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This article presents the results of a comprehensive toxicity assessment of brazzein and monellin, yeast-produced recombinant sweet-tasting proteins. Excessive sugar consumption is one of the leading dietary and nutritional problems in the world, resulting in health complications such as obesity, high blood pressure, and cardiovascular disease. Although artificial small-molecule sweeteners widely replace sugar in food, their safety and long-term health effects remain debatable. Many sweet-tasting proteins, including thaumatin, miraculin, pentadin, curculin, mabinlin, brazzein, and monellin have been found in tropical plants. These proteins, such as brazzein and monellin, are thousands-fold sweeter than sucrose. Multiple reports have presented preparations of recombinant sweet-tasting proteins. A thorough and comprehensive assessment of their toxicity and safety is necessary to introduce and apply sweet-tasting proteins in the food industry. We experimentally assessed acute, subchronic, and chronic toxicity effects, as well as allergenic and mutagenic properties of recombinant brazzein and monellin. Our study was performed on three mammalian species (mice, rats, and guinea pigs). Assessment of animals' physiological, biochemical, hematological, morphological, and behavioral indices allows us to assert that monellin and brazzein are safe and nontoxic for the mammalian organism, which opens vast opportunities for their application in the food industry as sugar alternatives. [ABSTRACT FROM AUTHOR]

This article presents the results of a comprehensive toxicity assessment of brazzein and monellin, yeast-produced recombinant sweet-tasting proteins. Excessive sugar consumption is one of the leading dietary and nutritional problems in the world, resulting in health complications such as obesity, high blood pressure, and cardiovascular disease. Although artificial small-molecule sweeteners widely replace sugar in food, their safety and long-term health effects remain debatable. Many sweet-tasting proteins, including thaumatin, miraculin, pentadin, curculin, mabinlin, brazzein, and monellin have been found in tropical plants. These proteins, such as brazzein and monellin, are thousands-fold sweeter than sucrose. Multiple reports have presented preparations of recombinant sweet-tasting proteins. A thorough and comprehensive assessment of their toxicity and safety is necessary to introduce and apply sweet-tasting proteins in the food industry. We experimentally assessed acute, subchronic, and chronic toxicity effects, as well as allergenic and mutagenic properties of recombinant brazzein and monellin. Our study was performed on three mammalian species (mice, rats, and guinea pigs). Assessment of animals’ physiological, biochemical, hematological, morphological, and behavioral indices allows us to assert that monellin and brazzein are safe and nontoxic for the mammalian organism, which opens vast opportunities for their application in the food industry as sugar alternatives.

Sweet proteins are the sweetest natural molecules. This aspect prompted several proposals for their use as food additives, mainly because the amounts to be added to food would be very small and safe for people suffering from sucrose-linked diseases. During studies of sweet proteins as food additives we found that their sweetness is affected by water salinity, while there is no influence on protein’s structure. Parallel tasting of small size sweeteners revealed no influence of the water quality. This result is explained by the interference of ionic strength with the mechanism of action of sweet proteins and provides an experimental validation of the wedge model for the interaction of proteins with the sweet receptor.

Sweet proteins are a class of proteins with the ability to elicit a sweet sensation in humans upon interaction with sweet taste receptor T1R2/T1R3. Single-chain Monellin, MNEI, is among the sweetest proteins known and it could replace sugar in many food and beverage recipes. Nonetheless, its use is limited by low stability and high aggregation propensity at neutral pH. To solve this inconvenience, we designed a new construct of MNEI, dubbed Mut9, which led to gains in both sweetness and stability. Mut9 showed an extraordinary stability in acidic and neutral environments, where we observed a melting temperature over 20 °C higher than that of MNEI. In addition, Mut9 resulted twice as sweet than MNEI. Both proteins were extensively characterized by biophysical and sensory analyses. Notably, Mut9 preserved its structure and function even after 10 min boiling, with the greatest differences being observed at pH 6.8, where it remained folded and sweet, whereas MNEI lost its structure and function. Finally, we performed a 6-month shelf-life assessment, and the data confirmed the greater stability of the new construct in a wide range of conditions. These data prove that Mut9 has an even greater potential for food and beverage applications than MNEI.

