Characterization of bioactive fatty acids and oxidative stability of microwave vacuum dried fish powder supplemented extruded product

Abstract

This study was aimed to determine the retention of bioactive fatty acids and oxidative stability of fish powder (FP) supplemented extruded products. The Labeo rohita fish meat was minced, microwave vacuum dried, and extruded. Product formulations containing FP (0-25%) and semolina (75-100%) were optimized using response surface methodology. The maximum bioactive fatty acids retention was 94.84% in the final product. The highest values of polyunsaturated fatty acids (PUFAs) were 87.97% and 85.66% at 25 °C for 30 and 60 days. Maximum TBARS values were 0.42 and 0.44 mg MDA/kg at 25 °C for 0 and 30 days, respectively. The highest carbonyl contents were 6.02, 7.67, 9.37 nmol/mg protein for 0, 30, and 60 days of storage at 25 °C, respectively. The moderate barrel exit temperature (125 °C) and feed moisture contents (20%) and high screw speed (150 rpm), FP (25%) exhibit the highest retention of PUFAs in a product.

Keywords:
fish powder; microwave vacuum drier; thermal extrusion; oxidative stability

1 Introduction

Fish has always been used as the richest source of essential PUFAs, including n-3 and n-6 which have beneficial effects on human health. PUFAs have the potential to address depression and other mental disorders (Ab Latif Wani & Ara, 2015). A docosahexaenoic fatty acid is also found abundantly in fish and essential nutrients for eye development, nervous system, and brain development of the fetus (Silva et al., 2015; Fu et al., 2017). The n-6 such as arachidonic acid and n-3 like eicosapentaenoic acid are have been proven precursors of lipid mediator signaling molecules, named “eicosanoids,” that play important roles in the regulation of inflammation (Patterson et al., 2012). The cross-sectional studies conducted on human beings have shown that the patients having a higher ratio of n-6 to n-3 demonstrated unpleasant moods while and lower has a significant effect on good mood by probably reducing the vulnerability to depression and cardiovascular diseases (CVDs) (Berger et al., 2017). Labeo rohita (rohu) is one of the prime species of fish cultivated in Asia. It has high commercial and nutritional values due to the high amount of PUFAs and white meat (Memon et al., 2011). Rohu belongs to the carp family and is abundantly found in freshwater lakes and rivers of Pakistan, Bangladesh, India, and Thailand. It is considered one of the most popular, crucial, and economic freshwater fish.

Drying is one of the ancient methods of preserving fish meat. The need for developing dried fish powder (FP) and the formation of functional and ready-to-eat foods is growing day by day (Shaviklo et al., 2013). The quality and safety characteristics of dried fish directly depend upon the source of raw material and process applied for drying purposes (Shaviklo et al., 2011, 2012). Novel processing is further helping to prepare FP which is helpful for the storage of fish for a longer period. The microwave vacuum drying (MVD) method is a combination of both microwave and vacuum drying as it has more uniform heat transferring potential and a more energy-saving process for all kinds of foodstuffs (Cui et al., 2008). While extrusion is a multi-functional and multi-purposes thermal and mechanical cooking process widely used in food industries to exhibit higher nutrition value containing essential amino acids and essential fatty acids along with pharmaceutical applications (Li et al., 2007). The geometry of extruders, food mix composition, and processing conditions play a vital role in physiochemical and nutritional changes in food material (Zhang et al., 2014; Shahzad et al., 2020). Research studies have reported that thermal extrusion causes complete inactivation of enzymes in food unlike that of high-temperature short-time processing methods. Most of the anti-nutritional substances are destroyed in extrusion processing. Proteins and starches processed through extrusion are more digestible and absorbable in the body than un-extruded products. It increases the bioavailability of amino acids and inhibits the activity of trypsin along with other growth-inhibiting substances (Singh et al., 2007; Ahmad et al., 2019).

The lipid oxidation is reduced even at high temperature and makes more absorbable in the human body. Lower temperature, residence time, and optimized mechanical shear give end product of higher quality retaining maximum omega fatty acids. Low lipid levels (less than 5%) aids in extrusion processing and greater product stability and expansion (Kouba & Mourot, 2011). During extrusion, most of the hydrolytic lipases are destroyed thus preventing catabolism of triglycerides into free fatty acids and glycerol. Short retention time inhibits lipid oxidation so maximum lipids get retained in the final product. Millard reaction products resulting in extrusion also act as antioxidants thus preventing lipid oxidation during extrusion (Kouba & Mourot, 2011). In this context, the basic purpose of this research was to examine the retention and oxidative stability of essential unsaturated fatty acids of FP supplemented extruded macaroni with high PUFAs contents.

2 Materials and methods

2.1 Procurement and preparation of sample

Rohu fish (weight of 1500 ± 150 g) was used as raw and experimental material for this research. Fish was purchased from local markets, Faisalabad-Pakistan. Head, fins, tail, and viscera of fish were removed following deboning and mincing then subjected to microwave vacuum drying in a microwave vacuum dryer.

2.2 Microwave Vacuum Drying (MVD)

A lab-scale microwave vacuum dryer (WZD2S, Nanking Sanle, China) was used for drying fish. It had a capacity of drying fish ranging from 8 to 12 kg of fish under different temperatures and pressure. It was optimized to the power of 1300W and operated at 2450 MHz frequency, 90 Pascal pressure for 4.5 hours at 50 °C. The dried minced powder was preserved in polyethylene bags.

2.3 Grinding of dried mince

After completion of the drying process, the dried mince fish was ground to powder form using a spinning blade grinder (Duronic CG250- 250W motor, UK).

2.4 Thermal extrusion optimization

The twin-screw extruder was used to extrude the products. It had a screw diameter of 36 mm with a length to diameter ratio of 24:1. It had a temperature control zone for thermal cooking with a temperature measuring probe and also had a screw speed controller as shown in the supplementary file (Wang et al., 2017). Optimization was performed using Box Behnken Design, RSM. Optimization conditions for BET were ranging from 100-150 °C, SS from 100-150 rpm, FFR from 30-90 kg/hr, FMC from 10-30% and FP from 0-25% (Imran & Anjum, 2014). Semolina (75-100%) was also used for making the final product. For the convenience of experimental design, coding was used which is presented in Table 1.

Table 1
Codes and actual levels of independent variables for optimization of response factors as determined by Box-Behnken design.

Independent variables	Units	Coded levels
		‒1	0	+1
Barrel Exit Temperature (BET)	°C	100	125	150
Feed Flow Rate (FFR)	kg/hr	30	60	90
Screw Speed (SS)	rpm	50	100	150
Feed Moisture Content (FMC)	%	10	20	30
Fish Powder (FP)	%	0	12.5	25

2.5 Preparation of sample for fatty acids analysis

Total lipids of FP and final product were estimated according to AOAC Method No. 923.07 (Ogbobe & Akano, 1993). Fatty acid methyl ester (FAME) of samples were prepared according to the method prescribed by (Carvalho & Malcata, 2005).

2.6 Gas chromatographic analysis

The FAME sample of 1.0 μL was injected with Helium (1 mL/min) as a carrier gas onto the column that was programmed for operating conditions such as column oven temperature 160 °C at 0 minutes with subsequent increase of 3 °C/min until 180 °C. The column oven temperature was increased from 180 °C to 220 °C at 1 °C/min and was held for 7.5 min at 220 °C. The split ratio was 50% with injector temperature of 240 °C and detector 250 °C. The peak areas and total fatty acids composition were calculated for each sample by retention time using Varian Chem Station software.

