Sweet protein brazzein as a promising sweetener
AbstractThe excessive consumption of sugar-containing foods contributes to the development of a number of diseases, including obesity, diabetes mellitus, etc. As a substitute for sugar, people with diabetes mellitus and obesity most often use sweeteners. Sweet proteins, in particular brazzein, are an alternative to synthetic sweeteners that have natural origin, are broken down in the intestines along with food proteins, and do not affect blood sugar and insulin levels.
The purpose of the review was to analyze the available data on the sweet protein brazzein, its physical and chemical properties, existing biotechnological methods of production, and prospects for application in the food industry in order to further develop an optimized heterologous expression system.
Material and methods. Google Scholar, Scopus, Web of Science, PubMed, RSCI and eLibrary.ru databases were used for collecting and analyzing literature. Search depth – 30 years.
Results. Numerous studies of the physical and chemical properties of brazzein have demonstrated its high potential for use in the food industry. In particular, a short amino acid sequence, thermal stability, the ability to maintain its structure and sweet properties in a wide pH range, hypoallergenicity, lack of genotoxicity, and an extremely high level of sweetness compared to sucrose allow us to conclude that its use is promising. Mutant variants of brazzein have been generated, the sweetest of which (with three amino acid substitutions H31R/E36D/E41A) exceeds sucrose sweetness by 22 500 times. To date, various systems for the expression of recombinant brazzein have already been developed, in which bacteria (Escherichia coli, Lactococcus lactis, Bacillus licheniformis), yeast (Komagataella phaffii, Kluyveromyces lactis, Saccharomyces cerevisiae), plants (Zea mays, Oryza sativa, Lactuca sativa, Nicotiana tabacum, Daucus carota) and animals (Mus musculus) have been used.
Conclusion. Due to its high sweetness, organoleptic properties and long history of human consumption, brazzein can be considered as a promising natural sweetener. Despite the short peptide sequence, the production of the recombinant protein faced a number of problems, including low protein yield (for example, it could only be detected in mouse milk by Western blot hybridization) and loss of sweetness. Thus, further optimization of the process is necessary for widespread brazzein use in the food industry, which includes the selection of an adequate producer and the use of extracellular expression systems to reduce the final cost of the product.
Keywords:sweeteners; brazzein; sweet proteins; sweet taste receptors; brazzein expression; brazzein mutant variants
Funding. The research was carried out at the expense of the research grant No. 94030690 from the Saint-Petersburg State University “Development of a method for targeted integration of a transgene into the ROSA26 locus to obtain humanized animals and create a test system for rapid genotyping using Cas nucleases”.
Conflict of interest. The authors declare no conflict of interest.
Contribution: Research design – Markova E.V.; collection and processing of material – Markova E.V. and Sopova Ju.V.; manuscript writing, editing, adoption of the final article, responsibility for the integrity of all parts of the article – all authors.
For citation: Markova E.V., Leonova E.I., Sopova Ju.V. Sweet protein brazzein as a promising sweetener. Voprosy pitaniia [Problems of Nutrition]. 2024; 93 (1): 61–71. DOI: https://doi.org/10.33029/0042-8833-2024-93-1-61-71 (in Russian)
References
