In a recent study published in the journal Foods, researchers comprehensively assessed the toxicity of monellin and brazzein, two recombinant, naturally occurring sweet-tasting proteins produced by yeast.
Study: Sweet-Tasting Natural Proteins Brazzein and Monellin: Safe Sugar Substitutes for the Food Industry. Image Credit: John B Hewitt/Shutterstock.com
Background
Monellin and brazzein, natural sweet-tasting proteins originally isolated from tropical plants and now produced recombinantly in yeast, have been widely used to replace sugar in food due to their high sweetness and potential health benefits. Unlike artificial small-molecule sweeteners, these proteins are thousands of times sweeter than sucrose and are protein-based, not chemically synthesized. Sweet-tasting proteins abundant in tropical plants offer advantages over sugar, including reduced risk of obesity, cardiovascular disease, and dental issues.
However, studies assessing their safety are limited. Brazzein is a heat-stable, water-soluble, non-caloric sweetener, while monellin has a more complex structure and is more sensitive to heat. Despite numerous publications on their structural configurations and modifications, a systematic study of their safety has yet to be conducted.
About the study
In the present study, researchers experimentally assessed the acute, sub-chronic, and chronic (150-day) toxicity and allergenic and mutagenic properties of recombinant monellin and brazzein.
The tested proteins were purified using chromatography, ultrafiltration, and lyophilization, and the microbiological purity of the sweet proteins was evaluated using bacteriological methods. Two experiments were conducted for each protein, with the animals kept under continuous observation for two weeks.
The equivalent dose (ED) of sucrose for guinea pigs (EDguinea pig) was calculated based on the relative sweetness of monellin and brazzein compared to sucrose, meaning that the amount of protein administered corresponded to the amount required to match the perceived sweetness of a standard sucrose dose for guinea pigs. This approach enabled the researchers to compare physiological effects at sweetness-equivalent doses, rather than weight-equivalent doses. Protein solutions and water were administered daily for 21 days in guinea pigs (sub-chronic toxicity) and 150 days in rats (chronic toxicity).
The team evaluated recombinant monellin and brazzein's acute and chronic toxicity on three mammalian species: guinea pigs, rats, and mice. Acute toxicity was assessed in rats and mice, chronic toxicity was evaluated in rats, and subchronic toxicity was evaluated in guinea pigs.
Acute toxicity assessments included weight gain, the general condition of animals, intoxication symptoms, the relative mass of main organs, and the pathomorphological examination of internal organs.
To assess sub-chronic and chronic toxicity, in addition to these tests, behavior, visible physiological functions, and diuresis volume were evaluated. Biochemical, hematological, and urine analyses were also performed.
For the sub-chronic toxicity study, guinea pigs were administered intragastrically with aqueous solutions of brazzein or monellin, while control group animals received distilled water. The study investigated the allergenic effects of monellin and brazzein on male albino guinea pigs and outbred male mice.
The animals were tested 10 to 12 days after sensitization using skin, conjunctival, and nasal tests, as well as the indirect mass cell degranulation reaction.
Additionally, the inflammatory response to concanavalin A was evaluated in mice. The results were evaluated after 15 minutes (immediate-type reaction) and 24 to 48 hours (delayed-type hypersensitivity). To investigate the mutagenicity of the proteins, the Ames test, bone marrow chromosomal aberration test, and micronucleus tests were performed in mice.
Results
The study on rats and mice revealed that monellin and brazzein are non-toxic and safe for mammalian organisms, opening vast opportunities for their application in the food industry as sugar alternatives.
The acute toxicity experiments demonstrated that the intragastrically administered proteins had an LD50 of over 5,000 mg/kg body weight. There were no intoxication or death-related symptoms observed for the protein doses tested in the experiments.
Intragastric injection of monellin or brazzein to rats at dosages ranging from 107 to 5,000 mg per kg of body weight and 71 to 5,000 mg per kg of body weight, respectively, had no impact on body weight or the coefficients for the mass of body organs. In acute toxicity tests conducted on murine animals, similar results were achieved.
Macroscopic examination of rats and mice organs following the administration of monellin or brazzein intragastrically showed no pathological alterations in the lungs, liver, heart, kidneys, brain, spleen, pancreas, lymph nodes, esophagus, thymus, small intestine, stomach, large intestine, ovaries, or testes.
The highest tested dosage of monellin or brazzein for mice and rats in the experiments was 5,000 mg per kg of body weight, near the maximal permissible dosage for intragastric delivery to these animals.
The lack of intoxication symptoms and death, as well as the impact on protein, weight, internal organ masses, and biochemical indices, demonstrated that regular intragastric infusion of monellin and brazzein for three weeks at the EDguinea pig dose and 10-fold EDguinea pig dose, respectively, had no toxic impact on the guinea pigs.
Macroscopic evaluation and the determination of internal organ mass coefficients in the experimental animals showed no pathological alterations. Regarding chronic toxicity, the relative gain in body weight among rats decreased toward the study termination, with no other toxic effects detected. This decrease in weight gain was attributed to a satiety effect, as both protein- and sucrose-treated rats had lower feed intake, rather than to toxicity.
Brazzein administration slightly but significantly reduced total protein and globulin levels in male rats and increased glucose concentration compared with a low control value.
In female rat sera, monellin administration at a dose of EDrat reduced fructosamine and cholesterol levels. A small but statistically significant increase in diuresis was observed in male rats at the highest tested dose, though this was not associated with any pathological changes and was not considered harmful by the study authors. Brazzein and monellin, at all tested concentrations, do not exhibit a mutagenic effect in vitro on S. typhimurium strains TA 100, TA 98, and TA 97, both in the presence and absence of a metabolic activation system. No cytogenetic activity was observed in bone marrow chromosomal aberration and micronucleus tests.
Conclusion
Overall, the study findings highlighted the safety of potential protein-based sweeteners, brazzein and monellin, in mammal experimental models, with no signs of intoxication, mortality, or inflammation. They also showed no allergenic or mutagenic properties. Some minor, statistically significant biochemical changes were observed, but all remained within physiological reference ranges, showed no consistent dose–response, and were considered not clinically significant by the investigators. Further research is needed to determine their safety in pregnant women and children, as well as to assess their impact on the fecal microbiota and animal metabolism.
Article Revisions
- May 30 2025 - In response to a user's comment, this article was reviewed and amended. The article was edited to accurately describe monellin and brazzein as recombinant, naturally occurring protein-based sweeteners, clarifying their origins in tropical plants and method of recombinant production. All references to “artificial small-molecule sweeteners” were corrected accordingly. The concept of “equivalent dose” (ED) was clarified throughout, with the introduction and consistent use of the proper scientific notation ED₍guinea pig₎ and ED₍rat₎. The rationale for using sweetness-equivalent, rather than weight-equivalent, dosing was explained for scientific clarity. Further methodological precision was added by specifying the 150-day chronic toxicity testing period in rats. The explanation of weight gain differences was revised to attribute reduced gain to satiety effects and lower feed intake, not toxicity. Additional detail was included regarding minor, statistically significant increases in diuresis at high doses in male rats, specifying that these changes were not considered harmful. The summary of biochemical findings was clarified to note that all observed changes remained within physiological ranges and showed no clinical significance. All changes are explained for editorial transparency and scientific accuracy. The user's comment can be found near the bottom of this page.
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