2.7 Retention of fatty acids

The retention of fatty acids was calculated according to the following Equation 1:

$\text{Fatty acids retention \%}=\frac{fattyacidspresentinproductafterextrusion}{fattyacidspresentinrawmaterial}X100$ (1)

2.8 Thiobarbituric acid reactive substances (TBARS) analysis

Product sample (5 g) was taken and homogenized with 11% trichloroacetic acid (TCA) for 1 min at 11000 rpm (Homogenizer: IKA, Wilmington, USA) and subsequently placed in the ice bath for 1.0 min and then homogenized for another 1.0 min. The homogenate was separated through Whatman No.1 and then added 1.0 mL of 20 mM thiobarbituric acid solution to 1.0 mL of filtrate following incubation for 20 hr in dark conditions at 25 °C. The absorbance value was determined at 532 nm through the UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The results were calculated as mg of MDA/kg.

2.9 Carbonyl contents analysis

The carbonyl contents were determined to calculate the oxidation of protein during extrusion and storage time. A sample of 3.0 g was homogenized with 0.15 M KCl (7.4 pH) at 7100 rpm for 90 seconds (Homogenizer: IKA, Wilmington, USA). The precipitation of protein present in the homogenate was done using 10% TCA and centrifuging at 5000 g, 4 °C for 5 minutes (ST 16R, Thermo Scientific, Wilmington, DE). The supernatant is discarded and then 10 mM 2, 4-dinitrophenylhydrazine (DNPH) in 2.0 N HCl was added in the precipitate and incubated at 25 °C for 1 hr in the dark with gentle vortexing after every 15 minutes. The substrate reacted with DNPH was then precipitated using TCA (10%) following centrifugation at 11000 g, 4 °C for 10 minutes. The precipitate was then washed three times using ethanol/ethyl acetate (1:1, v/v) solution and solubilized with guanidine hydrochloride (6M) in 20 mM sodium phosphate buffer having pH 6.5. Centrifuge again at 11000 g, 25 °C for 10 minutes to remove insoluble particles. Absorbance was estimated at 370 nm through the UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). Carbonyl contents were estimated through the absorbance coefficient for protein hydrazones. The obtained results were expressed as nmol of carbonyl/mg of protein.

2.10 Sensory evaluation

A trained panel of judges evaluated the product for sensory evaluation using a 9-point hedonic scale (Civille & Carr, 2015). Color, flavor, and overall acceptability were evaluated after product development and the same after a storage period of 60 days interval.

2.11 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 the probability of 0.05 through MATLAB® (Ver. 7.9.0) software (Mathworks, Inc., Natick, USA).

3 Results and discussion

3.1 Results

The powder obtained from fish under microwave vacuum drying contained 9.64 ± 32% total lipids that non-significantly changed during the drying process. The PUFAs that were determined and measured in this study were C18: 2 n-6, C18: 3 n-3, C20:3 n-7, C24:4 n-6, C25:5 n-3, and C22:6 n-3, while the MUFAs were C16:1 n-7, C18:1 n-9, and C20:1 n-9. The SFAs were C14:0, C15:0, C16:0, C17:0, C18:0, C19:0, and C20:0. The PUFAs, MUFAs, and SFAs were 14.30 ± 1.72, 27.60 ± 1.32 and 49.86 ± 2.37% in FP obtained from the drying process. The PUFA retention after different extrusion conditions is presented in Table 2. The highest PUFAs contents (607 ± 8 mg) were found in product with 25% FP extruded under BET (125 °C), SS (150 rpm), FFR (60 kg/hr), FMC (20%) conditions and minimum PUFAs (320 ± 2 mg) were present in a product without FP extruded under BET (125 °C), SS (50 rpm), FFR (60 kg/hr), FMC (20%) as shown in Table 2. The maximum PUFAs retention was 94.84% under optimizing conditions of BET (125 °C), SS (150 rpm), FFR (60 kg/hr), FMC (20%) and FP (25%) and in final products, while the minimum PUFAs retention (80%) was observed in a product at BET (125 °C), SS (50 rpm), FFR (60 kg/hr), FMC (20%) and FP (0%). The supplementary file shows how a response variable (PUFAs) relates to predictor variables. It shows that the response is connected to produce contour lines of constant responses.

Table 2
Effect of extrusion processing conditions on retention of polyunsaturated fatty acids at different storage intervals.

Extrusion processing run	Independent variables					Dependent variable
	Barrel Exit Temp. (BET)	Screw Speed (SS)	Feed Flow Rate (FFR)	Feed Moisture Content (FMC)	Fish Powder (FP)	PUFAs (mg/100 g)
						0 Day	30 Days	60 Days
1	150 (1)	150 (1)	60 (0)	20 (0)	12.5 (0)	444 ± 1cd	391 ± 3fg	385 ± 5fg
2	125 (0)	150 (1)	60 (0)	20 (0)	25 (1)	607 ± 8a	534 ± 6b	520 ± 2b
3 (C1)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	460 ± 6c	410 ± 3ef	401 ± 3f
4	150 (1)	100 (0)	60 (0)	30 (1)	12.5 (0)	408 ± 9ef	360 ± 2h	345 ± 4hi
5	100 (-1)	100 (0)	60 (0)	20 (0)	0 (-1)	380 ± 7g	340 ± 1i	329 ± 1j
6	100 (-1)	150 (1)	60 (0)	20 (0)	12.5 (0)	512 ± 9b	452 ± 2cd	440 ± 4d
7	125 (0)	150 (1)	30 (-1)	20 (0)	12.5 (0)	490 ± 8bc	433 ± 5de	424 ± 2e
8	125 (0)	50 (-1)	60 (0)	10 (-1)	12.5 (0)	409 ± 4ef	352 ± 3h	346 ± 4hi
9	150 (1)	100 (0)	60 (0)	20 (0)	0 (-1)	353 ± 7h	308 ± 6lm	303 ± 1m
10	125 (0)	100 (0)	60 (0)	30 (1)	25 (1)	539 ± 1b	471 ± 3c	461 ± 2c
11	125 (0)	100 (0)	90 (1)	10 (-1)	12.5 (0)	447 ± 6cd	386 ± 2fg	379 ± 5g
12 (C2)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	455 ± 3cd	401 ± 2f	391 ± 3fg
13	125 (0)	150 (1)	60 (0)	30 (1)	12.5 (0)	508 ± 5b	445 ± 5cd	436 ± 2d
14	125 (0)	100 (0)	60 (0)	10 (-1)	25 (1)	505 ± 9b	445 ± 2cd	436 ± 5d
15	125 (0)	100 (0)	30 (-1)	20 (0)	0 (-1)	360 ± 3h	319 ± 1k	311 ± 2ll
16	100 (-1)	100 (0)	60 (0)	10 (-1)	12.5 (0)	411 ± 5ef	360 ± 3h	354 ± 2h
17	100 (-1)	100 (0)	30 (-1)	20 (0)	12.5 (0)	427 ± 5de	371 ± 2gh	366 ± 3gh
18	150 (1)	100 (0)	90 (1)	20 (0)	12.5 (0)	394 ± 1fg	344 ± 2hi	338 ± 5i
19	125 (0)	50 (-1)	60 (0)	20 (0)	0 (-1)	320 ± 2k	285 ± 5n	277 ± 4o
20	125 (0)	100 (0)	60 (0)	10 (-1)	0 (-1)	363 ± 1gh	324 ± 1jk	316 ± 2kl
21	125 (0)	100 (0)	90 (1)	20 (0)	25 (1)	527 ± 3b	462 ± 3c	451 ± 5cd
22	125 (0)	150 (1)	60 (0)	20 (0)	0 (-1)	382 ± 5g	338 ± 2i	334 ± 1ij
23	125 (0)	100 (0)	90 (1)	30 (1)	12.5 (0)	465 ± 2c	419 ± 4e	397 ± 2f
24 (C3)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	459 ± 7c	405 ± 2f	390 ± 4fg
25	100 (-1)	100 (0)	90 (1)	20 (0)	12.5 (0)	433 ± 2de	387 ± 3fg	375 ± 2gh
26 (C4)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	452 ± 5cd	405 ± 2f	392 ± 4fg
27	125 (0)	50 (-1)	90 (1)	20 (0)	12.5 (0)	411 ± 1ef	369 ± 3gh	357 ± 6h
28	125 (0)	150 (1)	60 (0)	10 (-1)	12.5 (0)	466 ± 2c	409 ± 2ef	401 ± 4f
29	125 (0)	100 (0)	30 (-1)	20 (0)	25 (1)	514 ± 7b	459 ± 2c	438 ± 2d
30	125 (0)	100 (0)	60 (0)	30 (1)	0 (-1)	390 ± 2fg	343 ± 1hi	340 ± 1i
31 (C5)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	455 ± 3cd	405 ± 5f	394 ± 3fg
32	100 (-1)	100 (0)	60 (0)	30 (1)	12.5 (0)	449 ± 2cd	391 ± 3fg	387 ± 5fg
33	150 (1)	100 (0)	60 (0)	20 (0)	25 (1)	470 ± 1c	414 ± 3ef	405 ± 7f
34	125 (0)	50 (-1)	60 (0)	20 (0)	25 (1)	497 ± 4bc	436 ± 6d	422 ± 4e
35	100(-1)	50 (-1)	60 (0)	20 (0)	12.5 (0)	418 ± 2e	369 ± 2gh	357 ± 1h
36	150 (1)	100 (0)	30 (-1)	20 (0)	12.5 (0)	392 ± 1fg	344 ± 1hi	340 ± 2i
37	150 (1)	100 (0)	60 (0)	10 (-1)	12.5 (0)	403 ± 5f	358 ± 4h	348 ± 3hi
38	125 (0)	100 (0)	90 (1)	20 (0)	0 (-1)	348 ± 2hi	311 ± 3ll	301 ± 2m
39 (C6)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	447 ± 6cd	399 ± 2f	388 ± 5fg
40	100 (-1)	100 (0)	60 (0)	20 (0)	25 (1)	540 ± 3b	475 ± 2c	466 ± 5c
41	125 (0)	150 (1)	90 (1)	20 (0)	12.5 (0)	470 ± 4c	426 ± 1de	401 ± 1f
42	150 (1)	50 (-1)	60 (0)	20 (0)	12.5 (0)	388 ± 3fg	338 ± 5i	327 ± 3j
43	125 (0)	50 (-1)	30 (-1)	20 (0)	12.5 (0)	422 ± 1e	368 ± 2gh	366 ± 3gh
44	125 (0)	100 (0)	30 (-1)	10 (-1)	12.5 (0)	429 ± 5de	373 ± 3gh	363 ± 1gh
45	125 (0)	100 (0)	30 (-1)	30 (1)	12.5 (0)	466 ± 2c	409 ± 6ef	401 ± 5f
46	125 (0)	50 (-1)	60 (0)	30 (1)	12.5 (0)	419 ± 6e	364 ± 3gh	357 ± 2h

