Involvement of Acetobacter orientalis in the production of lactobionic acid in Caucasian yogurt (“Caspian Sea yogurt”) in Japan

Occurrence of lactobionic acid (LacA) in the Caucasian yogurt. (A) The Caucasian yogurt was analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (bold line). Lactobionic acid, maltobionic acid (MalA), and cellobionic acid (CelA) standards were also analyzed. (B) Mass spectrometry analysis of Caucasian yogurt. The signals at (m/z)⁻ = 357 and (m/z)⁻ = 341 corresponded to those of LacA and lactose, respectively.

Mass/mass analysis of purified lactobionic acid (LacA). A sample of LacA purified from Caucasian yogurt was subjected to mass spectrometry analysis. The LacA signal [(m/z)⁻ = 357] was trapped and fragmented (A). The LacA standard was also fragmented under the same conditions (B). The m/z values of the major fragments are indicated above.

Production of lactobionic acid (LacA) in various layers of Caucasian yogurt. After fermentation, the yogurt was frozen and cut into 4 layers, as indicated in the figure. Each layer was treated as described in the Materials and Methods section, and the LacA concentration was measured by high-performance anion-exchange chromatography with pulsed amperometric detection.

Time course of lactobionic acid (LacA) accumulation in the upper layer of the Caucasian yogurt. The yogurt (10mL) was prepared in a 100-mL flask (thick, 0.5cm) at 27°C. Lactobionic acid (closed circle) and pH (open circle) were measured after various periods of fermentation.

Estimation of daily intake of lactobionic acid (LacA). The Caucasian yogurt (approximately 700mL) was prepared with a yogurt maker. After 12h of fermentation at 27°C, the yogurt was stored at 4°C. One hundred grams of yogurt was sampled at 12h and every 24h thereafter (36, 60, 84, 108, 132h) to measure the LacA concentration by high-performance anion-exchange chromatography with pulsed amperometric detection. Results are means±standard deviation (n = 3).

Lactobionic acid (LacA) production by resting cells cultured in yeast extract-polypepton-glucose-lactose (YPGL) liquid medium and milk. Acetobacter orientalis was cultured in 2.0mL of YPGL liquid medium (lane 3, 4) and milk (lane 5, 6) at 27°C for 2 d with (lane 4, 6) and without (lane 5, 7) shaking. After washing, the cells were reacted with 2% lactose and 0.026 g/mL of CaCO₃ at 27°C with shaking (240 oscillation/min). After 7h of shaking, the reaction mixture was analyzed by thin-layer chromatography. Lanes 1 and 2 are the LacA and lactose standard, respectively.

Time course of lactobionic acid (LacA) production in washed Acetobacter orientalis cells. Acetobacter orientalis was cultured in yeast extract-polypepton-glucose-lactose liquid medium at 27°C for 2 d. The washed A. orientalis cells collected from 10mL of culture broth were shaken (240 oscillations/min) with 2% lactose at 27°C. Calcium carbonate (1/2 equimolar amount of lactose) was added for neutralization. The concentration of LacA was measured by high-performance anion-exchange chromatography with pulsed amperometric detection.

Substrate specificities of Acetobacter orientalis cells. Acetobacter orientalis was cultured in yeast extract-polypepton-glucose-lactose liquid medium at 27°C for 2 d. Collected and washed cells were mixed with each sugar (10mM) and incubated at 27°C for 4h with shaking (240 oscillations/min). The reaction mixtures were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection. The amount of each aldonic acid from the respective substrates was calculated by using LacA (disaccharide) and d-gluconic acid (monosaccharide) as standards.