Determinação dos isômeros lactulose e lactose em leite UHT para a avaliação dos processos térmico e hidrólise enzimática por eletroforese capilar de zona com detecção no ultravioleta

Abstract

The UHT (ultra-high temperature) milk is the main product in the lactose-free dairy category, also known as “lactose-hydrolysed”. The feasibility of an analytical method for monitoring the UHT and lactose hydrolysis processes is necessary for both the industry, as it allows standardization and cost reduction, as well as for the regulatory agencies, to verify the adequacy of the product in the current legislation. The main goal of this thesis was to optimize and apply the capillary zone electrophoresis with indirect detection at ultraviolet region (CZEUV) for conventional UHT (regular content of lactose – REG) and lactose-hydrolysed (LH) milk to monitoring the UHT process and the enzymatic hydrolysis of lactose by determining lactulose and lactose isomers, respectively. For this, the optimization of instrumental parameters such as injection pressure, applied tension and temperature, as well as different sample preparations using acidic and organic agents were done. Quantification limits of 9.4 mg 100 mL-1 and 0.024 g 100 mL-1 for lactulose and lactose, respectively, intra-laboratory variability from 0.83 to 3.60% and recovery range between 86 and 102% were obtained. The improvement of sample preparation using trichloroacetic acid, as well as the adjustment of the instrumental parameters provided an electrophoretic run of 12 minutes. The determination of the isomers was achieved in 116 minutes using the single point standard addition quantification method. At the same time, the thermal indicators 5-hydroxylmethyl-2- furaldehyde (HMF) and the Whey Protein Nitrogen Index (WPNI) were also assessed by spectrophotometry and turbidimetry, respectively. The low lactose content of LH milk did not influence the WPNI index profile, and it can be used as a thermal indicator for REG and LH matrices. The HMF index was significantly higher in LH milk, and can distinguish the type of matrix (13.1 and 41.1 µmol L-1 , for REG and LH milk, respectively). LH samples showed lower lactulose levels than REG ones (13.0 and 35.6 mg 100 mL-1 , respectively). There were no differences between the electrophoretic and cryoscopic methods used to monitor in batch and aseptic hydrolysis processes, despite the CZE-UV method be ore sensitive. The optimization of the CZE-UV method allowed determining the lactulose and lactose isomers. Therefore, this method can be applied to monitor the UHT thermal processing, mainly for REG milk, and as a tool for inspection and control of the enzymatic hydrolysis process for LH milk.