• C₁, C₂, C₃, C₄, C₅, C₆ are the centre points where extrusion processing was applied. Different superscripts letters indicate the significance difference (p ≤ 0.05) within the rows and columns. BET = Barrel Exit Temp; SS = Screw Speed; FFR = Feed Flow Rate; FMC = Feed Moisture Content; FP = Fish Powder; PUFAs = polyunsaturated fatty acids.

The effect of thermal processing on MUFAs and SFAs are shown in Table 3. The results show that the maximum MUFAs retention was 96.48% under optimizing conditions of BET (125 °C), SS (150 rpm), FFR (60 kg/hr), FMC (20%) and FP (25%) in final products while minimum MUFAs retention (66%) was observed at 125 °C BET, 50 rpm SS, 60 kg/hr FFR, 20% FMC and 0% FP. Maximum SFAs contents (1.55 ± 0.007%) were observed in a final product that was extruded under conditions of BET (150 °C), SS (100 rpm), FFR (60 kg/hr), FMC (20%) and FP (25%). The minimum SFAs contents were found to be 0.214% under 100 °C BET, 100 rpm SS, 60 kg/hr FFR, 20% FMC and 0% FP. BET was the most independent parameter affecting the retention of PUFAs. A trend in PUFAs decrease was observed with increasing temperature and decreasing moisture content while an increasing trend in PUFAs was observed with increasing SS and FP as shown in Figure 1.

Table 3
Effect of extrusion processing conditions on retention of SFAs and MUFAs at different storage intervals.