1. Beauchamp G.K. Why do we like sweet taste: a bitter tale? Physiol. Behav. 2016; 164: 432–7. DOI: https://doi.org/10.1016/J.PHYSBEH.2016.05.007
2. Beauchamp G.K., Mennella J.A. Flavor perception in human infants: development and functional significance. Digestion. 2011; 83 (suppl 1): 1–6. DOI: https://doi.org/10.1159/000323397
3. Kendig M.D. Cognitive and behavioural effects of sugar consumption in rodents. A review. Appetite. 2014; 80: 41–54. DOI: https://doi.org/10.1016/j.appet.2014.04.028
4. Han P., Bagenna B., Fu M. The sweet taste signalling pathways in the oral cavity and the gastrointestinal tract affect human appetite and food intake: a review. Int J Food Sci Nutr. 2019; 70 (2): 125–35. DOI: https://doi.org/10.1080/09637486.2018.1492522
5. Gutierrez R., Fonseca E., Simon S.A. The neuroscience of sugars in taste, gut-reward, feeding circuits, and obesity. Cell Mol Life Sci. 2020; 77 (18): 3469–502. DOI: https://doi.org/10.1007/s00018-020-03458-2
6. Stanhope K.L. Sugar consumption, metabolic disease and obesity: the state of the controversy. Crit Rev Clin Lab Sci. 2016; 53 (1): 52–67. DOI: https://doi.org/10.3109/10408363.2015.1084990
7. Kholmatova K., Krettek A., Leon D.A., Malyutina S., Cook S., Hopstock L.A., et al. Obesity prevalence and associated socio-demographic characteristics and health behaviors in Russia and Norway. Int J Environ Res Public Health. 2022; 19 (15): 9428. DOI: https://doi.org/10.3390/ijerph19159428
8. Jacques A., Chaaya N., Beecher K., Ali S.A., Belmer A., Bartlett S. The impact of sugar consumption on stress driven, emotional and addictive behaviors. Neurosci Biobehav Rev. 2019; 103: 178–99. DOI: https://doi.org/10.1016/j.neubiorev.2019.05.021
9. Yan R.R., Chan C.B., Louie J.C.Y. Current WHO recommendation to reduce free sugar intake from all sources to below 10% of daily energy intake for supporting overall health is not well supported by available evidence. Am J Clin Nutr. 2022; 116 (1): 15–39. DOI: https://doi.org/10.1093/ajcn/nqac084
10. Joveini H., Sharifi N., Meymary B.K., Mehri A., Shahrabadi R., Rahmanian V., et al. The effect of empowerment program to reduce sugar consumption based on the multi-theory model on body mass index and abdominal obesity in Iranian women. BMC Womens Health. 2023; 23 (1): 207. DOI: https://doi.org/10.1186/s12905-023-02361-9
11. Warshaw H., Edelman S.V. Practical strategies to help reduce added sugars consumption to support glycemic and weight management goals. Clin Diabetes. 2021; 39 (1): 45–56. DOI: https://doi.org/10.2337/cd20-0034
12. Temussi P.A. Sweet, bitter and umami receptors: a complex relationship. Trends Biochem Sci. 2009; 34 (6): 296–302. DOI: https://doi.org/10.1016/j.tibs.2009.02.005
13. Jiang P., Cui M., Zhao B., Snyder L.A., Benard L.M., Osman R., et al. Identification of the cyclamate interaction site within the transmembrane domain of the human sweet taste receptor subunit T1R3. J Biol Chem. 2005; 280 (40): 34 296–305. DOI: https://doi.org/10.1074/jbc.M505255200
14. Assadi-Porter F.M., Maillet E.L., Radek J.T., Quijada J., Markley J.L., Max M. Key amino acid residues involved in multi-point binding interactions between brazzein, a sweet protein, and the T1R2-T1R3 human sweet receptor. J Mol Biol. 2010; 398 (4): 584–99. DOI: https://doi.org/10.1016/j.jmb.2010.03.017
15. Nango E., Akiyama S., Maki-Yonekura S., Ashikawa Y., Kusakabe Y., Krayukhina E., et al. Taste substance binding elicits conformational change of taste receptor T1r heterodimer extracellular domains. Sci Rep. 2016; 6 (1): 25745. DOI: https://doi.org/10.1038/srep25745
