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Beta-lactoglobulin (beta-LG) was treated with high hydrostatic pressure (HHP) at 600 MPa and 50 degrees C for selected times as long as 64 min. The intrinsic tryptophan fluorescence of beta-LG indicated that HHP treatment conditions induced a conformational change. HHP treatment conditions also promote a 3-fold increase in the extrinsic fluorescence of 1-anilinonaphthalene-8-sulfonate and a 2.6-fold decrease for cis-paraneric acid, suggesting an increase in accessible aromatic hydrophobicity and a decrease in aliphatic hydrophobicity. Far-ultraviolet circular dichroism (CD) spectra reveal that the secondary structure of beta-LG converts from native beta-sheets to non-native alpha-helices following HHP treatment, whereas near-ultraviolet CD spectra reveal that the native tertiary structure of beta-LG essentially disappears. Urea titrations reveal that native beta-LG unfolds cooperatively, but the pressure-treated molecule unfolds noncooperatively. The noncooperative state is stable for 3 months at 5 degrees C. The nonaccessible free thiol group of cysteine121 in native beta-LG became reactive to Ellman's reagent after adequate HHP treatment. Gel electrophoresis with and without beta-mercaptoethanol provided evidence that the exposed thiol group was lost concomitant with the formation of S-S-linked beta-LG dimers. Overall, these results suggest that HHP treatments induce beta-LG into hydrophobic molten globule structures that remain stable for at least 3 months.

Ultrasound in the 20-1000 kHz range show unique propagation characteristics in fluid media and possess energy that can break down fruit matrices to facilitate the extraction of valuable bioactive compounds. Red raspberries carry significant amounts of specific antioxidants, including ellagitannins and anthocyanins that are important for human health. The objective of this study was to investigate the effects of ultrasound frequencies associated with cavitation (20 kHz) and microstreaming (490 and 986 kHz) on total antioxidant activity (AOA), total phenolics content (TPC), and total monomeric anthocyanin content (ACY) of red raspberry puree prepared from crushed berries. The pureed fruit was subjected to high-intensity (20 kHz) and higher frequency-low intensity (490 and 986 kHz) ultrasound for 30 min. The temperature of treated purees increased to a maximum of 56 °C with 986 kHz. Sonication at 20 and 490 kHz significantly (p<0.05) affected the AOA, ACY, and TPC of red raspberry puree, while 986 kHz had no significant effect on ACY and AOA (p<0.05). In all cases, ultrasound treatment had significant and positive effect on at least one of the measured parameters up to 30 min. Sonication beyond 10 min (and up to 30 min) using 20 kHz either produced no change or caused a drop in AOA and ACY. However, for 986 and 20 kHz, TPC, increased by 10% and 9.5%, respectively after 30 min (p<0.05) compared to the control. At 20 kHz, AOA and ACY increased by 17.3% and 12.6% after 10 min. It was demonstrated that 20 kHz ultrasound treatment, when limited to 10 min, was the most effective for extraction of bioactive compounds in red raspberry compared to 490 and 986 kHz although the effect could be similar at the higher frequencies if different amplitudes are used.

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