Extrusion processing run	Independent variables					Dependent variables
	Barrel Exit Temp. (BET)	Screw Speed (SS)	Feed Flow Rate (FFR)	Feed Moisture Content (FMC)	Fish Powder Content (FP)	SFAs (mg/100 g product)			MUFAs (mg/100 g product)
						0 Day	30 Days	60 Days	0 Day	30 Days	60 Days
1	150 (1)	150 (1)	60 (0)	20 (0)	12.5 (0)	827 ± 2kl	836 ± 3k	846 ± 5jk	375 ± 1H	333 ± 3J	350 ± 2I
2	125 (0)	150 (1)	60 (0)	20 (0)	25 (1)	1379 ± 7g	1388 ± 9g	1411 ± 2fg	714 ± 3A	640 ± 2C	672 ± 6AB
3 (C1)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	805 ± 4m	816 ± 4lm	839 ± 1jk	386 ± 2GH	342 ± 4IJ	350 ± 3I
4	150 (1)	100 (0)	60 (0)	30 (1)	12.5 (0)	860 ± 6j	880 ± 3i	889 ± 3hi	353 ± 2I	311 ± 1K	333 ± 2J
5	100 (-1)	100 (0)	60 (0)	20 (0)	0 (-1)	214 ± 1s	217 ± 5rs	219 ± 1rs	91 ± 1O	79 ± 1OP	89 ± 1O
6	100 (-1)	150 (1)	60 (0)	20 (0)	12.5 (0)	777 ± 3no	784 ± 1n	793 ± 5mn	437 ± 1FG	387 ± 5GH	410 ± 5G
7	125 (0)	150 (1)	30 (-1)	20 (0)	12.5 (0)	783 ± 5n	795 ± 3mn	805 ± 4m	405 ± 3G	357 ± 3I	378 ± 2H
8	125 (0)	50 (-1)	60 (0)	10 (-1)	12.5 (0)	866 ± 2ij	872 ± 6ij	889 ± 3hi	349 ± 2I	306 ± 2KL	323 ± 3JK
9	150 (1)	100 (0)	60 (0)	20 (0)	0 (-1)	268 ± 2q	275 ± 1pq	277 ± 1pq	83 ± 1O	68 ± 1P	75 ± 2OP
10	125 (0)	100 (0)	60 (0)	30 (1)	25 (1)	1469 ± 5ef	1489 ± 8de	1514 ± 7d	677 ± 5AB	603 ± 6DE	631 ± 4CD
11	125 (0)	100 (0)	90 (1)	10 (-1)	12.5 (0)	820 ± 6l	844 ± 2jk	847 ± 3jk	374 ± 2H	333 ± 3J	352 ± 2I
12 (C2)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	816 ± 1lm	826 ± 4kl	842 ± 5jk	371 ± 3H	330 ± 2J	349 ± 1I
13	125 (0)	150 (1)	60 (0)	30 (1)	12.5 (0)	777 ± 3no	785 ± 5n	787 ± 4n	410 ± 1G	369 ± 1H	387 ± 3GH
14	125 (0)	100 (0)	60 (0)	10 (-1)	25 (1)	1523 ± 9cd	1554 ± 9bc	1569 ± 1b	652 ± 5BC	585 ± 2E	614 ± 5D
15	125 (0)	100 (0)	30 (-1)	20 (0)	0 (-1)	238 ± 7r	234 ± 2r	236 ± 2r	81 ± 1O	72 ± 2OP	75 ± 2OP
16	100 (-1)	100 (0)	60 (0)	10 (-1)	12.5 (0)	844 ± 4jk	856 ± 1j	859 ± 5j	355 ± 4I	310 ± 5K	337 ± 1J
17	100 (-1)	100 (0)	30 (-1)	20 (0)	12.5 (0)	827 ± 2kl	836 ± 4k	847 ± 6jk	364 ± 2HI	320 ± 3JK	342 ± 1IJ
18	150 (1)	100 (0)	90 (1)	20 (0)	12.5 (0)	871 ± 5ij	884 ± 2hi	896 ± 3h	348 ± 3I	306 ± 4KL	323 ± 3JK
19	125 (0)	50 (-1)	60 (0)	20 (0)	0 (-1)	277 ± 3pq	275 ± 5pq	278 ± 1pq	66 ± 1P	54 ± 1Q	56 ± 3Q
20	125 (0)	100 (0)	60 (0)	10 (-1)	0 (-1)	219 ± 1rs	214 ± 2s	216 ± 1s	92 ± 1O	78 ± 2OP	85 ± 5O
21	125 (0)	100 (0)	90 (1)	20 (0)	25 (1)	1492 ± 8de	1523 ± 7cd	1534 ± 9c	666 ± 6B	590 ± 5E	627 ± 7CD
22	125 (0)	150 (1)	60 (0)	20 (0)	0 (-1)	218 ± 2rs	214 ± 2s	219 ± 1rs	105 ± 1N	89 ± 2O	95 ± 3O
23	125 (0)	100 (0)	90 (1)	30 (1)	12.5 (0)	849 ± 5jk	856 ± 2j	868 ± 5ij	375 ± 3H	333 ± 2J	346 ± 2IJ
24 (C3)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	815 ± 4lm	826 ± 4kl	840 ± 3jk	384 ± 2GH	340 ± 2IJ	359 ± 3HI
25	100 (-1)	100 (0)	90 (1)	20 (0)	12.5 (0)	807 ± 8m	816 ± 7lm	828 ± 2kl	379 ± 2H	330 ± 3J	349 ± 4I
26 (C4)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	816 ± 5lm	826 ± 5kl	841 ± 3jk	378 ± 1H	333 ± 1J	349 ± 2I
27	125 (0)	50 (-1)	90 (1)	20 (0)	12.5 (0)	879 ± 2i	887 ± 2hi	892 ± 2hi	366 ± 1HI	324 ± 2JK	340 ± 1IJ
28	125 (0)	150 (1)	60 (0)	10 (-1)	12.5 (0)	843 ± 6jk	858 ± 1j	865 ± 5ij	383 ± 3GH	342 ± 3IJ	359 ± 3HI
29	125 (0)	100 (0)	30 (-1)	20 (0)	25 (1)	1480 ± 9e	1513 ± 7d	1526 ± 8cd	657 ± 5BC	585 ± 5E	614 ± 2D
30	125 (0)	100 (0)	60 (0)	30 (1)	0 (-1)	219 ± 1rs	214 ± 3s	216 ± 1s	111 ± 2N	98 ± 2O	105 ± 2N
31 (C5)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	812 ± 3lm	816 ± 2lm	843 ± 3jk	375 ± 4H	332 ± 3J	350 ± 4I
32	100 (-1)	100 (0)	60 (0)	30 (1)	12.5 (0)	787 ± 2n	805 ± 4m	813 ± 2lm	387 ± 3GH	340 ± 5IJ	359 ± 5HI
33	150 (1)	100 (0)	60 (0)	20 (0)	25 (1)	1555 ± 7bc	1581 ± 3ab	1598 ± 9a	626 ± 2CD	558 ± 3F	589 ± 5E
34	125 (0)	50 (-1)	60 (0)	20 (0)	25 (1)	1548 ± 8bc	1570 ± 3b	1586 ± 6ab	613 ± 1D	552 ± 7F	576 ± 6EF
35	100(-1)	50 (-1)	60 (0)	20 (0)	12.5 (0)	869 ± 2ij	872 ± 5ij	889 ± 2hi	344 ± 2IJ	306 ± 2KL	323 ± 2JK
36	150 (1)	100 (0)	30 (-1)	20 (0)	12.5 (0)	870 ± 5ij	874 ± 1ij	896 ± 5h	355 ± 1I	315 ± 1K	335 ± 2J
37	150 (1)	100 (0)	60 (0)	10 (-1)	12.5 (0)	854 ± 1j	867 ± 3ij	880 ± 4i	366 ± 4HI	324 ± 2JK	342 ± 3IJ
38	125 (0)	100 (0)	90 (1)	20 (0)	0 (-1)	227 ± 3r	224 ± 2r	226 ± 2r	73 ± 1OP	63 ± 1P	66 ± 1P
39 (C6)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	813 ± 6lm	820 ± 5l	847 ± 2jk	386 ± 2GH	340 ± 1IJ	362 ± 1HI
40	100 (-1)	100 (0)	60 (0)	20 (0)	25 (1)	1420 ± 4fg	1444 ± 5f	1462 ± 6ef	679 ± 2AB	603 ± 3DE	631 ± 3CD
41	125 (0)	150 (1)	90 (1)	20 (0)	12.5 (0)	688 ± 6p	693 ± 2op	705 ± 2o	306 ± 1KL	273 ± 2M	285 ± 2L
42	150 (1)	50 (-1)	60 (0)	20 (0)	12.5 (0)	885 ± 2hi	897 ± 1	906 ± 6gh	322 ± 4JK	288 ± 2L	304 ± 5KL
43	125 (0)	50 (-1)	30 (-1)	20 (0)	12.5 (0)	844 ± 5jk	856 ± 3j	865 ± 3ij	362 ± 4HI	324 ± 3JK	342 ± 3IJ
44	125 (0)	100 (0)	30 (-1)	10 (-1)	12.5 (0)	849 ± 2jk	858 ± 2j	865 ± 3ij	390 ± 3GH	348 ± 4I	365 ± 1HI
45	125 (0)	100 (0)	30 (-1)	30 (1)	12.5 (0)	801 ± 2m	819 ± 5l	821 ± 1l	404 ± 1G	361 ± 1HI	378 ± 3H
46	125 (0)	50 (-1)	60 (0)	30 (1)	12.5 (0)	866 ± 5ij	877 ± 2i	885 ± 4hi	357 ± 2I	315 ± 2K	337 ± 2J

• C₁, C₂, C₃, C_4, C₅, C₆ are the centre points where extrusion processing was applied. Different superscripts letters ^(a-s) indicate the significance difference (p ≤ 0.05) within the rows and columns within SFAs. Different superscripts letters ^(A-Q) indicate the significance difference (p ≤ 0.05) within the rows and columns within MUFAs. BET = Barrel Exit Temp; SS = Screw Speed; FFR = Feed Flow Rate; FMC = Feed Moisture Content; FP = Fish Powder; SFAs = saturated fatty acids; MUFAs = monounsaturated fatty acids.

Figure 1
Mutual interaction effects of BET (100-150 °C), SS (50-150 rpm), FFR (30-90 kg/hour), FMC (10-30%) and FP (0-25%) on PUFAs retention during thermal extrusion in functional macaroni production.

Highest values of PUFAs retention (87.97% and 85.66%) at a storage temperature of 25 °C for 30 and 60 days were observed in a product extruded under processing conditions of BET (125 °C), SS (150rpm), FFR (60 kg/hr), FMC (20%) and FP (25%). While the minimum PUFAs values (85.80% and 83.87%) were observed at 25 °C storage temperature for 30 and 60 days in product processed at BET (125 °C), SS (150 rpm), FFR (90 kg/hr) and FMC (20%) and FP (12.5%) as shown in Table 2. The minimum value of MUFAs (55 ± 1 and 56 ± 1 mg) at 30 and 60 days storage were found at 125 °C BET, 50 rpm SS, 60 kg/hr FFR, 20% FMC and 0% FP and minimum SFAs (214 ± 2 and 216 ± 1 mg) at 30 and 60 days storage were present at 125 °C BET, 100 rpm SS, 60 kg/hr FFR, 30% FMC and 0% FP as shown in Table 3.