16. Ahmad R., Dalziel J.E. G protein-coupled receptors in taste physiology and pharmacology. Front Pharmacol. 2020; 11: 587664. DOI: https://doi.org/10.3389/fphar.2020.587664
17. Dutta Banik D., Martin L.E., Freichel M., Torregrossa A.M., Medler K.F. TRPM4 and TRPM5 are both required for normal signaling in taste receptor cells. Proc Natl Acad Sci USA. 2018; 115 (4): E772–81. DOI: https://doi.org/10.1073/pnas.1718802115
18. Jeong J.-Y., Cha Y.K., Ahn S.R., Shin J., Choi Y., Park T.H., et al. Ultrasensitive bioelectronic tongue based on the Venus Flytrap Domain of a human sweet taste receptor. ACS Appl Mater Interfaces. 2022; 14 (2): 2478–87. DOI: https://doi.org/10.1021/acsami.1c17349
19. Mahmood A.A.R., Al-Juboori S.B. A review: saccharin discovery, synthesis, and applications. Ibn AL-Haitham J Pure Appl Sci. 2020; 33 (2): 43–61. DOI: https://doi.org/10.30526/33.2.2442
20. Yusuf E.H. An overview of biotransformation for the sustainability of sweet-tasting proteins as natural sugar replacers. Chem Proc. 2022; 8 (1): 85. DOI: https://doi.org/10.3390/ecsoc-25-11640
21. Delfi M., Emendato A., Temussi P.A., Picone D. Striking dependence of protein sweetness on water quality: the role of the ionic strength. Front Mol Biosci. 2021; 8: 705102. DOI: https://doi.org/10.3389/fmolb.2021.705102
22. Kim H., Kang J., Hong S., Jo S., Noh H., Kang B.H., et al. 3M-brazzein as a natural sugar substitute attenuates obesity, metabolic disorder, and inflammation. J Agric Food Chem. 2020; 68 (7): 2183–2192. DOI: https://doi.org/10.1021/acs.jafc.0c00317
23. Bilal M., Ji L., Xu S., Zhang Y., Iqbal H.M.N., Cheng H. Bioprospecting and biotechnological insights into sweet-tasting proteins by microbial hosts – a review. Bioengineered. 2022; 13 (4): 9815–28. DOI: https://doi.org/10.1080/21655979.2022.2061147
24. Nagata K., Hongo N., Kameda Y., Yamamura A., Sasaki H., Lee W.C., et al. The structure of brazzein, a sweet-tasting protein from the wild African plant Pentadiplandra brazzeana. Acta Cryst. 2013; 69 (4): 642–7. DOI: https://doi.org/10.1107/S0907444913001005
25. Kohmura M., Ota M., Izawa H., Ming D., Hellekant G., Ariyoshi Y. Assignment of the disulfide bonds in the sweet protein brazzein. Biopolymers. 1996; 38 (4): 553–6. DOI: https://doi.org/10.1002/(SICI)1097-0282(199604)38:4%3C553::AID-BIP10%3E3.0.CO;2-B
26. Lynch B., Wang T., Vo T., Tafazoli S., Ryder J. Safety evaluation of oubli fruit sweet protein (brazzein) derived from Komagataella phaffii, intended for use as a sweetener in food and beverages. Toxicol Res Appl. 2023; 7: 1–21. DOI: https://doi.org/10.1177/23978473231151258
27. Assadi-Porter F.M., Aceti D.J, Cheng H., Markley J.L. Efficient production of recombinant brazzein, a small, heat-stable, sweet-tasting protein of plant origin. Arch Biochem Biophys. 2000; 376 (2): 252–8. DOI: https://doi.org/10.1006/abbi.2000.1725
28. Singarapu K.K. Tonelli M., Markley J.L., Assadi-Porter F.M. Structure-function relationships of brazzein variants with altered interactions with the human sweet taste receptor. Protein Sci. 2016; 25 (3): 711–9. DOI: https://doi.org/10.1002/pro.2870
29. Assadi-Porter F.M., Aceti D.J., Markley J.L. Sweetness determinant sites of brazzein, a small, heat-stable, sweet-tasting protein. Arch Biochem Biophys. 2000; 376 (2): 259–65. DOI: https://doi.org/10.1006/abbi.2000.1726
30. Zhao X., Wang C., Zheng Y., Liu B. New insight into the structure-activity relationship of sweet-tasting proteins: protein sector and its role for sweet properties. Front Nutr. 2021; 8: 691368. DOI: https://doi.org/10.3389/fnut.2021.691368
