4065. Published 2023 Nov 8. doi:10.3390/foods12224065.

Kazemi-Nasab A, Shahpiri A. Expression of Brazzein, a Small Sweet-Tasting Protein in Saccharomyces cerevisiae: An Introduction for Production of Sweet Yeasts. Protein Pept Lett. 2020;27(10):945-952. doi:10.2174/0929866527666200331134431.

Shi Tianyuan, Cao Guoqiang. Research progress of gene engineering expression of sweet protein [J]. Food Industry Science and Technology, 2023,44(2):453 − 459. doi: 10.13386/j.issn1002-0306.2022020262.

Saraiva A, Carrascosa C, Ramos F, et al. Brazzein and Monellin: Chemical Analysis, Food Industry Applications, Safety and Quality Control, Nutritional Profile and Health Impacts. Foods. 2023;12(10):1943. Published 2023 May 10. doi:10.3390/foods12101943

Assadi-Porter FM, Abildgaard F, Blad H, Cornilescu CC, Markley JL. Brazzein, a small, sweet protein: effects of mutations on its structure, dynamics and functional properties. Chem Senses. 2005;30 Suppl 1:i90-i91. doi:10.1093/chemse/bjh128