Oxidative stability of the product was measured by determining the TBARS and carbonyl contents are presented in Table 4. The highest TBARS values at processing conditions of BET (125 °C), SS (150 rpm), FFR (90 kg/hr) and FMC (20%) and FP (12.5%) was 0.42 and 0.44 mg MDA/kg at 25 °C storage temperature for 0 and 30 days respectively while 0.55 mg MDA/kg at 60 days storage were in product extruded at BET (125 °C), SS (100 rpm), FFR (30%), FMC (20%) and FP (25%). The highest carbonyl contents of 6.02, 7.67, 9.37 nmol/mg protein were observed in product extruded under BET (125 °C), SS (100 rpm), FFR (60 kg/hr), FMC (30%) and FP (25%) for 0, 30 and 60 days of storage at 25 °C. The sensory evaluation score for products is shown in Table 5. The results for color, flavor and overall acceptability have shown that the product extruded at 125 °C BET, 150 rpm SS, 60 kg/hr FFR, 20% FMC and 25% FP have the gain highest score (7.30 ± 0.50 and 6.59 ± 0.65), (7.04 ± 0.56, 6.54 ± 0.32) for color and flavor at 0 and 60 days storage, respectively. The product extruded at 125 °C BET, 150 rpm SS, 60 kg/hr FFR, 30% FMC and 12.5% FP have the best overall acceptability score.

Table 4
Effect of thermal extrusion processing conditions on TRARS and carbonyl contents of product at 25 °C for different storage intervals.

Extrusion processing run	Independent variables					TBARS (mg MDA/kg)			Carbonyl contents (nmol/mg protein)
	Barrel Exit Temp. (BET)	Screw Speed (SS)	Feed Flow Rate (FFR)	Feed Moisture Content (FMC)	Fish Powder Content (FP)	Storage time (days)			Storage time (days)
						0	30	60	0	30	60
1	150 (1)	150 (1)	60 (0)	20 (0)	12.5 (0)	0.27 ± 0.01de	0.29 ± 0.02de	0.34 ± 0.02d	3.99 ± 0.10R	5.64 ± 0.05L	7.27 ± 0.19EF
2	125 (0)	150 (1)	60 (0)	20 (0)	25 (1)	0.42 ± 0.04c	0.44 ± 0.03bc	0.51 ± 0.07ab	5.91 ± 0.15IJ	7.56 ± 0.08D	9.27 ± 0.07B
3 (C1)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	0.26 ± 0.02de	0.31 ± 0.02d	0.33 ± 0.02d	3.93 ± 0.08R	5.54 ± 0.12LM	7.10 ± 0.13FG
4	150 (1)	100 (0)	60 (0)	30 (1)	12.5 (0)	0.24 ± 0.01e	0.26 ± 0.01de	0.30 ± 0.04d	4.08 ± 0.09QR	5.67 ± 0.13KL	7.32 ± 0.11EF
5	100 (-1)	100 (0)	60 (0)	20 (0)	0 (-1)	0.10 ± 0.01fg	0.11 ± 0.01f	0.13 ± 0.01f	2.89 ± 0.07V	4.06 ± 0.17QR	5.19 ± 0.12NO
± 6	100 (-1)	150 (1)	60 (0)	20 (0)	12.5 (0)	0.29 ± 0.02de	0.32 ± 0.02d	0.37 ± 0.05cd	3.87 ± 0.08RS	5.46 ± 0.14M	7.10 ± 0.15FG
7	125 (0)	150 (1)	30 (-1)	20 (0)	12.5 (0)	0.28 ± 0.02de	0.31 ± 0.04d	0.35 ± 0.07d	4.12 ± 0.11Q	5.73 ± 0.16K	7.37 ± 0.13E
08	125 (0)	50 (-1)	60 (0)	10 (-1)	12.5 (0)	0.23 ± 0.03e	0.25 ± 0.01e	0.27 ± 0.01de	4.21 ± 0.11PQ	5.80 ± 0.11JK	7.47 ± 0.16D
9	150 (1)	100 (0)	60 (0)	20 (0)	0 (-1)	0.09 ± 0.01fg	0.10 ± 0.01fg	0.13 ± 0.01f	3.11 ± 0.10U	4.47 ± 0.13O	5.81 ± 0.12JK
10	125 (0)	100 (0)	60 (0)	30 (1)	25 (1)	0.38 ± 0.04cd	0.41 ± 0.04c	0.47 ± 0.06b	6.02 ± 0.13I	7.67 ± 0.14CD	9.37 ± 0.15A
11	125 (0)	100 (0)	90 (1)	10 (-1)	12.5 (0)	0.24 ± 0.01e	0.27 ± 0.02de	0.32 ± 0.03d	3.84 ± 0.09S	5.43 ± 0.17MN	7.09 ± 0.12FG
12 (C2)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	0.27 ± 0.05de	0.30 ± 0.03d	0.33 ± 0.02d	3.90 ± 0.12RS	5.51 ± 0.12LM	7.11 ± 0.16FG
13	125 (0)	150 (1)	60 (0)	30 (1)	12.5 (0)	0.29 ± 0.02de	0.31 ± 0.01d	0.35 ± 0.05d	3.63 ± 0.14T	5.23 ± 0.14N	6.89 ± 0.12H
14	125 (0)	100 (0)	60 (0)	10 (-1)	25 (1)	0.40 ± 0.06c	0.43 ± 0.04bc	0.54 ± 0.03a	5.87 ± 0.13J	7.53 ± 0.10D	9.23 ± 0.15BC
15	125 (0)	100 (0)	30 (-1)	20 (0)	0 (-1)	0.10 ± 0.01fg	0.12 ± 0.01f	0.13 ± 0.01f	3.02 ± 0.10UV	4.20 ± 0.09Q	5.39 ± 0.11MN
16	100 (-1)	100 (0)	60 (0)	10 (-1)	12.5 (0)	0.28 ± 0.03de	0.30 ± 0.01d	0.33 ± 0.02d	3.77 ± 0.09ST	5.37 ± 0.08MN	7.03 ± 0.09G
017	100 (-1)	100 (0)	30 (-1)	20 (0)	12.5 (0)	0.27 ± 0.01de	0.30 ± 0.02d	0.34 ± 0.04d	3.88 ± 0.16RS	5.47 ± 0.16M	7.11 ± 0.15FG
