Impact of thermal extrusion and microwave vacuum drying on fatty acids profile during fish powder preparation

Abstract The current study aimed to optimize the process for ready‐to‐eat extruded fish powder preparation and to ascertain the impact of two methods on fatty acid profiles. For the investigation, Labeo rohita (Rohu) fish was first minced, extruded, and microwave vacuum‐dried. The results show that the yield for extruded fish powder (EFP) fluctuated from 22.32% to 29.07%. The maximum retention for docosahexaenoic acid (DHA), arachidonic acid (AA), and eicosapentaenoic acid (EPA) was 3.24 ± 0.08 g/100 g lipids, 2.74 ± 0.05 g/100 g lipids, and 1.24 ± 0.09 g/100 g lipids, by using different extrusion parameters. Moreover, nonsignificant changes were observed during 0 days, and 1 and 3 months of storage (at 4°C and 25°C) for DHA, AA, and EPA, whereas significant results were recorded for the samples stored for 6 months at 25°C. Also, the maximum peroxide value (PV) and thiobarbituric acid reactive substance values (TBARS) were 1.72 ± 0.04 meq/kg fat and 0.135 ± 0.008 mg malondialdehyde/kg fat. It is anticipated from the outcomes that the study will be helpful to prepare value‐added food products in future studies.


| INTRODUC TI ON
Fish is among the most common and nutritious seafood consumed throughout the globe. In Pakistan, the total fish production in the year 2017-2018 was estimated as 482,000 million tons (Govt. of Pakistan, 2017). Among different fish varieties, Rohu is mostly consumed in Pakistan and India (Latif & Faheem, 2020), due to its nutritional and sensorial attributes. Numerous studies have proved the bioactive composition of fish meat which helps in preventing cardiovascular ailments, atherosclerosis, acts as antithrombotic, and functions against arrhythmia (Šimat et al., 2020;Ucak et al., 2021).
However, being perishable and high-fat content, the seafood is enlisted in a highly perishable food class. Therefore, various processing technologies are being used to develop fish products for long-term consumption and storage. In addition to other sea processed products, fish powder (FP) consumption is increasing and it acts as an important ingredient for enriched foodstuff (Manzoor et al., 2021;Shaviklo et al., 2010). FP is reported to carry polyunsaturated fatty acids (PUFAs) including docosahexaenoic acid (DHA), arachidonic acid (AA), and eicosapentaenoic acid (EPA). The health-promoting fatty acid profile acts as antithrombotic and hypocholesterolemic, and performs other physiological actions (Tocher, 2015). Furthermore, DHA is helpful for brain development (Echeverría et al., 2017) and eye retina of newborn children (Jacques et al., 2011). Similarly, AA acts as a precursor in products such as anandamide, isoprostanes, and epoxides (Tallima & El Ridi, 2018). The National Institute of the United States recommended 650 mg of fish fatty acids daily (Chew et al., 2015).
However, PUFAs are highly susceptible to oxidative which can render the quality of fish and make it unfit for consumption. Novel processing approaches helped to prepare FP, having a longer shelf life. Extrusion is one of the processing technologies in which high temperature and smear force are used to produce a product with improved biochemical characteristics (Ahmad et al., 2020). This study aimed to process Rohu fish into dried extruded fish powder (EFP), with limited oxidation of bioactive fatty acids.

| Procurement and preparation of fish meat
Rohu fish (1,400 ± 150 g) was purchased from the local market of Faisalabad, Pakistan. Fish guts (fins, head, and tail) were removed following deboning and mincing. A spinning blade grinder (Duronic CG250-250W motor, UK) was used for mincing, then exposed to thermal extrusion, and finally, microwave vacuum-dried to form FP as the final product.

| Thermal extrusion
The twin-screw extruder was used for thermal extrusion  Hunan Fumake Engineering Technology Co., Ltd., Hunan, China). It had a screw width of 36 mm with a length to distance proportion of 24:1 and kick the bucket measurement of 36 × 4 mm. It had controlled zones for thermal cooking with a temperature estimating test and a screw speed controller. The Box-Behnken design was used for the optimization. The conditions of barrel external temperature (BET) for the optimization ranging from 100 to 150°C, screw speed (SS) of 50-150 rpm, feed flow rate (FFR) of 30-90 kg/hr, feed moisture content (FMC) of 10%-30%, and FP 0%-25%. Coding techniques were used for the experimental design shown in Table 1.

| Microwave vacuum drying
For the drying of samples, a small-scale vacuum dryer (WZD2S; Nanjing Sanle, China) was employed. The equipment had the potential to dry 8-12 kg fish samples in controlled temperature and pressure. The dryer was adjusted to 1,300 W power, the frequency was set to 2,450 MHz frequency, and a sample was kept at 90 Pascal pressure for 4.5 hr (50°C). For keeping the quality of n-3 and n-6 fatty acids in the finished product, low temperature and pressure were used in the study.

| Grinding and yield
Grinding was done to produce a homogenous particle size of powder using a grinder (Duronic CG250-250W motor, UK). The dried powder was successfully packed in polyethylene bags. The yield was calculated according to the AOAC method (923.07) (Latimer, 2012).

