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Project

Insight into non-enzymatic browning in shelf-stable orange juice during storage

Non-enzymatic browning (NEB) is an important quality defect during storage of shelf-stable fruit juices including orange juice. NEB in pasteurized shelf-stable orange juice is hypothesized to be due to chemical reactions involving the degradation of ascorbic acid and/or sugars. Nevertheless, the mechanism of NEB is still not fully elucidated. Until today, NEB is a shelf-life limiting factor of shelf-stable fruit juices. Therefore, there is a need for more research on NEB to gain a better understanding of this phenomenon.

This PhD work aimed to obtain more insight into NEB of pasteurized shelf-stable orange juice during storage using different approaches. In a first approach, a fractionation procedure followed by characterization was applied to investigate the changes occurring in different orange juice fractions such as serum, cloud, and pulp during storage and their relative contribution to NEB. As a first fractionation experiment, plain commercial pasteurized shelf-stable orange juice was stored for 15 weeks at 42 °C. After storage, different fractions of orange juice, namely serum, cloud, and pulp were separated by centrifugation followed by 70% ethanol precipitation, and further analyzed. Results showed that brown compounds formed during storage were present in all three fractions. In the orange juice serum, the brown compounds might be present in a free state; while in the orange juice insoluble fractions (cloud and pulp), the brown compounds were hypothesized to be associated with protein moieties (e.g., arabinogalactan proteins, protein-pectin complexes). However, as the molecular weight, the neutral sugar composition and the protein content of these fractions was not altered significantly by the presence of these brown compounds, they might account for only a small part of the insoluble fractions. In addition, the changes in soluble compounds present in the orange juice serum including ascorbic acid, sugars, furfural and 5-hydroxymethylfurfural (HMF) were highly correlated with the development of brown color during storage of the orange juice. As a second fractionation experiment, commercial plain orange juice was fractionated into different fractions containing soluble and insoluble compounds prior to storage. Thereafter, the plain orange juice and its derived fractions was pasteurized to achieve shelf-stable products and subsequently stored at 42 °C. The brown color development during storage of the fractions and the plain orange juice was investigated and the changes in different NEB-related attributes (i.e., ascorbic acid degradation, changes in sucrose, glucose and fructose content, and formation of furfural and HMF) were kinetically modelled. It was observed that those fractions containing soluble compounds (such as ascorbic acid, sugars) of orange juice turned brown, whereas, those fractions containing only insoluble compounds (such as polysaccharides) did not. Hence, our results suggested that soluble compounds of orange juice played an important role in NEB unlike the insoluble compounds. In addition, the estimated kinetic parameters showed that the brown color development, the changes in sugars, furfural, and HMF during storage of the soluble compound-containing fractions were similar to those of the plain orange juice. Therefore, the fraction containing only soluble compounds derived from the orange juice could be used as a highly relevant and adequate juice-based model system of reduced complexity for further NEB studies.

In a second approach, the aforementioned orange juice-based model system was used to study the effect of pH and addition of some NEB precursors such as ascorbic acid, fructose, and arginine on NEB. It was observed that lowering the pH of the juice-based model system from 3.8 to 1.5 accelerated the degradation of ascorbic acid, the hydrolysis of sucrose to glucose and fructose, and the formation of furfural and HMF during storage at 42 °C for eight weeks. This resulted in a higher browning intensity which became more pronounced towards the end of storage in the lower pH sample. Likewise, adding more ascorbic acid and fructose largely increased the formation of furfural and HMF, respectively, and resulted in a higher browning intensity. Addition of arginine to the orange juice-based model system, on the other hand, did not significantly alter the browning during storage. The latter observation might be because the natural presence of amino acids in the juice model system is more than enough for the browning reactions to take place so that excess addition of arginine does not further accelerate NEB. In conclusion, lowering the pH of the orange juice-based model system or addition of ascorbic acid or fructose but not arginine will enhance browning during prolonged storage.

As a third approach to gain further insight into NEB, the last part of this PhD work investigated the potential of 1H-NMR fingerprinting to study NEB in model systems and commercial orange juice. Different model systems with increasing complexity of different NEB precursors were formulated in citric acid buffer. As precursors, ascorbic acid, three sugars (sucrose, glucose, and fructose) and six main amino acids (proline, arginine, asparagine, aspartic acid, serine, and glutamic acid) were selected based on the composition of real orange juice. The model systems were pasteurized and stored at 42 °C for 16 weeks. The results of browning development of the model systems showed that the presence of ascorbic acid was essential for browning in the initial stage of storage. The browning intensity of the model systems containing ascorbic acid was enhanced in the presence of sugars and amino acids. In addition, sugars and amino acids played an important role in the browning in the later stage of storage. 1H-NMR fingerprinting was applied on (1) a simplified model system containing only ascorbic acid, (2) a complex model system containing ascorbic acid, sugars, and amino acids, and (3) plain commercial orange juice. In the model system containing ascorbic acid, the degradation of ascorbic acid to some intermediates such as xylonic acid, acetic acid, and erythrulose was the reaction pathway causing the major changes during storage. In a more complex model system containing ascorbic acid, sugars, and amino acids and the commercial orange juice, the hydrolysis of sucrose to glucose and fructose was identified as the main reaction leading to the differences in the samples along storage. These three sugars dominated the NMR spectra of the complex model system and the orange juice. Because of this, several important compounds for NEB such as ascorbic acid and its degradation products were not visible. Other more advanced NMR experiments such as 13C-NMR analysis and two-dimensional NMR analyses should be applied in future research to identify unknown compounds from NEB reactions.

In conclusion, a better understanding of NEB in orange juice during storage was obtained through the characterization of the changes in different juice fractions. Multiple reactions seem to be responsible for the browning of orange juice: ascorbic acid degradation is the most important pathway and the presence of sugars and amino acids enhances the browning. Besides the presence and concentration of NEB precursors, NEB can be affected by food intrinsic factors such as pH. Lowering of the natural pH of orange juice (e.g., to 1.5) accelerated the browning during storage.

1H-NMR fingerprinting is a nondestructive and fast technique to screen the changes in the orange juice and model systems induced by storage. Nevertheless, to gain mechanistic insight into NEB reactions using this technique, it should be combined with other more advanced NMR experiments and/or LC-MS/MS (liquid chromatography- tandem mass spectrometry) analyses.

Date:21 Sep 2015 →  15 Jun 2020
Keywords:Orange juice, Non-enzymatic browning, Storage
Disciplines:Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering, Food sciences and (bio)technology
Project type:PhD project