Thaumatin – an intensely sweet-tasting protein
Thaumatin is a sweet-tasting protein with an intensity approximately 1,600 – 2,000 times greater than sucrose (it is even about 7 times sweeter than stevia). Some sources say that it is approximately 3000 times sweeter than sucrose. The thaumatin protein consists of a single polypeptide chain of 207 residues. It elicits a sweetness 100,000 times higher than that of sucrose on a molar basis even at a low concentration of 50 nM.
Thaumatin comes from the West African Katemfe fruit Thaumatococcus daniellii. Thaumatococcus daniellii is a large flowering herb that can grow up to 4 m high and has large, papery leaves up to 46 centimeters long. It is commonly found in the rainforests of West Africa. The fruit was called ‘katemfe’ or ‘miraculous fruit of Sudan’. They are also known as miracle fruit, miracle berry, Yoruba soft cane, and African serendipity berry. Its fruits are red, triangular, approx. 4 cm in diameter, covered with a soft and fleshy red aril, which is the part that contains thaumatin, and inside they contain up to 3 shiny black seeds surrounded by a transparent jelly. In West Africa, the aril is traditionally used for sweetening bread, over-fermented palm wine and sour food. T. daniellii is used medicinally in the Ivory Coast and Congo as a laxative, emetic, and for pulmonary problems.
The intense sweetness of the fruit of Thaumatococcus daniellii was first described by a British surgeon in the Pharmaceutical Journal (Daniell, 1855).
Interestingly, thaumatins only taste sweet to Old World Monkeys and Apes, including humans, although rats can experience the flavour enhancement of thaumatins and pig weight gain is greater if Talin is added to their feed. Thaumatin is approved as a flavor enhancer. and as a sweetener in many countries.
Thaumatin is a low-calorie protein sweetener, which makes it a solution to help brands make low-calorie products. It has an energy content of 17 kJ/g (4 kcal/g).
From a chemical point of view, it is not a carbohydrate, but a mixture of closely related proteins that consist of a combination of many amino acids. The chemical composition of thaumatin consists of a long chain of peptides and has the chemical formula C49 H78 N14 O19 S. Five isoforms of the protein have been found in the fruit, namely thaumatins I, II, III, a and b, all of which are sweet. Thaumatin I and II are the main forms. The principal protein is thaumatin 1, while thaumatin 2 comprises no more than 45% of the mix. Thaumatins I and II are each composed of 207 amino acids with eight intramolecular disulfide bonds. Thaumatin is a 22 kDa sweet protein that was isolated from the arils of the katemfe fruit of Thaumatococcus daniellii Benth, comparable to casein. These are proteins with isoelectric points in the range of 11.5–12.5. There are no unusual side chains, atypical peptide linkages, or end-groups.
Extensive disulfide cross-linking confers to thaumatin thermal stability, resistance to denaturation, and maintenance of the tertiary structure of the polypeptide chain, which is critical to thaumatin’s technical function. Cleavage of just one disulfide bridge results in a loss of sweet taste.
Thaumatin consists of a single chain of normal amino acids, including Alanine, Aspartic Acid, Glutamic Acid, Glycine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine and Tyrosine.
The results showed that these amino acids play significant roles in thaumatin sweetness. Of these amino acid residues, Lys67 and Arg82 were particularly important for eliciting the sweetness.
The amount of thaumatins in arils of mature fruit is 30–55 mg/g of fresh weight (about 50% of total soluble protein), varying within a wide range depending on the degree of fruit maturity and origin.
The production of thaumatin evolved from the traditional plant extraction to a more relevant microbial production today. Thaumatin was produced from the fruits of Thaumatococcus danielli through aqueous extraction. Conventionally, the fruit is ruptured and extracted with water or a dilute aqueous buffer. A process for the extraction of thaumatin from the fruit of T. daniellii, comprises the steps of skinning the fruit, drying the pulp and mechanically separating the arils from the remainder of the fruit, powdering or otherwise comminuting the dried aril and extracting the resultant powder with water in the absence of added salts. The extract is concentrated by reverse osmosis or ion exchange chromatography, and then freeze-dried to produce thaumatin of high purity.
Currently, biotechnological production methods such as plant cell culture or microbial fermentation are used to meet the needs for commercial-scale production of natural sweeteners. The process involves fermentation of the culture medium under controlled conditions using bacteria or yeast fungi, which may take from a few days to a few weeks, depending on the conditions and the microorganisms used.
The availability of thaumatin of plant origin is very limited, and it is notoriously difficult to produce by recombinant DNA methods. Production has been attempted with Escherichia coli, Bacillus subtilis, Streptomyces lividans, Saccharomyces cerevisiae, and Aspergillus oryzae. A synthetic gene for thaumatin II with fungal codon usage has been synthesized, but expression in Aspergillus niger gave poor yields.
The usage of transgenic plants has also been tested in a number of studies. The attempts to produce sweet-proteins especially, thaumatin in potatoes, strawberries, etc. showed a lower level of accumulation. A sufficient amount of the recombinant thaumatin is achievable from transgenic barley and tomato. The invention relates to transgenic plants from barley containing more than 2 grams of thaumatin in 1 kg of kernel material. Both methods mentioned above allow you to obtain a high-quality product that is suitable for use as a food additive. One kg of fruit can produce 6 g of thaumatin. This makes it relatively expensive compared to other artificial sweeteners and not as widely used. It is an odourless, cream-colored powder. The taste characteristics are the slow onset of sweetness and a sweet aftertaste. It is soluble in water, and solutions of more than 60% concentration can be made. Thaumatin is stable in freeze-dried form. Thaumatin has good water solubility and stability during heating even in an acidic condition. Its sweetness is perceived more slowly than sucrose and lingers longer with a licorice-like after-taste. It must be blended with other sweeteners or sucrose to achieve a desirable taste. Solutions of thaumatin are stable throughout a wide range of pH (2.5–10). It is heat- and pH-stable and synergistic when combined with other low-calorie sweeteners. Thaumatin has a wide range of applications in food and drinks.
