Frozen ‘Beicun’ blueberry fruits were classified based on three key temperatures: the glass transition temperature (T_g’) at their maximum cryo-concentration, the intersection temperature (T_g”) between the freezing curve and the glass transition temperature curve, and the characteristic freezing endpoint temperature (T_m’) into three states: rubbery (T > T_m’), partially frozen concentrated (T_g” < T < T_m’), and glassy (T < T_g”). Blueberry fruits were quickly frozen in liquid nitrogen at −120 ℃ and separated into two storage groups. One group, without any state changes, was stored under rubbery (−18 ℃, T > T_m’), partially frozen concentrated (−40 ℃, T_g”< T < T_m’), or glassy state (−80 ℃, T < T_g”) conditions. The other group, with state changes, was initially stored at the glassy state temperature (−80 ℃) and then switched to the rubbery (−30 ℃, T > T_m’), partially frozen concentrated (−40 ℃, T_g” < T < T_m’), or lower glassy state temperature (−50 ℃, T_g’ < T < T_g”; −60 ℃, T < T_g’). Besides, a control group was set up without any temperature changes. Juice loss, hardness, nutrient contents, membrane integrity, and enzyme activities were determined to explore the effects of different freezing states and state changes on the quality of frozen blueberry fruits. The results indicated that frozen blueberry fruits in the glassy state exhibited the best quality. State transitions resulted in more pronounced quality deterioration in blueberry fruits; greater temperature changes led to higher juice loss, hardness reduction and nutrient loss, more serious cell integrity damage, and higher activities of peroxidase and polyphenol oxidase. Therefore, glassy state freezing (at temperatures below T_g’) could better maintain the quality of blueberry fruits; however, it is crucial to avoid state transitions caused by temperature fluctuations during frozen storage.

Here Maillard reaction time-temperature indicator (TTI) was prepared to observe the quality and remaining shelf life of mulberry during cold chain storage and transportation. The catalysts were used the xylose and glycine as the substrates and K₂HPO₄. The color change pattern of TTI was explored to adjust the concentration ratio of xylose, glycine, and K₂HPO₄. The results showed that the glycine concentration was 2.00 mol/L, when the xylose concentration was 1.00 mol/L. The attention of K₂HPO₄ was 1.00 mol/L, and the absorbance of TTI was higher. There were more stages of discoloration and more uniform discoloration. In addition, the intrinsic mechanism of the color change of TTI was also explored by FTIR and UV-visible absorption spectroscopy. The overall change trend of the spectra was slight with the long storage time of TTI. Still, there was no noticeable change in the FTIR spectra except for the weak difference in peak positions and intensities. The generation of new peaks was absence in the spectrograms. There was the benign effect of the mixture of xylose, glycine, whereas the K₂HPO₄ solution was produced a soothing effect. Meanwhile, C-O, C-H, C=O, C=C, C=N, amide I, amide II, amide III, C-N, N-H, and C-H were involved in the formation of melanoidin. The UV-vis absorption spectra showed that the absorbance change of TTI increased with the increase of storage time. The higher the temperature was, the shorter the time was required for the appearance of the characteristic peaks of color. The hardness, total soluble solids, titratable acid, and vitamin C in mulberries showed the different decreasing trends with the extension of storage time during refrigerated storage. By contrast, the weight loss rate and anthocyanins showed the increasing trends. Mulberry storage at ice temperature (-1℃) was favorable to maintain the quality of mulberry, which was used as the long-term refrigerated storage. The activation energies of 36.08, 40.42, 43.35, 38.28, 43.72, and 40.41 kJ/mol were calculated from the weight loss, anthocyanin, hardness, total soluble solids, titratable acid and vitamin C contents of mulberry at 25, 15, 10, 4 and -1 °C, respectively. The activation energy of the TTI was fitted to be 40.13 kJ/mol by the Arrhenius equation. There was no more than 25 kJ/mol difference in the calculated activation energy between each quality indicator and the TTI. As such, the quality change of mulberry at different ambient temperatures was more accurately matched with the time-temperature indicator. A chain-breaking simulation experiment was designed to verify the TTI monitoring under temperature fluctuation. Some changes were observed in the TTI color for the mulberry quality under different chain-breaking situations. The results showed that the abuse of temperature was accelerated the color change of TTI, even for the deterioration of mulberry quality. When the TTI color reached the endpoint, the mulberries began to decay. Furthermore, the change in TTI color was consistent with the shift in the mulberry quality. In addition, the kinetic analysis was performed by equivalent temperature. The difference between the comparable temperature values of TTI and each index of mulberry was calculated to be less than 1℃, thus verifying the monitoring ability of TTI. The TTI can be expected to monitor quality of mulberry for the long shelf life.