The plants described in this review are a source of natural high-intensity sweeteners, which can be used in food and by the pharmaceutical industry in the future. Most of the plants are still not approved for use, even though they are traditionally used in countries where they appear naturally. Ten of the herein described intense sweeteners are characterized by a much higher sweetness in relation to sucrose. The highest values were received for miraculin, obtained from Synsepalum dulcificum (400,000 times sweeter than sucrose, induced by citric acid); thaumatin (1,600 to 3,000 times sweeter), monatin (1,200 - 3,000) and pentadin (500 to 2,000 times sweeter). Some of these substances can also modify the taste, like changing sour into sweet taste (miraculin and neoculin). The most widely used sweeteners are steviol glycosides and thaumatin, which have been admitted for use as a sweetener in the European Union, while in the US, they have the GRAS status (thaumatin as a food enhancer). Mogroside obtained from Siraitia grosvenorii (called Luo Han Guo) is not approved for use in the EU, but was granted GRAS status in the US by the FDA. This gives a chance that it will soon be approved as a novel food or food additive in the European Union. [ABSTRACT FROM AUTHOR]

Cystatins are small proteins, typically composed of 100–120 amino acids, which together with similar proteins devoid of inhibitory properties, belong to a cystatin ‘superfamily’. Cystatins can do more than just inhibit proteases: two important aspects described here are aggregation properties linked to misfolding diseases and the unique ability of monellin, a plant cystatin, to elicit sweet taste. The explanation of the puzzling phenomenon of ‘sweet proteins’ required an in-depth structural study of monellin, also regarding the causes of the high thermal stability of its single chain structure. The detailed mechanisms by which cystatins aggregate could be relevant in the study of misfolding diseases involving cystatins. They are reviewed here with emphasis on 3D domain swapping, typical of aggregating cystatins. While studying monellin, we noticed that it aggregates in a conventional way, probably through the cross-β spine mechanism. However, several cystatins derived from oryzacystatin_I to emulate the taste behavior of monellin aggregate via different mechanisms.

A few proteins, discovered mainly in tropical fruits, have a distinct sweet taste. These proteins have played an important role towards a molecular understanding of the mechanisms of taste. Owing to the huge difference in size, between most sweeteners and sweet proteins, it was believed that they must interact with a different receptor from that of small molecular weight sweeteners. Recent modelling studies have shown that the single sweet taste receptor has multiple active sites and that the mechanism of interaction of sweet proteins is intrinsically different from that of small sweeteners. Small molecular weight sweeteners occupy small receptor cavities inside two subdomains of the receptor, whereas sweet proteins can interact with the sweet receptor according to a mechanism called the ‘wedge model’ in which they bind to a large external cavity. This review describes these mechanisms and outlines a history of sweet proteins. [ABSTRACT FROM AUTHOR]

The mechanism of interaction of sweet proteins with the T1R2-T1R3 sweet taste receptor has not yet been elucidated. Low molecular mass sweeteners and sweet proteins interact with the same receptor, the human T1R2-T1R3 receptor. The presence on the surface of the proteins of ‘sweet fingers’, i.e. protruding features with chemical groups similar to those of low molecular mass sweeteners that can probe the active site of the receptor, would be consistent with a single mechanism for the two classes of compounds. We have synthesized three cyclic peptides corresponding to the best potential ‘sweet fingers’ of brazzein, monellin and thaumatin, the sweet proteins whose structures are well characterized. NMR data show that all three peptides have a clear tendency, in aqueous solution, to assume hairpin conformations consistent with the conformation of the same sequences in the parent proteins. The peptide corresponding to the only possible loop of brazzein, c[CFYDEKRNLQC(37–47)], exists in solution in a well ordered hairpin conformation very similar to that of the same sequence in the parent protein. However, none of the peptides has a sweet taste. This finding strongly suggests that sweet proteins recognize a binding site different from the one that binds small molecular mass sweeteners. The data of the present work support an alternative mechanism of interaction, the ‘wedge model’, recently proposed for sweet proteins [Temussi, P. A. (2002) FEBS Lett. 526, 1–3.]. [ABSTRACT FROM AUTHOR]