31. Poursalim M., Shasaltaneh M.D., Jafarian V., Salehabadi H. The novel anti-cancer feature of brazzein through activating of hTLR5 by integration of biological evaluation: molecular docking and molecular dynamics simulation. Sci Rep. 2022; 12 (1): 21979. DOI: https://doi.org/10.1038/s41598-022-26487-2
32. Lee J.W., Cha J.E., Jo H.J., Kong K.H. Multiple mutations of the critical amino acid residues for the sweetness of the sweet-tasting protein, brazzein. Food Chem. 2013; 138 (2–3): 1370–3. DOI: https://doi.org/10.1016/j.foodchem.2012.10.140
33. Jin Z. Danilova V., Assadi-Porter F.M., Markley J.L., Hellekant G. Monkey electrophysiological and human psychophysical responses to mutants of the sweet protein brazzein: delineating brazzein sweetness. Chem Senses. 2003; 28 (6): 491–8. DOI: https://doi.org/10.1093/chemse/28.6.491
34. Jafari S.S., Jafarian V., Khalifeh K., Ghanavatian P., Shirdel S.A. The effect of charge alteration and flexibility on the function and structural stability of sweet-tasting brazzein. RSC Advances. 2016; 6 (64): 59834–41. DOI: https://doi.org/10.1039/c6ra12626j
35. Yoon S.Y., Kong J.N., Jo D.H., Kong K.H. Residue mutations in the sweetness loops for the sweet-tasting protein brazzein. Food Chem. 2011; 129 (4): 1327–30. DOI: https://doi.org/10.1016/j.foodchem.2011.06.054
36. Ghanavatian P., Khalifeh K., Jafarian V. Structural features and activity of brazzein and its mutants upon substitution of a surfaced exposed alanine. Biochimie. 2016; 131: 20–8. DOI: https://doi.org/10.1016/j.biochi.2016.09.006
37. Walters D.E. Cragin T., Jin Z., Rumbley J.N., Hellekant G. Design and evaluation of new analogs of the sweet protein brazzein. Chem Senses. 2009; 34 (8): 679–83. DOI: https://doi.org/10.1093/chemse/bjp048
38. Lim J.K. Jang J.C., Kong J.N., Kim M.C., Kong K.H. Importance of Glu53 in the C-terminal region of brazzein, a sweet-tasting protein. J Sci Food Agric. 2016; 96 (9): 3202–6. DOI: https://doi.org/10.1002/jsfa.7501
39. Lu R., Li X., Hu J., Zhang Y., Wang Y., Jin L. Expression of a triple mutational des-pGlu brazzein in transgenic mouse milk. FEBS Open Bio. 2022; 12 (7): 1336–43. DOI: https://doi.org/10.1002/2211-5463.13411
40. Ota M., Kohmura M., Ariyoshit Y. Synthesis and characterization of the sweet protein brazzein. Biopolymers. 1996; 39 (1): 95–101. DOI: https://doi.org/10.1002/(sici)1097-0282(199607)39:1<95::aid-bip10>3.0.co;2-b
41. Berlec A., Jevnikar Z., Majhenic A.C., Rogelj I., Strukelj B. Expression of the sweet-tasting plant protein brazzein in Escherichia coli and Lactococcus lactis: a path toward sweet lactic acid bacteria. Appl Microbiol Biotechnol. 2006; 73 (1): 158–65. DOI: https://doi.org/10.1007/s00253-006-0438-y
42. Berlec A., Tompa G., Slapar N., Fonović U.P., Rogelj I., Strukelj B. Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis. Lett Appl Microbiol. 2008; 46 (2): 227–31. DOI: https://doi.org/10.1111/j.1472-765X.2007.02297.x
43. Jafarian V., Bagheri K., Zarei J., Karami S., Ghanavatian P. Improved expression of recombinant sweet-tasting brazzein using codon optimization and host change as new strategies. Food Biotechnol. 2020; 34 (1): 62–76. DOI: https://doi.org/10.1080/08905436.2019.1711113
44. Lamphear B.J., Barker D.K., Brooks C.A., Delaney D.E., Lane J.R., Beifuss K., et al. Expression of the sweet protein brazzein in maize for production of a new commercial sweetener. Plant Biotechnol J. 2005; 3 (1): 103–14. DOI: https://doi.org/10.1111/j.1467-7652.2004.00105.x
45. Lee Y.R., Akter S., Lee I.H., Jung Y.J., Park S.Y., Cho Y.-G., et al. Stable expression of brazzein protein, a new type of alternative sweetener in transgenic rice. J Plant Biotechnol. 2018; 45 (1): 63–70. DOI: https://doi.org/10.5010/JPB.2018.45.1.063
