Summary of video content

The most abundant residues generated in the filleting processes of fish species, including aquaculture ones, are heads, trimmings, frames, skins, and viscera.

These wastes are commonly used for the production of silages or fish meals and fish oils of variable quality. However, they are materials rich in proteins that can be used as substrates for the production of enzymatic hydrolysates of remarkable nutritional and functional properties.

The first step necessary to advance in the process of hydrolysis, ensuring better homogeneity, is by grinding the substrate/residue to be processed.

Using a fully equipped 5 L reactor with strict control of pH, temperature, stirring and reagent addition, the substrate is first introduced into the reactor and mixed with the volume of water adequate to maximize hydrolysis performance.

The operating conditions are set at optimal values and the process begins when the proteolytic enzyme (protease), selected for each type of substrate, is added to the reactor at a defined concentration.

Working with this type of pH-stat reactor we can online monitor the degree of hydrolysis of the process.

This is due to the consumption of alkalis, to maintain the pH in the pre-established conditions, which is directly proportional to the breakdown of the peptide bonds present in the protein substrate.

Once the hydrolysis time has been reached, the content of the reactor is passed throughout a filter or sieve with a suitable pore size to separate non-enzymatically digested substrates, basically remains of bones, from the liquid pre-hydrolysate.

This liquid fraction is then centrifuged at high speed to help in the separation of the lipid phase from the protein-rich aqueous phase. The fat fraction is finally recovered after a short decantation time of the supernatant. The oil yield is a function of the type of substrate and the species used. With low-fat species and substrates, this step of oil recovery can be omitted.

The liquid hydrolysate free of high concentration of oil is quickly heat-treated (90-95°C for 10-15 min) in order to achieve total inactivation of the protease and thus end the hydrolysis process.

Although the hydrolysate obtained can be used in its liquid form for many applications, the most habitual way for conservation and use is as a solid.

For this, a drying step is necessary, which is commonly carried out by lyophilization or spray drying. In the first case, after freezing the sample and applying an intense vacuum in the drying chamber, the hydrolysate is dehydrated by means of a sublimation process.
Finally, the dry hydrolysate (FPH) generated is ground, vacuum packed, and stored under refrigeration in order to avoid oxidation and contamination.

The production of the hydrolysates on a pilot plant scale generally follows the same sequence of operation already described.

The fish wastes, once crushed, are introduced into a 300-liter pH-stat reactor to which is mixed with the volume of water necessary to maintain the most suitable solid: liquid ratio for hydrolysis.

After optimal temperature and pH conditions are established, the enzyme is also added to the reactor.

The concentration of protease, time of catalysis, and operational conditions are selected at the levels that maximise the proteolytic activity of the enzyme and leads to a degree of hydrolysis, chemical composition, and a peptide profile suitable for the end use of the hydrolysate.

After the recovery of the oil in a tricanter and the deactivation of the enzyme, an intensive concentration step is required.

Starting, as in this case, with a massive volume of hydrolysate (around 250 liters), the drying stage is very energy-demand and time-consuming if a significant reduction of the hydrolysate volume is not achieved.

A concentration step in a vacuum evaporator allows us a decrease in volume of 5 to 10 times in a reasonable operating time.

Finally, a final step of drying by means of a spray dryer leads us to obtain the final FPH of fish wastes.

The most relevant analytical determination that must be carried out to characterise the liquid hydrolysates, is by means of the quantification of soluble protein content using, for example, the method of Lowry.

This protocol combines the reaction of copper ions with peptide bonds in an alkaline medium together with the oxidation of aromatic residues of proteins.

The intensity of the blue-purple color generated is dependent on the protein concentration present and directly quantifiable by visible spectrophotometry at 750 nanometers.

The data of absorbance are then transformed into protein values using a calibration curve of a commercial protein measured with the same analytical protocol.

Once the dry hydrolysate has been obtained, its proximal composition must be determined, this is, the quantification of the presence of moisture, ash, organic matter, total fat, and total protein.

Solid samples are weighed in tubs, dried in an oven at 105ºC for one day, reweighed to determine the moisture content, and dried again in a muffle at 550ºC to subsequently determine ash and organic matter by gravimetry.

The presence of fats in the hydrolysate that could not be separated by combining centrifugation and decantation procedures are analyzed by Soxhlet extraction with organic solvents and are also quantified by gravimetry.

For its part, total protein is based on the quantification of total nitrogen by Kjeldahl digestion, reaction with salicylate/cyanurate, spectrophotometric measurement, calibration against an ammonium sulfate curve, and transformation into total protein using a conversion factor.

Knowing the average molecular weight of the hydrolysates together with the size distribution of the peptides present is extremely important to guide the type of application and also correlate the potential bioactivities of FPH.

For this, we use gel permeation chromatography. The sample of hydrolysate is injected into an HPLC, passing through a set of protein-specific size exclusion columns, and analysed simultaneously with a refractive index detector and a double angle light scattering detector.

The times and volumes of elution of the sample are proportional to the molecular size of the protein material.