Alpha 2 4 lsc plus

Microstructural images of the ice cream samples were created by scanning electron microscopy (SEM) at an acceleration voltage of 10 kV and high beam current (CamScan MV2300, EO Elektronen-Optik-Service GmbH, Dortmund, Germany). Frozen samples were prepared for microstructure observation with a rotational vacuum freeze-dryer, ALPHA 2–4 LSCplus (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany), at temperatures lower than the glass transmission temperature to protect the actual structure of the ice cream [28 (link)]. Then, the samples were prepared for SEM by polishing, and they were sputter-coated with a thin layer of gold. Measurements were taken with a secondary electron (SE) detection system, and photographs were taken at various levels of magnification [29 (link)].

The enzymatic hydrolysis was performed 10 times, with an amount of 1 kg minced raw material each time. Hydrolysis experiments were performed in 4 L closed glass vessels placed in a water bath at 52 °C. Warm (50 °C) distilled water was added to fish mince in a 1:1 ratio. The mixture was stirred at 150 rpm with an overhead stirrer. When the temperature of the mixture was 50 °C, the enzymes Papain F6 and Bromelain 400 (Enzybel International S.A., Villers-le-Bouillet, Belgium) were added at levels of 0.05% (w/w) (0.1% in total). After 60 min of hydrolysis, bones were removed by filtering the hydrolysate through a sieve before the enzymes were inactivated by heating up to 90 °C for 10 min in a microwave oven. The mixture was cooled down up to 30 °C before being transferred to 1 L centrifugation bottles and then centrifuged at 4100 g at 4 °C for 30 min. The liquid fraction (lipids and water-soluble proteins) was separated from the insoluble fraction. The liquid fraction was placed in a separatory funnel and allowed to settle, and then separated into oil and water-soluble proteins. The water-soluble protein phase representing fish protein hydrolysate (FPH) was collected and dried in the laboratory vacuum freeze-dryer Alpha 2–4 LSC Plus, equipped with a LyoCube 4–8 chamber (Martin Christ GmbH, Germany) as described below.

Vacuum-freeze drying was performed by the following procedure. The liquid samples were frozen at −24 ± 1.0 °C with the average thickness 0.010 ± 0.003 m. When the freezing process was completed and the temperature in the core of the sample reached the value of −24 °C. The samples were transferred into the laboratory vacuum freeze-dryer Alpha 2–4 LSC Plus, equipped with a LyoCube 4–8 chamber (Martin Christ GmbH, Germany). The drying pressure was set at 0.3 mbar. The temperature of the samples was slowly increased (0.4 °C min⁻¹) from −24.0 to 10.0 °C during 48 h. As a result, the moisture content in the dried samples was in the range between 2.0 and 3.0% w.b. Then the dried samples were milled into powder by ball mill Pulverisette 6 (Fritsch, Germany) and vacuum-packed (50 mbar) in low density polyethylene bags.

The chemical characterization of the AcUF was performed after lyophilization (Alpha 2-4 LSC plus, Christ, Osterode am Harz, Germany).

In order to obtain the samples for LC-MS analysis, liquid extracts (the samples obtained under the optimal extraction conditions in every employed extraction technique for achieving the highest polyphenol content) were dried. Additionally, the samples for FT-IR spectroscopy, antimicrobial, and skin regeneration potential analyses were lyophilized. The ethanol from the extracts was evaporated using Heizbad Hei-VAP (Heidolph, Schwabach, Germany) at 40–50 °C, a pressure of 50 mbar, and a rotation speed of 150 rpm. Subsequently, the sample was frozen in the freezer, at −80 °C for 1 h, and freeze-dried at −75 °C and pressure of 0.011 mbar for 24 h and at −65 °C and pressure of 0.054 mbar for one additional hour (Alpha 2-4 LSCplus, Christ, Osterode am Harz, Germany).

The optimized formulations were freeze-dried using a laboratory freeze-drier (Alpha 2-4 LSC Plus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). The following conditions were used for the preparation of the vaccine formulations under sterilized conditions: (1) freezing at −80 °C for one hour; (2) freezing at −50 °C for 20 min; (3) primary drying at −15 °C for 2 h and then 0 °C for 1 h; (4) secondary drying at 25 °C for 30 min at 0.100 mbar.