There is an increasing public awareness about the danger of dietary sugars with respect to their caloric contribution to the diet and the rise of overweight throughout the world. Therefore, low-calorie sugar substitutes are of high interest to replace sugar in foods and beverages. A promising alternative to natural sugars and artificial sweeteners is the fructose derivative 5-keto-D-fructose (5-KF), which is produced by several Gluconobacter species. A prerequisite before 5-KF can be used as a sweetener is to test whether the compound is degradable by microorganisms and whether it is metabolized by the human microbiota. We identified different environmental bacteria (Tatumella morbirosei, Gluconobacter japonicus LMG 26773, Gluconobacter japonicus LMG 1281, and Clostridium pasteurianum) that were able to grow with 5-KF as a substrate. Furthermore, Gluconobacter oxydans 621H could use 5-KF as a carbon and energy source in the stationary growth phase. The enzymes involved in the utilization of 5-KF were heterologously overproduced in Escherichia coli, purified and characterized. The enzymes were referred to as 5-KF reductases and belong to three unrelated enzymatic classes with highly different amino acid sequences, activities, and structural properties. Furthermore, we could show that 15 members of the most common and abundant intestinal bacteria cannot degrade 5-KF, indicating that this sugar derivative is not a suitable growth substrate for prokaryotes in the human intestine. Key points • Some environmental bacteria are able to use 5-KF as an energy and carbon source. • Four 5-KF reductases were identified, belonging to three different protein families. • Many gut bacteria cannot degrade 5-KF.

5-Keto-D-fructose (5-KF) is a natural diketone occurring in micromolar concentrations in honey, white wine, and vinegar. The oxidation of D-fructose to 5-KF is catalyzed by the membrane-bound fructose dehydrogenase complex found in several acetic acid bacteria. Since 5-KF has a sweetening power comparable to fructose and is presumably calorie-free, there is great interest in making the diketone commercially available as a new sugar substitute. Based on a genetically modified variant of the acetic acid bacterium Gluconobacter oxydans 621H, an efficient process for the microbial production of 5-KF was recently developed. However, data on the toxicology of the compound are completely lacking to date. Therefore, this study aimed to investigate the effect of 5-KF on the viability of prokaryotic and eukaryotic cells. It was found that the compound significantly inhibited the growth of the gram-positive and gram-negative model organisms Bacillus subtilis and Escherichia coli in a concentration-dependent manner. Furthermore, cell viability assays confirmed severe cytotoxicity of 5-KF toward the colon cancer cell line HT-29. Since these effects already occurred at concentrations of 5 mM, the use of 5-KF in the food sector should be avoided. The studies performed revealed that in the presence of amines, 5-KF promoted a strong Maillard reaction. The inherent reactivity of 5-KF as well as the Maillard products formed could be the trigger for the observed inhibition of prokaryotic and eukaryotic cells.