| Preparation of sample for fatty acid (FA) analysis
PUFAs of EFP were calculated according to AOAC,Method No. 923.07 (Latimer, 2012). Fatty acid methyl esters (FAME) were synthesized by following the method proposed by Carvalho et al. (2005). In the tube (16 × 150 mm), 1 g sample was taken in tube (16 × 150 mm) and then added 10 ml of hexane containing 0.1% butylated hydroxyl toluene. After that mixed by shaking and placed in an ultrasound water bath for 5 min. It was centrifuged at 1,500 g. The hexane was heated at 60°C to evaporate it from the sample mixture. Aspirated sample with nitrogen for 10 s, in 50 mg of sample, 1.0 ml toluene, and 2.0 ml boron trichloride-methanol. The samples were heated further at 60°C for 10 min. Finally, 2.0 ml of hexane and 2.0 ml of cooled water were added to a test tube for the extraction of FAME.
The moisture was removed by adding anhydrous sodium sulfate, and dehydrated FAME was transferred to a volumetric flask (10 ml) and made up the volume with hexane.

| Gas chromatographic assessment
For GC analysis, 1.0 μl FAME sample was introduced with helium (as carrier gas) with a speed of 1 ml/min, whereas the column oven temperature was adjusted to 160°C with a gradual increase in 3°C/ min until it reaches 180°C. Additionally, the column oven temperature was raised from 180°C to 220°C at 1°C/min speed. At 220°C, it was kept for 7.5 min; furthermore, the split ratio was set at 50% with injector temperature of 240°C and detector temperature of 250°C.

| Retention of FAs
For FA retention calculations, the following equation was employed:

| Peroxide value analysis
A sample of 5 g FP was taken in a conical flask. 30 ml of acetic acid, 20 ml chloroform, and 1 ml potassium iodide solution were added to it and stayed in a dark place for 30 min. 1 ml of starch solution (1%) and 50 ml of water were mixed into the solution. The final solution was then titrated with sodium thiosulfate solution (0.1 N) until the colorless endpoint is obtained (Ranganna, 1986). The following equation was used to determine the PV of FP.

| Thiobarbituric acid reactive substance (TBARS) test
For TBARS analysis, 5 g of sample was first homogenized with trichloroacetic acid (11%) for 1 min (at 5,400 g) using a homogenizer (IKA, Wilmington, USA). Then, the sample was kept in ice for 1 min followed by homogenization for 1 min again. Further, separation of homogenate was performed with Whatman's No. 1 and mixed 1 ml (20 mM) thiobarbituric acid followed by incubation for 20 hr (at 25°C) in a darkroom. Finally, the absorbance was ascertained using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan) at 532 nm wavelength. The obtained results were calculated in mg of MDA/kg.

| Statistical analysis
The optimization of microwave and extrusion conditions was performed through RSM using Design-Expert 11 software of statistics.
The experiment analyses were conducted through the method of Montgomery (1991). All experiments were conducted in triplicates and average values were considered as mean values. The significance of values was calculated statistically through mean using analysis of variance (ANOVA) at a probability of 0.05.

| Yield and proximate composition of fish and EFP
The effects of thermal extrusion conditions of BET, SS, FFR, and FMC were examined for EFP preparation, yield, and retention of DHA, EPA, and AA. The average yield for EFP as a result of different operating conditions differed from 22.32% to 29.07%. Whereas the total fat content was 9.62 ± 0.43% and the total crude protein was 67.28 ± 1.09% in EFP, the values for CHO, moisture, and ash were 11.55 ± 0.78%, 4.52 ± 0.31%, and 6.93 ± 0.90%, respectively ( Table 2). The fatty acid profile of fish meat and EFP is presented in

| Oxidative stability of EFP
The maximum value observed for TBARS at 0 days with 150 BET and 10 FMC was 0.135 ± 0.008 and the minimum value examined at 100 BET and 30 FMC was 0.006 ± 0.001 as shown in Table 8. The values for TBARS at 4°C and 25°C were significantly different in 1, 3, and 6 months at different processing conditions (Table 8).

| D ISCUSS I ON
In addition to other health benefits, fish and fish products are vital for cardiac health and the proper functions of the eye. Fish is a good source of PUFAs (DHA, AA, and EPA) which have gained consumer attention in the last three decades. Therefore, we processed fish into powder which was a colorless and odor product. Protein content dominates (75%) in the product with essential minerals and fat content; therefore, the EFP can be incorporated in food items as a source of protein, minerals, and fat. One type of fish differs from others due to the presence of long-chain PUFAs, DHA, or configuration of the fatty acids profile.
However, the design of the dryer, liquid material, operating conditions, and fish composition are responsible for the end product and dried powder (Deis, 1997). Keeping in view the importance of PUFAs for human health, we also studied their storage stability at two different temperatures (4°C and 25°C) for 1, 3, and 6 months with different processing conditions.
Some recent studies were also reported on the nutritional significance of fish and fish products. For instance, Abbey et al. (2017) prepared FP from different species and determined 28 to 80 g/100 g crude proteins, 4.7 to 11.5/100 g crude fat, 3.4 to 14.2 g ash content, and 3.5 to 8.4 (g/100 g) moisture content, which were comparable with our findings. Also, carbohydrate content ranged from 5.39 to 7.53 g/100 g, which was not comparable to our findings. The difference in results might be due to different processing conditions and types of fish used.