18	150 (1)	100 (0)	90 (1)	20 (0)	12.5 (0)	0.24 ± 0.02e	0.26 ± 0.01de	0.30 ± 0.02d	4.27 ± 0.12P	5.86 ± 0.12J	7.54 ± 0.18D
19	125 (0)	50 (-1)	60 (0)	20 (0)	0 (-1)	0.06 ± 0.01fg	0.07 ± 0.01fg	0.09 ± 0.01fg	3.11 ± 0.08U	4.29 ± 0.11P	5.49 ± 0.15LM
20	125 (0)	100 (0)	60 (0)	10 (-1)	0 (-1)	0.07 ± 0.01fg	0.09 ± 0.02fg	0.10 ± 0.01fg	2.94 ± 0.10V	4.07 ± 0.13QR	5.28 ± 0.12N
21	125 (0)	100 (0)	90 (1)	20 (0)	25 (1)	0.35 ± 0.03d	0.39 ± 0.07cd	0.53 ± 0.08ab	5.78 ± 0.11JK	7.42 ± 0.18DE	9.14 ± 0.14C
0.22	125 (0)	150 (1)	60 (0)	20 (0)	0 (-1)	0.11 ± 0.01f	0.13 ± 0.01f	0.15 ± 0.01f	2.98 ± 0.12V	4.17 ± 0.12Q	5.37 ± 0.13MN
23	125 (0)	100 (0)	90 (1)	30 (1)	12.5 (0)	0.23 ± 0.01e	0.25 ± 0.02e	0.28 ± 0.01de	3.78 ± 0.13ST	5.37 ± 0.11MN	7.04 ± 0.12G
24 (C3)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	0.27 ± 0.03de	0.30 ± 0.02d	0.33 ± 0.03d	3.92 ± 0.11R	5.51 ± 0.15LM	7.09 ± 0.21FG
25	100 (-1)	100 (0)	90 (1)	20 (0)	12.5 (0)	0.24 ± 0.02e	0.27 ± 0.01de	0.31 ± 0.02d	3.96 ± 0.12R	5.58 ± 0.14L	7.23 ± 0.16F
26 (C4)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	0.26 ± 0.03de	0.31 ± 0.03d	0.34 ± 0.04d	3.91 ± 0.09R	5.51 ± 0.11LM	7.11 ± 0.13FG
27	125 (0)	50 (-1)	90 (1)	20 (0)	12.5 (0)	0.21 ± 0.01ef	0.23 ± 0.01e	0.27 ± 0.02de	4.10 ± 0.10QR	5.71 ± 0.19KL	7.36 ± 0.17E
28	125 (0)	150 (1)	60 (0)	10 (-1)	12.5 (0)	0.28 ± 0.02de	0.32 ± 0.02d	0.36 ± 0.03cd	3.84 ± 0.13S	5.45 ± 0.14M	7.10 ± 0.17FG
29	125 (0)	100 (0)	30 (-1)	20 (0)	25 (1)	0.38 ± 0.03cd	0.43 ± 0.05bc	0.55 ± 0.05a	5.76 ± 0.17K	7.42 ± 0.15DE	9.12 ± 0.19C
30	125 (0)	100 (0)	60 (0)	30 (1)	0 (-1)	0.06 ± 0.01fg	0.07 ± 0.01fg	0.10 ± 0.01fg	2.86 ± 0.11V	4.04 ± 0.12QR	5.25 ± 0.13N
31 (C5)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	0.26 ± 0.03de	0.30 ± 0.03d	0.34 ± 0.05d	3.91 ± 0.14R	5.51 ± 0.10LM	7.11 ± 0.09FG
32	100 (-1)	100 (0)	60 (0)	30 (1)	12.5 (0)	0.26 ± 0.02de	0.29 ± 0.02de	0.33 ± 0.02d	3.79 ± 0.12ST	5.39 ± 0.12MN	7.05 ± 0.16G
33	150 (1)	100 (0)	60 (0)	20 (0)	25 (1)	0.32 ± 0.03d	0.39 ± 0.05cd	0.48 ± 0.04b	5.97 ± 0.18I	7.63 ± 0.11CD	9.33 ± 0.23AB
34	125 (0)	50 (-1)	60 (0)	20 (0)	25 (1)	0.31 ± 0.05d	0.37 ± 0.03cd	0.46 ± 0.04b	5.86 ± 0.13J	7.52 ± 0.16D	9.21 ± 0.22BC
35	100 (-1)	50 (-1)	60 (0)	20 (0)	12.5 (0)	0.25 ± 0.01e	0.29 ± 0.02de	0.34 ± 0.02d	4.16 ± 0.17Q	5.75 ± 0.15K	7.41 ± 0.11DE
36	150 (1)	100 (0)	30 (-1)	20 (0)	12.5 (0)	0.26 ± 0.02de	0.29 ± 0.01de	0.35 ± 0.04d	4.22 ± 0.09PQ	5.82 ± 0.14J	7.48 ± 0.09D
37	150 (1)	100 (0)	60 (0)	10 (-1)	12.5 (0)	0.25 ± 0.02e	0.28 ± 0.01de	0.33 ± 0.02d	4.30 ± 0.10P	5.92 ± 0.18IJ	7.60 ± 0.11CD
38	125 (0)	100 (0)	90 (1)	20 (0)	0 (-1)	0.09 ± 0.01fg	0.10 ± 0.01fg	0.12 ± 0.01f	3.06 ± 0.11UV	4.26 ± 0.09PQ	5.81 ± 0.12JK
39 (C6)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	0.27 ± 0.01de	0.31 ± 0.02d	0.34 ± 0.03d	3.89 ± 0.10RS	5.50 ± 0.17LM	7.10 ± 0.18FG
40	100 (-1)	100 (0)	60 (0)	20 (0)	25 (1)	0.37 ± 0.02cd	0.42 ± 0.05c	0.54 ± 0.04a	5.92 ± 0.18IJ	7.58 ± 0.09CD	9.28 ± 0.22B
41	125 (0)	150 (1)	90 (1)	20 (0)	12.5 (0)	0.28 ± 0.03de	0.30 ± 0.01d	0.33 ± 0.02d	3.73 ± 0.16ST	5.34 ± 0.13N	6.99 ± 0.12GH
42	150 (1)	50 (-1)	60 (0)	20 (0)	12.5 (0)	0.22 ± 0.01ef	0.25 ± 0.02e	0.29 ± 0.05de	4.41 ± 0.11O	6.01 ± 0.12I	7.67 ± 0.13CD
43	125 (0)	50 (-1)	30 (-1)	20 (0)	12.5 (0)	0.24 ± 0.02e	0.27 ± 0.01de	0.30 ± 0.02d	4.17 ± 0.14Q	5.78 ± 0.14JK	7.43 ± 0.17DE
44	125 (0)	100 (0)	30 (-1)	10 (-1)	12.5 (0)	0.26 ± 0.01de	0.29 ± 0.03de	0.33 ± 0.04d	4.02 ± 0.17R	5.63 ± 0.13L	7.27 ± 0.10EF
45	125 (0)	100 (0)	30 (-1)	30 (1)	12.5 (0)	0.25 ± 0.02e	0.28 ± 0.01de	0.32 ± 0.02d	3.82 ± 0.10S	5.43 ± 0.19MN	7.08 ± 0.19FG
46	125 (0)	50 (-1)	60 (0)	30 (1)	12.5 (0)	0.22 ± 0.01ef	0.24 ± 0.02e	0.28 ± 0.01de	4.04 ± 0.11QR	5.63 ± 0.17L	7.29 ± 0.20EF

