The reduction of tetrazolium XTT with the Maillard reaction system containing various sugars or aldehydes and glycine was monitored and compared with the conventional indices of the Maillard reaction such as nitroblue tetrazolium (NBT) and dichlorophenolindophenol (DPI) reducibility, free hydroxymethylfurfural and browning. The change in XTT reducibility against the monosaccharide-glycine system showed a similar behavior as that of NBT and DPI reducibility, indicating that the Alnadori product and reductones formed in the Maillard reaction may be involved in the reduction of XTT. In contrast, the XTT reducibility aga consisting of lactose, Iactulose or cellobiose showed a different behavior from those indices. In the system, the reduction of XTT was caused by the substances which have a low reducibility of NBT. Such a property was recognized in some model systems, including the condensation reaction of D-glucosamine, that can form nitrogen-containing heterocyclic compounds such as pyrazinium and pyridinium. The condensation product of D-glucosamine rapidly lost the XTT reducibility with the addition of Cu2+. The phenomenon was also observed in the UHT-treated milk, and the decrease in the XTT reducibility during the storage was suggested to be due to the presence of Cu2+ in milk.

Agricu/ture, Kochi University, Monobe B-200, Nankoku Co., Ltd., 1-2l-3, Sakae-cho, Higashimurayama, Tokyo 783, Japa n 189, Japan UHT-treated milk reduced 3'-{1-L(phenylamino)-carbonylJ-3,1~tetrazolium}-bis(4hmethoxy-6-nitro)benzenesulfonic acid hydrate (XTT). The reducibility was caused by the Maillard reaction intermediate between milk protein and lactose. First, the assay procedure of the XTT reduction was optimized using the heated model solution containing casein and lactose and UHT-treated milk. When the XTT reduction assay was applied to two kinds of UHT-treated milks under optimum conditions, the ability of each milk sample to reduce XTT significantly reflected the extent of the heat treatment as well as the evaluation method using the hydroxymethylfurfural (HMF) value. In contrast to the HMF value, the ability of UHT-treated milk gradually decreased depending upon the storage period and temperature. These results suggest not only that the present XTT assay is applicable to estimate the extent of heat treatment but also that the reducibility of the milk sample heat-treated under a given condition can serve to estimate the storage period if the storage temperature is known or vice versa. The method is much simpler (only mixing of the XTT solution containing menadione with milk sample) and quicker (about 20 min) with a satisfactory reproducibility than the conventional methods to estimate the extent of the Maillard reaction such as the lactulose or HMF determination.

and A spectrophotometric microtiter-based assay was established for quantification of the ability of UHT-treated milk to reduce 3'-{1-L(phenylamino)-carbonyl]-3,4~tetrazolium}-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate (XTT). Under the optimized conditions, 40 pl of a milk sample was mixed with OO pl of 0.5 mM XTr solution (pH 7.0) saturated with menadione in each well of a 96-well nricrotiter plate. The formed XTT formazan in the reaction mixture was determined by an increase in the absorbance difference between that at 492 nm and at 600 nm during the incubation for 20 min at ambient temperature. The use of the absorbance at 600 um as a reference wavelength permitted the determination of XTT formazan without any interference from the turbidity caused by the addition of the milk sample. The present microtiter plate assay could differentiate two kinds of UHT-treated milk with a different extent of thermal treatment. In addition, the reducibility of the UHT-treated milk gradually decreased during storage dependent on the period or temperature. The method was very convenient and economical; it is promising as a method of estimating the extent of the heat treatment of milk and the storage conditions.

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