Brazzein, a sweet protein with outstanding potential

 This article is also available in French: 

More than a mere sense, taste is a tool for assessing food quality and detecting potentially energy-rich molecules. The physiological role of sweetness causes us to appreciate and consume foods that are rich in sugars such as sucrose, glucose, fructose, galactose, lactose and starch. Natural in humans, attraction to sugar is even prevalent throughout animal kingdom, even as far as insects including ants and butterflies.

As part of an overall effort to improve and promote consumer health, the food industry is using less sugar to offer finished products with a lower sugar content. The World Health Organization recommends that adults and children eat less than 50 grams of sugar per day (12 teaspoons) to reduce the risk of  overweight or obesity. According to a CREDOC study (CCAF 2010), the French consumed an average of 100 grams of sugar per day - twice as much as the recommended intake.

There are two distinct strategies for reducing sugar intake. The first is to substitute sugars with sweeteners made with natural or synthetic molecules that have a sweet taste but no or few calories. The second is to reduce the amount of sugar but increase sweetness with flavour enhancers. Sweeteners include aspartame, sucralose, cyclamate and saccharin. Unfortunately, they have a different, not always pleasant, taste to table sugar.

There are, however, other natural molecules that are still rare in human nutrition including certain proteins. One example is brazzein with its strong sweet taste. Brazzein is made from the fruit of a climbing African shrub (Pentadiplandra brazzeana) that is consumed by local populations and monkeys in Gabon. It is extremely sweet but contains no sugar. Brazzein is 500 to 2,000 times sweeter than table sugar and has very valuable properties for the food industry, being stable, easy to digest and with a potential to be used in blends.

INRA research director at the Dijon Centre for Food and Taste Sciences, Loïc Briand gives us his view of this sweet-tasting protein, the complexities for production and significant advantages.  

Brazzein

A special sweet protein

"Proteins usually have little flavour. For example, pure milk protein or egg white have no flavour," he says. Brazzein is one of the few proteins to have potent sweet flavour. What’s more, it has unusual physicochemical properties. "Being very stable at high temperatures, it can be heated at 80°C for 4 hours without deterioration. It resists pH variations and is active in acidic pH, which is potentially useful for beverages or dairy products. The flavour of brazzein is very similar to the flavour of sugar. It is also digested in the stomach, which may help in the fight against diabetes," Loïc says.

A special protein but difficult to source

Brazzein is found inside the fruit of an African shrub that is rare and difficult to grow. As a result, industrial-scale extraction is not possible. But Loïc Briand and his team have found a solution: "Using genetic engineering, we can produce recombinant forms of pichia pastoris yeasts to produce brazzein.” The DNA sequence of a yeast can be modified to produce a protein in its culture medium that it could not previously synthesise. This is called a recombinant protein. “To obtain large quantities, we had to optimise the expression system. We conducted a lot of research into strains and growing conditions. The process must be optimised because it is much more expensive than stevia and aspartame, for example, which are abundant.”  

A controversial but beneficial production

Genetic recombination produces a molecule derived from a genetically modified microorganism. Significant regulatory constraints apply. Products derived from GMOs are covered by EC Regulation no. 258/97 dated 27 January 1997 for new foods and food ingredients. The restricted use of genetically modified microorganisms is governed by directive 90/219/EEC as amended by directive 98/81/EC by the committee for research and industry. Applications must be filed with the European Food Safety Authority (EFSA) to assess the safety of the compound. "Products derived from GMOs are a concern in some countries, including France. It is less the case in the USA and China," Loïc adds.

The protein can be improved using genetic engineering. "As a result, we are able to produce variants that are sweeter than original brazzein," he says. The next step would be to “produce variants of brazzein, with zero sugar content, that increase sweetness when blended with fructose for example.” In that case, brazzein would no longer be a sweetener. It would be an “an ingredient that could be found in beverages”.

Sweet taste

How is it detected?

The tongue is covered with taste buds. They contain receptors that express taste. "Sweet-tasting receptors in humans are composed of two subunits, T1R2 and T1R3, that assemble to form a receptor," Loïc explains. "Sweetness is first detected when sweet molecules dissolve in saliva. The molecules activate the receptor that activates in turn a biochemical signalling cascade called transduction, which stimulates a neurological message in the brain.” 

The synergy between sugar & brazzein

"We discovered that brazzein was able to create synergies with sugar. Adding a small amount of brazzein to fructose results in a more intense sweet taste.” Why? The activation mechanism of the sweet taste receptor is not the same in sugar and brazzein. As Loïc explains, "Conventional sugar and sweeteners are small molecules. Brazzein is 50 times larger." It is therefore impossible for the activation mechanism to be the same, even if we don’t yet understand it completely. By using sugar and brazzein, "we activate both mechanisms which enhances the effect," he concludes. "The next step is to understand the brazzein mechanism."

Pet Food

In addition to their use for human consumption, sweet-flavoured proteins can also be used for pet food. “Brazzein could be used to develop flavoursome products at reduced cost for all animals, ranging from pets to livestock,” Loïc says. "For example, calves and pigs sometimes have difficulty feeding when they are weaned. This may be a useful solution to help them make the transition from liquids to solids." Use of this type of protein can therefore also be studied for use in animal feed.

Keywords

Taste, flavour, sugar, protein, brazzein, plant, sweetener, genetic engineering, yeast, health, diet

Further reading

• L’amertume sentinelle du système immunitaire - article publié dans la revue Science n° 473 – Mars 2017 à télécharger ici 
• Belloir, C., Savistchenko, J., Neiers, F., Taylor, A.J., McGrane, S., and Briand, L. (2017a). Biophysical and functional characterization of the N-terminal domain of the cat T1R1 umami taste receptor expressed in Escherichia coli. PloS One 12, e0187051.
• Belloir, C., Neiers, F., and Briand, L. (2017b). Sweeteners and sweetness enhancers. Curr. Opin. Clin. Nutr. Metab. Care 20, 279–285.
• Poirier, N., Roudnitzky, N., Brockhoff, A., Belloir, C., Maison, M., Thomas-Danguin, T., Meyerhof, W., and Briand, L. (2012). Efficient production and characterization of the sweet-tasting brazzein secreted by the yeast Pichia pastoris. Agric. Food Chem. 60, 9807–9814.