• C₁, C₂, C₃, C₄, C₅, C₆ are the center points where extrusion processing was applied. Values with same superscript letters (^a-fg) show the homogenous group within the rows and columns for TBARS values (p ≤ 0.05). Values with same superscript letters (^A-V) show the homogenous group within the rows and columns carbonyl contents (p ≤ 0.05). BET = Barrel Exit Temp; SS = Screw Speed; FFR = Feed Flow Rate; FMC = Feed Moisture Content; FP = Fish Powder; TBARS = Thiobarbituric acid reactive substance; MDA= malondialdehyde.

Table 5
Effect of thermal extrusion processing conditions on the sensory attributes of product at 25 °C during storage intervals.

Extrusion processing run	Independent variables					Sensory attributes
	Barrel Exit Temp. (BET)	Screw Speed (SS)	Feed Flow Rate (FFR)	Feed Moisture Content (FMC)	Fish Powder Content (FP)	Color		Flavor		Overall acceptability
						0	60	0	60	0	60
1	150 (1)	150 (1)	60 (0)	20 (0)	12.5 (0)	6.92 ± 0.43bc	6.22 ± 0.39fg	6.33 ± 0.37de	5.73 ± 0.45gh	6.80 ± 0.40b	6.21 ± 0.65de
2	125 (0)	150 (1)	60 (0)	20 (0)	25 (1)	7.30 ± 0.50a	6.59 ± 0.65d	7.04 ± 0.56a	6.54 ± 0.32c	6.44 ± 0.81cd	5.91 ± 0.45f
3 (C1)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	6.20 ± 0.37fg	5.60 ± 0.48i	6.28 ± 0.45de	5.79 ± 0.60gh	6.84 ± 0.65ab	6.25 ± 0.36de
4	150 (1)	100 (0)	60 (0)	30 (1)	12.5 (0)	6.30 ± 0.39ef	5.66 ± 0.55i	6.25 ± 0.33e	5.71 ± 0.56gh	6.77 ± 0.45b	6.22 ± 0.74de
5	100 (-1)	100 (0)	60 (0)	20 (0)	0 (-1)	5.33 ± 0.31jk	4.91 ± 0.27lm	5.82 ± 025g	5.45 ± 0.35i	6.44 ± 0.33cd	6.07 ± 0.39ef
6	100 (-1)	150 (1)	60 (0)	20 (0)	12.5 (0)	6.73 ± 0.45cd	6.17 ± 0.43fg	6.29 ± 0.47de	5.83 ± 0.55g	6.82 ± 0.70ab	6.37 ± 0.71d
7	125 (0)	150 (1)	30 (-1)	20 (0)	12.5 (0)	6.39 ± 0.43ef	5.88 ± 0.54h	6.46 ± 0.55cd	6.05 ± 0.45f	6.93 ± 0.74a	6.51 ± 0.47cd
8	125 (0)	50 (-1)	60 (0)	10 (-1)	12.5 (0)	6.12 ± 0.38fg	5.41 ± 0.60j	6.21 ± 0.38e	5.62 ± 0.60h	6.72 ± 0.58bc	6.13 ± 0.52e
9	150 (1)	100 (0)	60 (0)	20 (0)	0 (-1)	5.21 ± 0.55k	4.72 ± 0.42m	5.94 ± 0.40fg	5.51 ± 0.45hi	6.43 ± 0.56cd	6.02 ± 0.26ef
10	125 (0)	100 (0)	60 (0)	30 (1)	25 (1)	7.10 ± 0.55b	6.43 ± 0.71ef	7.02 ± 0.62a	6.53 ± 0.80c	6.36 ± 0.66d	5.87 ± 0.45fg
11	125 (0)	100 (0)	90 (1)	10 (-1)	12.5 (0)	6.10 ± 0.38fg	5.47 ± 0.48j	6.32 ± 0.39de	5.79 ± 0.32gh	6.79 ± 0.65b	6.27 ± 0.80de
12 (C2)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	6.18 ± 0.88fg	5.59 ± 0.41ij	6.34 ± 0.46de	5.87 ± 0.34fg	6.84 ± 0.49ab	6.37 ± 0.96d
13	125 (0)	150 (1)	60 (0)	30 (1)	12.5 (0)	6.88 ± 0.42cd	6.43 ± 0.60ef	6.48 ± 0.50cd	6.13 ± 0.48ef	6.99 ± 0.55a	6.66 ± 0.91bc
14	125 (0)	100 (0)	60 (0)	10 (-1)	25 (1)	6.90 ± 0.53c	6.12 ± 0.55fg	6.83 ± 0.51b	6.24 ± 0.88e	6.29 ± 0.75de	5.71 ± 0.62gh
15	125 (0)	100 (0)	30 (-1)	20 (0)	0 (-1)	5.40 ± 0.96j	4.98 ± 0.33l	5.91 ± 0.96fg	5.53 ± 0.37hi	6.59 ± 0.58c	6.22 ± 0.35de
16	100 (-1)	100 (0)	60 (0)	10 (-1)	12.5 (0)	6.41 ± 0.38ef	5.84 ± 0.37h	6.18 ± 0.36ef	5.72 ± 0.56gh	6.66 ± 0.65bc	6.24 ± 0.72de
17	100 (-1)	100 (0)	30 (-1)	20 (0)	12.5 (0)	6.50 ± 0.91e	5.92 ± 0.57gh	6.23 ± 0.48e	5.83 ± 0.47g	6.70 ± 0.46bc	6.34 ± 0.38d
18	150 (1)	100 (0)	90 (1)	20 (0)	12.5 (0)	6.20 ± 0.39fg	5.53 ± 0.48ij	6.22 ± 0.88e	5.65 ± 0.88h	6.69 ± 0.70bc	6.09 ± 0.27ef
19	125 (0)	50 (-1)	60 (0)	20 (0)	0 (-1)	5.21 ± 0.40k	4.72 ± 0.33m	5.85 ± 0.28g	5.43 ± 0.52i	6.52 ± 0.51cd	6.11 ± 0.74ef
20	125 (0)	100 (0)	60 (0)	10 (-1)	0 (-1)	5.32 ± 0.88jk	4.93 ± 0.27l	5.89 ± 0.88fg	5.54 ± 0.91hi	6.57 ± 0.42c	6.21 ± 0.48de
21	125 (0)	100 (0)	90 (1)	20 (0)	25 (1)	6.98 ± 0.56bc	6.23 ± 0.71fg	6.94 ± 0.67ab	6.39 ± 0.78d	6.36 ± 0.71d	5.78 ± 0.56g
22	125 (0)	150 (1)	60 (0)	20 (0)	0 (-1)	5.42 ± 0.41j	5.01 ± 0.50l	6.06 ± 0.29ef	5.67 ± 0.96gh	6.69 ± 0.49bc	6.29 ± 0.52de
23	125 (0)	100 (0)	90 (1)	30 (1)	12.5 (0)	6.30 ± 0.44ef	5.84 ± 0.39h	6.43 ± 0.36d	6.07 ± 0.60ef	6.89 ± 0.59ab	6.51 ± 0.37cd
24 (C3)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	6.27 ± 0.39f	5.80 ± 0.56hi	6.31 ± 0.47de	5.96 ± 0.54fg	6.84 ± 0.72ab	6.45 ± 0.84cd
25	100 (-1)	100 (0)	90 (1)	20 (0)	12.5 (0)	6.52 ± 0.96de	6.05 ± 0.37g	6.16 ± 0.88ef	5.80 ± 0.36gh	6.68 ± 0.57bc	6.30 ± 0.36de
26 (C4)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	6.21 ± 0.34fg	5.54 ± 0.96ij	6.35 ± 0.96de	5.78 ± 0.54gh	6.75 ± 0.62b	6.23 ± 0.69de
27	125 (0)	50 (-1)	90 (1)	20 (0)	12.5 (0)	6.03 ± 0.39gh	5.37 ± 0.36jk	6.24 ± 0.33e	5.69 ± 0.55gh	6.73 ± 0.58bc	6.24 ± 0.58de
28	125 (0)	150 (1)	60 (0)	10 (-1)	12.5 (0)	6.37 ± 0.43ef	5.89 ± 0.55h	6.41 ± 0.39d	6.02 ± 0.51f	6.91 ± 0.65ab	6.51 ± 0.85cd
29	125 (0)	100 (0)	30 (-1)	20 (0)	25 (1)	6.90 ± 0.96c	6.14 ± 0.66fg	6.89 ± 0.66ab	6.33 ± 0.69de	6.33 ± 0.55d	5.78 ± 0.37g
30	125 (0)	100 (0)	60 (0)	30 (1)	0 (-1)	5.47 ± 0.43j	5.09 ± 0.41kl	6.04 ± 0.34f	5.67 ± 0.49gh	6.64 ± 0.60bc	6.27 ± 0.64de
31 (C5)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	6.33 ± 0.41ef	5.80 ± 0.36hi	6.29 ± 0.47de	5.87 ± 0.96fg	6.83 ± 0.51ab	6.41 ± 0.38cd
32	100 (-1)	100 (0)	60 (0)	30 (1)	12.5 (0)	6.62 ± 0.39d	6.11 ± 0.47fg	6.23 ± 0.37e	5.82 ± 0.54g	6.74 ± 0.88bc	6.33 ± 0.90d
33	150 (1)	100 (0)	60 (0)	20 (0)	25 (1)	6.75 ± 0.63cd	6.08 ± 0.59g	6.79 ± 0.61b	6.31 ± 0.75de	6.21 ± 0.96de	5.72 ± 0.73gh
34	125 (0)	50 (-1)	60 (0)	20 (0)	25 (1)	6.73 ± 0.54cd	5.91 ± 0.65gh	6.87 ± 0.57ab	6.24 ± 0.80e	6.24 ± 0.79de	5.74 ± 0.18g
35	100 (-1)	50 (-1)	60 (0)	20 (0)	12.5 (0)	6.30 ± 0.96ef	5.75 ± 0.96hi	6.12 ± 0.33ef	5.67 ± 0.37gh	6.61 ± 0.70bc	6.16 ± 0.88e
36	150 (1)	100 (0)	30 (-1)	20 (0)	12.5 (0)	6.22 ± 0.46fg	5.56 ± 0.43ij	6.20 ± 0.45ef	5.64 ± 0.39h	6.72 ± 0.47bc	6.17 ± 0.58e
37	150 (1)	100 (0)	60 (0)	10 (-1)	12.5 (0)	6.11 ± 0.40fg	5.49 ± 0.39ij	6.14 ± 0.50ef	5.61 ± 0.51h	6.64 ± 0.63bc	6.18 ± 0.61e
38	125 (0)	100 (0)	90 (1)	20 (0)	0 (-1)	5.33 ± 0.36jk	4.99 ± 0.47l	5.82 ± 0.37g	5.53 ± 0.36hi	6.61 ± 0.54bc	6.32 ± 0.49de
39 (C6)	125 (0)	100 (0)	60 (0)	20 (0)	12.5 (0)	6.07 ± 0.38g	5.46 ± 0.51j	6.35 ± 0.91de	5.85 ± 0.50g	6.87 ± 0.69ab	6.32 ± 0.83de
40	100 (-1)	100 (0)	60 (0)	20 (0)	25 (1)	6.81 ± 0.88cd	6.11 ± 0.91fg	6.82 ± 0.67b	6.31 ± 0.67de	6.21 ± 0.60de	5.73 ± 0.46g
41	125 (0)	150 (1)	90 (1)	20 (0)	12.5 (0)	6.47 ± 0.45e	5.96 ± 0.50gh	6.46 ± 0.51cd	6.05 ± 0.44f	6.94 ± 0.67a	6.50 ± 0.74cd
42	150 (1)	50 (-1)	60 (0)	20 (0)	12.5 (0)	6.42 ± 0.37ef	5.81 ± 0.54hi	6.11 ± 0.35ef	5.59 ± 0.88hi	6.59 ± 0.61c	6.06 ± 0.39ef
43	125 (0)	50 (-1)	30 (-1)	20 (0)	12.5 (0)	6.02 ± 0.91gh	5.45 ± 0.38j	6.27 ± 0.44de	5.81 ± 0.49g	6.72 ± 0.57bc	6.23 ± 0.62de
44	125 (0)	100 (0)	30 (-1)	10 (-1)	12.5 (0)	6.14 ± 0.88fg	5.62 ± 0.44i	6.23 ± 0.32e	5.82 ± 0.51g	6.79 ± 0.52b	6.36 ± 0.38d
45	125 (0)	100 (0)	30 (-1)	30 (1)	12.5 (0)	6.35 ± 0.44ef	5.90 ± 0.52gh	6.36 ± 0.48de	6.02 ± 0.55f	6.92 ± 0.71a	6.57 ± 0.88c
46	125 (0)	50 (-1)	60 (0)	30 (1)	12.5 (0)	6.13 ± 0.37fg	5.54 ± 0.54ij	6.31 ± 0.88de	5.83 ± 0.60g	6.80 ± 0.66b	6.31 ± 0.91de