A series of experiments were carried out to explore the effects of combined immersion freeze-drying protectants (trehalose CaCl₂₎ and ultra-low temperature freeze-thaw pretreatment on the active substances and quality of blueberries. Directly dried blueberries were taken as the control group. Three groups were selected as the ordinary freeze-thaw (-20℃) , ultra-low temperature rapid freeze-thaw (-80 ℃), and ultrasound-assisted freeze-drying protectants immersion freeze-thaw (-20 ℃) , and (-80 ℃). A systematic comparison was implemented to determine various indicators of blueberry, including the active substances (PPO and POD enzyme activity), nutrients (VC, anthocyanins, total phenols, and flavonoids), and texture characteristics (hardness and chewiness). The experimental results show that the higher quality of blueberries was protected in the combination of a single freeze-drying protectant immersion or ultra-low temperature treatment, compared with the single one. Especially, the hardness was effectively maintained for blueberries that soaked and frozen at -80℃, while the retention of nutrients was significantly improved with less duration in vacuum freeze-drying. Three-factor three-level Box Behnken design was adopted to clarify the effects of soaking time, freeze-thaw frequency and freezing time on the comprehensive indicators of blueberries. It was found that the soaking time was 3.7 h, and the single freezing time was 4.2 d when the number of freeze-thaw cycles was 2. The highest comprehensive indicators of blueberries were achieved in the retention of nutrients. Feature and sum normalizations were performed on the experimental data. All data was between (0, 1) and the sum was 1. The lipid membrane interaction of calcium pectinate and trehalose was formed by the interaction between Ca²⁺ and the cell wall. There was a great reduction in the structural damage caused by freeze-thaw. At the same time, the finer ice crystals were formed in the ultra-low temperature environments. The quality of freeze-dried blueberries was significantly improved when combined with the freeze-dried protective agents. The freeze-drying protectant impregnation pretreatment and ultra-low temperature freeze-thaw were combined to improve the quality of vacuum freeze-drying blueberries. A systematic investigation was implemented to clarify the effects of immersion time, freeze-thaw frequency, and freezing time on the quality of dried products. The optimal parameters were then determined for the vacuum freeze-drying pretreatment. In summary, the combination of immersion freeze-drying protectant (trehalose CaCl₂) and ultra-low temperature freeze-thaw pretreatment can be expected to improve the active substances and quality of blueberries. The pretreatment conditions and parameters were optimized to successfully improve the nutrient retention and quality of blueberries. The findings can provide useful and practical references for the processing of blueberry products. The application scope can be further expanded for vacuum freeze-drying in the field of food processing.

The glass transition temperature T_g’ of ‘Northcountry’ blueberries was determined according to its state diagram. Using T_g’ as the freezing end point, blueberries were frozen at −80 ℃ using an ultra-low temperature freezer (RF−80 ℃), or at −80, −100 or −120 ℃ by direct immersion in liquid nitrogen (LN−80 ℃, LN−100 ℃, and LN−120 ℃, respectively). Freezing curves, juice loss, hardness, nutrient contents, cell membrane integrity, enzyme activities and microstructure were measured to investigate the effects of liquid nitrogen quick freezing combined with T_g’ on the quality characteristics of blueberries. The results showed that the T_g’ of blueberries was −52.55 ℃; compared with RF-80 ℃, the quality of liquid nitrogen frozen blueberries was better maintained. The freezing time of liquid nitrogen quick freezing was significantly shortened, the frozen fruit lost less juice after thawing, and the hardness was better maintained. The contents of soluble solids, titratable acid, ascorbic acid and anthocyanin were closer to those of fresh fruit, the relative conductivity and malondialdehyde (MDA) content were lower, the integrity of the cell membrane was less broken, the activities of peroxidase (POD) and polyphenol oxidase (PPO) were lower, and the microstructural compactness and integrity were stronger. The lower the ambient temperature of liquid nitrogen quick freezing, the better the freezing effect, and LN-120 ℃ minimized the quality deterioration of frozen blueberries. In conclusion, liquid nitrogen quick freezing significantly increased the freezing rate, and its combined with T_g’ helped to maintain good quality of frozen blueberries. The results of this study can provide a theoretical basis for the selection of blueberry freezing conditions.