The design of safe sweeteners is very important for people who are affected by diabetes, hyperlipemia, and caries and other diseases that are linked to the consumption of sugars. Sweet proteins, which are found in several tropical plants, are many times sweeter than sucrose on a molar basis. A good understanding of their structure-function relationship can complement traditional SAR studies on small molecular weight sweeteners and thus help in the design of safe sweeteners. However, there is virtually no sequence homology and very little structural similarity among known sweet proteins. Studies on mutants of monellin, the best characterized of sweet proteins, proved not decisive in the localization of the main interaction points of monellin with its receptor. Accordingly, we resorted to an unbiased approach to restrict the search of likely areas of interaction on the surface of a typical sweet protein. It has been recently shown that an accurate survey of the surface of proteins by appropriate paramagnetic probes may locate interaction points on protein surface. Here we report the survey of the surface of MNEI, a single chain monellin, by means of a paramagnetic probe, and a direct assessment of bound water based on an application of ePHOGSY, an NMR experiment that is ideally suited to detect interactions of small ligands to a protein. Detailed surface mapping reveals the presence, on the surface of MNEI, of interaction points that include residues previously predicted by ELISA tests and by mutagenesis. [ABSTRACT FROM AUTHOR]

Owing to various undesirable health effects of sugar overconsumption, joint efforts are being made by industrial sectors and regulatory authorities to reduce sugar consumption practices, worldwide. Artificial sweeteners are considered potential substitutes in several products, e.g., sugar alcohols (polyols), high-fructose corn syrup, powdered drink mixes, and other beverages. Nevertheless, their long-standing health effects continue to be debatable. Consequently, growing interest has been shifted in producing non-caloric sweetenersfrom renewable resources to meet consumers' dietary requirements. Except for the lysozyme protein, various sweet proteins including thaumatin, mabinlin, brazzein, monellin, miraculin, pentadin, and curculin have been identified in tropical plants. Given the high cost and challenging extortion of natural resources, producing these sweet proteins using engineered microbial hosts, such as Yarrowia lipolytica, Pichia pastoris, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Pichia methanolica, Saccharomyces cerevisiae, and Kluyveromyces lactis represents an appealing choice. Engineering techniques can be applied for large-scale biosynthesis of proteins, which can be used in biopharmaceutical, food, diagnostic, and medicine industries. Nevertheless, extensive work needs to be undertaken to address technical challenges in microbial production of sweet-tasting proteins in bulk. This review spotlights historical aspects, physicochemical properties (taste, safety, stability, solubility, and cost), and recombinant biosynthesis of sweet proteins. Moreover, future opportunities for process improvement based on metabolic engineering strategies are also discussed.

Sweet proteins are a class of proteins with the ability to elicit a sweet sensation in humans upon interaction with sweet taste receptor T1R2/T1R3. Single-chain Monellin, MNEI, is among the sweetest proteins known and it could replace sugar in many food and beverage recipes. Nonetheless, its use is limited by low stability and high aggregation propensity at neutral pH. To solve this inconvenience, we designed a new construct of MNEI, dubbed Mut9, which led to gains in both sweetness and stability. Mut9 showed an extraordinary stability in acidic and neutral environments, where we observed a melting temperature over 20 °C higher than that of MNEI. In addition, Mut9 resulted twice as sweet than MNEI. Both proteins were extensively characterized by biophysical and sensory analyses. Notably, Mut9 preserved its structure and function even after 10 min boiling, with the greatest differences being observed at pH 6.8, where it remained folded and sweet, whereas MNEI lost its structure and function. Finally, we performed a 6-month shelf-life assessment, and the data confirmed the greater stability of the new construct in a wide range of conditions. These data prove that Mut9 has an even greater potential for food and beverage applications than MNEI. [ABSTRACT FROM AUTHOR]

The article announces that scientists Jing-Ke Weng and Li-Qing Chen received the Tansley Medal 2013 from the journal "New Phytologist."