• C₁, C₂, C₃, C₄, C₅, C₆ are the center points where extrusion processing was applied. Different superscripts letters indicate the significance (p ≤ 0.05) within for color, flavor and overall acceptability. BET = Barrel Exit Temp; SS = Screw Speed; FFR = Feed Flow Rate; FMC = Feed Moisture Content; FP = Fish Powder.

3.2 Discussion

Fish and fish products are consumed as the major source of PUFAs, however, in low-income countries, due to high postharvest losses, this vital source of micronutrients and essential fatty acids are rarely available and affordable to the communities. In fishery sector, the post-harvest losses increases from 10 to 12 million tons per year also have a low shelf life (14-16 days) under ice storage condition (Nowsad et al., 2015). In Pakistan, most of the fish are consumed by roasting and frying technology that has few hours of storage that further leads to CVDs (Gadiraju et al., 2015). So this study was designed to use FP to develop fish powder supplemented macaroni with high PUFAs and better oxidative stability. Thus PUFAs optimized fished powder was obtained through MVD. In MVD the PUFAs non-significantly changed and remained unaffected from their original composition and contents. In MVD, due to a higher vacuum degree, the boiling temperature of the water is relatively lowered which results in easily drying of fish mince and could improve the quality of the finished product (Hearn et al., 1987). The addition of FP not only increases the PUFAs and MUFAs but also the protein contents of the final product in functional macaroni. The PUFAs and MUFAs increased from 0.4 to 0.64% and 0.1 to 0.74%, respectively, providing a significant contribution to the recommended intakes of unsaturated fatty acids. Thus, extrusion processing plays an important role in the retention of PUFAs and MUFAs and their oxidative stability. It was reported that twin-screw extrusion with a high temperature of 180 °C and a high SS of 300 rpm did not significantly change the polyenoic fatty acids (Zadernowski et al., 1997). The BET, SS along FP addition have a highly significant effect on the retention of polyenoic fatty acids and particular PUFAs while FMC affects slight significance whereas the FFR does not significantly influence them. It can also be substantiated that the mutual influence of BET and FMC was slightly significant because the increase in BET at high FMC negatively affects the retention of PUFAs and MUFAs.

It was observed that SFAs significantly increased during the extrusion process. The higher temperature results in the conversion of double bonds from Cis to Trans fatty acids (Camire, 2000). These changes further occur during storage conditions leading to a change in the composition of fatty acids and oxidation of the product. It was observed that the better retention of TBARS values and carbonyl contents were detected in products with a high quantity of PUFAs and MUFAs. Changes in composition were rapid in the first 30 days as compared to the next 30 days of storage at 25 °C. Björck & Asp (1983) also found that with the increase in an extrusion temperature decrease in fat content occurred and this statement is comparable to current findings. This might be due to the expulsion of fumes at higher temperatures from fats. So, the change in parameters like BET, SS, and FMC also affect the number of fats on different levels. BET and FMC are both the variables that mainly affect the composition retention and storage stability of PUFA’s. Fitzpatrick et al. (2004) stated that storage conditions may influence the levels of fat and the same results are noticed in this study. With the change in BET, SS and FMC also change the levels of fat. The high temperature not only inactivates the lipase enzymes but also facilities the binding of lipids with carbohydrates contents that ultimately affect the oxidative stability of lipid in extruded products (Thachil et al., 2014).

4 Conclusion

The moderate BET (125 °C) with high SS (150 rpm), moderate FMC (20%), and high FP (25%) facilitate the highest retention of PUFAs and MUFAs in end products. The Highest PUFAs retention for short time storage takes place in product extruded at moderate BET with moderate SS high FFR and moderate FP. Moderate conditions have been recommended as the best option for good quality products with maximum retention of long-chain PUFAs and MUFAs and storage stability. Moreover, results from this work will aid in the formulation of healthier food products supplemented with fish dried powder and may address a critical industrial demand in terms of formulation options. Further studies can be conducted to determine the maximal shelf life of products supplemented with fish PUFAs along with protein for value addition and treatment of nutritional disorders through their absorption, metabolism, and distribution pattern into biological tissues.

• Practical Application: Fish powder can be applied for the production of value-added foods and food fortification has supported the food industry to investigate various approaches for producing fish powder from multiple raw materials.

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Publication Dates

• Publication in this collection
  22 Feb 2021
• Date of issue
  2022

History

• Received
  19 July 2020
• Accepted
  27 Aug 2020