Thaumatin, an intensely sweet protein, crystallizes rapidly in the presence of tartrate ions. The ease with which crystals form has led to the use of thaumatin over the past decade as a model system for the study of protein crystallization. The protein is heat stable to 70 °C but loses its sweetness at higher temperatures.
The first isolation of thaumatin from the native plant was achieved by van der Wel and Loeve (1972). It has been used in Japan since 1979, and in the US it is used in chewing gum. In Canada, Thaumatin is approved for use as a flavour enhancer. It was licensed for use in pharmaceuticals and food in the UK in 1983, except for baby food. It is an approved high-intensity sweetener and flavour enhancer in most countries. Thaumatin is exceptionally sweet. Aqueous solutions of thaumatin range from 1300 times sweeter than sucrose, when compared at a 13% concentration, to about 5500 times sweeter when compared to sucrose. The taste bud responds to a thaumatin stimulus. The threshold value of the sweetness of thaumatin to humans is around 10-8 mol/l. However, its sweetness is perceived more slowly than sucrose and lingers longer with a licorice-like after-taste. It must be blended with other sweeteners or sucrose to achieve a desirable taste. Thaumatin masks the bitter, unpleasant taste of sodium, potassium, and iron ions. Thaumatin is a taste-modifying protein that functions as a natural sweetener or flavor enhancer. It may enhance the aroma and improve the flavors. To achieve a taste closer to that of sucrose, thaumatin must be blended with other intense sweeteners or with sugars. Synergism has been observed with saccharin, acesulfame-K, and stevioside, but not with aspartame and cyclamate.
Thaumatin can be used as a sweetener in chocolate milk, hot chocolate made with dairy, strawberry milk, milkshakes, and ice cream. Thaumatin, a sweetness enhancer, can reduce the quantity of added sugar in food and beverages.
Thaumatin offers a few benefits, including its natural origin and its ability to provide intense sweetness without the need for a large quantity. This makes it a suitable alternative to artificial sweeteners for those seeking a more natural option. Additionally, it can help to mask off-flavors and improve the taste profile of various food products. However, because of its high cost, and limited availability, it is used on a small scale. In some cases, thaumatin’s unique flavor profile may be perceived as artificial, and it leaves a licorice-like aftertaste. Thaumatin is an approved sweetener in the EU (E957) and the USA. In the United States, Thaumatin is classified as GRAS (Generally Recognised as Safe) by the FDA (Food and Drug Administration) and it is generally recognized as safe as a flavouring agent.
Thaumatins are allowed for use in multiple foods and beverages at application rates up to 200 mg/kg. Thaumatin is nontoxic and makes an insignificant contribution to normal protein intake. Therefore the WHO JEFCA in 1985 specified it to have no ADI. Thaumatin won’t cause any spike or even a rise in human blood sugar level. It has a zero glycemic index, thus it is very suitable for diabetics.
Thaumatin is considered and classified as a nonnutritive sweetener, and it is recommended as an alternative to sugar for those who cannot consume nutritional sweeteners. Thaumatin has many great benefits, such as its low-calorie content, its glycemic index of zero, its plant-based protein content, and its lack of bitter aftertaste. Thaumatin showed no mutagenic or teratogenic effects and no allergenic effects were noted.
Thaumatin was considered acceptable owing to its lack of toxicity, combined with its being readily digested into normal food components. Due to its intense sweetness, the amounts used are small. This natural sweetener is known to possess antihypertensive as well as anti-inflammatory properties. Thaumatin is a protein, therefore it can cause digestive problems in some people, especially those with allergies or intolerances to certain proteins.
While thaumatin is considered to be safe for food use, the protein shares structural similarities with allergens found in apple and kiwi fruit and is resistant to gastric digestion. These properties raise the potential that thaumatin could also be allergenic. So far, there has been no evidence that thaumatin can provoke an allergic reaction.
Thaumatin at a recommended use range of 1 to 5 mg/kg complete feed is safe for all animal species with a considerable margin of safety. With respect to this margin of safety, thaumatin can be administered simultaneously via feed or water for drinking. Since the natural production of thaumatin is often too expensive, it may increase food and beverage manufacturing costs. Generally speaking, thaumatin is perceived to be more expensive than traditional sugar substitutes. However, it is exceptionally sweet, so smaller amounts are needed to achieve the same level of sweetness as sugar in food. Apart from being a low-calorie sweetener, thaumatin can also act as a flavor modifier in food applications. Hence, such unique properties of this sweet protein make it an attractive ingredient for the food industry. It is important to consider that the cost-effectiveness of using thaumatin will also depend on factors such as the specific composition of the product, the desired level of sweetness, and the intended use of the product.
Nowadays, thaumatin has widely been used not only as a natural sweetener but also as a substance for flavor enhancers as well as masking unpleasant tastes in the food and pharmaceutical industries.
Authors:
dr hab. Małgorzata Ziarno, prof. SGGW, SGGW
dr inż. Dorota Zaręba, ZSG
The article was also published in Forum Mleczarskie