Fructose

Example 9

NEBT7EL-pA06238 was grown on LB with 50 μg/ml kanamycin. A 600 ml culture of TB_kan50 was inoculated with NEBT7EL-pA06238 and incubated overnight at 37° C. at 200 rpm. The next morning, a 10 L fermentor was prepared with 9.5 L of TB and then inoculated with 500 ml of the overnight culture. The culture was grown at 37° C. The pH was maintained at 6.2 with NaOH and the dO₂ was maintained ≥20%. After 2 hours of growth, the temperature was dropped to 25° C. The culture was grown for an additional 1 hour with the OD₆₀₀ around 7. IPTG was added to a final concentration of 1 mM and CoCl₂ was added to 25 μM. Additional CoCl₂ was added 1 and 2 hours after induction to bring the final concentration to 300 μM. The cells were grown for 20 hours at which point the fermentor was chilled to 10° C. and the cells were harvested by centrifugation. The cell pellet was stored at −80° C. until use.

The cell pellet from the fermentation was lysed by stirring in buffer with lysozyme and DNAse. Cell debris was removed by centrifugation and the supernatant was filtered through a 0.45 micron filter. Filtered supernatant was incubated with Ni-NTA agarose resin and then enzyme was eluted with imidazole. Purified FC4E pA06238 was immobilized onto 5.25 grams of ECR8204F resin using the standard published protocol from Purolite.

The immobilized enzyme was loaded into a 11×300 mm glass fixed bed reactor and run for approximately 200 h at constant temperature (60° C.) with a constant feed composition of 30 wt % fructose+70 wt % aqueous buffer solution (20 mM KPO₄, 50 mM NaCl, 300 uM CoCl₂). Feed rate was held constant at 140 uL/min throughout the run. The fixed bed reaction reached a maximal conversion of approximately 30% tagatose and had a half-life of −50 hours (FIG. 15).

Example 14

An adult patient with dietary fructose intolerance presents with one or more of symptoms such as abdominal bloating, flatulence, pain, distension, diarrhea and nausea. Treatment with the preparation of the invention is initiated by the clinician at an effective dose, which mitigates fructose-induced symptoms. Assessment of symptoms and testing are periodically performed. The dose of the treatment is adjusted as required by the clinician in attendance to manage symptoms of the dietary fructose-related condition. The subject may be treated with other drugs concurrently and may or may not be under restricted diet. Treatment with the preparation of the present invention is able to mitigate one or more symptoms related to dietary fructose.

EXAMPLE 8

Diet Cookies

Flour (50.0%), margarine (30.0%) fructose (10.0%), maltitol (8.0%), whole milk (1.0%), salt (0.2%), baking powder (0.15%), vanillin (0.1%) and different glucosyl Stevia compositions (0.03%) were kneaded well in dough-mixing machine. The obtained dough was molded and baked in oven at 200° C. for 15 minutes. Glucosyl Stevia compositions were by represented by Samples 1a, 2a, and 3, obtained according to EXAMPLES 3, 4, and 5, respectively; with Sample 4 being a commercial β-amylase treated product (containing only mono- and di-α-1,4-glucosyl-derivatives of steviol glycosides).

The sensory properties were evaluated by 20 panelists. The best results were obtained in samples prepared by high purity short-chain glucosyl Stevia compositions (containing four or less α-1,4-glucosyl residues) derivatives (Samples 1a and 2a). The panelists noted rounded and complete flavor profile and mouthfeel in cookies prepared with Samples 1a and 2a.