46. Jung Y.J., Kang K.K. Stable expression and characterization of brazzein, thaumatin and miraculin genes related to sweet protein in transgenic lettuce. J Plant Biotechnol. 2018; 45 (3): 257–65. DOI: https://doi.org/10.5010/JPB.2018.45.3.257
47. Choi H.E., Lee J.I., Jo S.Y., Chae Y.C., Lee J.H., Sun H.J., et al. Functional expression of the sweet-tasting protein brazzein in transgenic tobacco. Food Sci Technol. 2022; 42: e40521. DOI: https://doi.org/10.1590/fst.40521
48. Han J.E., Lee H., Ho T.-T., Park S.-Y. Brazzein protein production in transgenic carrot cells using air-lift bioreactor culture. Plant Biotechnol Rep. 2022; 16 (2): 161–71. DOI: https://doi.org/10.1007/s11816-022-00743-3
49. Han J.-E., Park Y.-J., Lee H., Jeong Y.-J., Park S.-Y. Increased brazzein expression by abiotic stress and bioreactor culture system for the production of sweet protein, brazzein. Plant Biotechnol Rep. 2020; 14 (4): 459–66. DOI: https://doi.org/10.1007/s11816-020-00625-6
50. Yan S., Song H., Pang D., Zou Q., Li L., Yan Q., et al. Expression of plant sweet protein brazzein in the milk of transgenic mice. PLoS One. 2013; 8 (10): 1–8. DOI: https://doi.org/10.1371/journal.pone.0076769
51. Neiers F., Naumer C., Krohn M., Briand L. The recent development of a sweet-tasting brazzein and its potential industrial applications. In: J.M. Merillon, K. Ramawat (eds). Sweeteners. Reference Series in Phytochemistry. Cham: Springer, 2016: 1–20. DOI: https://doi.org/10.1007/978-3-319-26478-3_2-1 ISBN978-3-319-26478-3.
52. Poirier N., Roudnitzky N., Brockhoff A., Belloir C., Maison M., Thomas-Danguin T., et al. Efficient production and characterization of the sweet-tasting brazzein secreted by the yeast Pichia pastoris. J Agric Food Chem. 2012; 60 (39): 9807–14. DOI: https://doi.org/10.1021/jf301600m
53. Jo H.J., Noh J.S., Kong K.H. Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis. Protein Expr Purif. 2013; 90 (2): 84–9. DOI: https://doi.org/10.1016/j.pep.2013.05.001
54. Yun C.R., Kong J.N., Chung J.H., Kim M.C., Kong K.H. Improved secretory production of the sweet-tasting protein, brazzein, in Kluyveromyces lactis. J Agric Food Chem. 2016; 64 (32): 6312–6. DOI: https://doi.org/10.1021/acs.jafc.6b02446
55. 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): 945–52. DOI: https://doi.org/10.2174/0929866527666200331134431
56. Kazlovsky I.S., Bel’skaya I.V., Zinchenko А.I. Biosynthesis of brazzein using the bacterial cell-free protein synthesis system. Doklady natsional’noy akademii nauk Belarusi [Reports of the National Academy of Sciences of Belarus]. 2020; 64 (1): 71–7. DOI: https://doi.org/10.29235/1561-8323-2020-64-1-71-77 (in Russian)
57. Assadi-Porter F.M., Patry S., Markley J.L. Efficient and rapid protein expression and purification of small high disulfide containing sweet protein brazzein in E. coli. Protein Expr Purif. 2008; 58 (2): 263–8. DOI: https://doi.org/10.1016/j.pep.2007.11.009
58. Hung C.Y., Cheng L.H., Yeh C.M. Functional expression of recombinant sweet-tasting protein brazzein by Escherichia coli and Bacillus licheniformis. Food Biotechnol. 2019; 33 (3): 251–71. DOI: https://doi.org/10.1080/08905436.2019.1618323
59. Park S.W., Kang B.H., Lee H.M., Lee S.J., Kim H.S., Choi H.W., et al. Efficient brazzein production in yeast (Kluyveromyces lactis) using a chemically defined medium. Bioprocess Biosyst Eng. 2021; 44 (4): 913–25. DOI: https://doi.org/10.1007/s00449-020-02499-y
60. Lee H.M., Park S.W., Lee S.J., Kong K.H. Optimized production and quantification of the tryptophan-deficient sweet-tasting protein brazzein in Kluyveromyces lactis. Prep Biochem Biotechnol. 2019; 49 (8): 790–9. DOI: https://doi.org/10.1080/10826068.2019.1621892