Fatty acids profile, antioxidant activity, lipid oxidation, induction period, and sensory properties of burgers produced from blends of fish and mango kernel oils

ABSTRACT

In this study, five different types of fish burgers were produced using 80% ground fish meat, 10% lipid, 6% spices mixture and 4% water. Burgers (5 mm thickness and 90 g weight) were produced using 100% fish oil (control), 75% fish oil and 25% mango kernel oil (MKO) (T₁), 50% fish oil and 50% MKO (T₂₎, 25% fish oil and 75% MKO (T₃), 0% fish oil and 100% MKO (T₄). Burgers were packaged in polyethylene bags and stored at −16 to −18°C for 90 days.   The mangiferin, quercetin, catechin, chlorogenic and caffeic acid in MKO were 1381, 87, 512, and 973 mg/100 g. Total antioxidant capacity of control, T₁, T₂, T₃, and T₄ were 18.97, 31.47, 47.69, 65.37, and 78.82%. GC-MS analysis showed that ALA, EPA, and DHA) in T₂ were 0.85, 3.62, and 4.19%. In controlled storage, loss of ALA, EPA, and DHA was 55, 14, and 11%. At T₂ concentration, antioxidant compounds of MKO significantly altered the lipid oxidation in burgers. After storage in T₂, loss of ALA, EPA, and DHA was 3.6, 1.98, and 1.71%. POV of 90 days stored control, T₁, T₂, T₃, and T₄ was 3.42, 1.55, 0.63, 0.41, and 0.31 (MeqO₂/kg). Induction period of fish oil, MKO, in all levels was 2.45, 64.28, 7.33, 13.42, 18.39, and 62.84 hrs. Color flavor and texture score of 90 days stored T₂ was 91, 90, and 86% of total score. MKO and fish oil can be used at 50:50 concentrations to formulate fish burgers of acceptable sensory properties.

KEYWORDS:

• Fish oil
• Mango kernel oil
• burgers, fatty acids composition
• lipid oxidation

Introduction

Fish is the main constituent of human food having high-quality protein, minerals such as phosphorus, selenium, zinc, iodine, calcium, fluorine, iron, and several other bioactive compounds.^[1] Fish oil is a good source of omega-3 essential polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6). Omega-3 fatty acids possesses cardiac, hepato protective, anti-inflammatory, anti-obesity, anti-carcinogenic, anti-diabetic, anti-thrombotic, hypotensive, cholesterol effects, and so on. According to FDA, regular intake of 2 g omega-3 fatty acids can significantly reduce the risks of metabolic diseases and sudden deaths, which are very common in low-socio economic sectors of the developing and underdeveloped countries.^[2,3] Presently fish oil is used for the production of bread, cookies, processed cheese, yogurt, cheddar, mozzarella cheese and salad oils.^[4] Due to significant changes in lifestyles, food consumption patterns are also changed. Consumption of fast foods such as pizza, fried chicken and burgers are increasing in almost every country of the world.^[5] Consumers have great deal of knowledge of nutrition and interaction of metabolic diseases with the consumption of traditional foods. Therefore, food industries have extensively focused on the development of functional burgers. About 5–20% fat is used in the formulation of burgers to provide desired juiciness, flavor and textural attributes. From textural point of view, the most significant role of fat in burgers is to act as a binding, tendering, shaping and softening agent.^[6] Body fat and partially hydrogenated oils are used in the formulation of burgers, these fats contain lot of cholesterol, saturated and harmful trans fatty acids. Partially hydrogenated fats are highly dangerous for cardiovascular system due to the existence of trans fatty acids. Therefore, it is direly needed to find an alternate source of fat that can be used to replace body fats and partially hydrogenated oils with no issue of trans fatty acids and cholesterol. Mango kernel oil (MKO) is derived from the seed of mango fruit, oil is edible with 10–12% yield. MKO has 45% unsaturated fats and 55% saturated fats. Gas chromatographic analysis of MKO showed that major fatty acids were stearic acid, palmitic acid, and oleic acid with a melting point 32–36°C. It is solid at room temperature and can be used as an alternate of palm oil for several food applications.^[7] Fish oil is a rich source of omega-3 fatty acids, MKO has superior functional properties and oxidative stability therefore, fat portion of burgers can be formulated from blends of mango kernel oil and fish oil. Due to vast consumption, presence of fat, burgers can be used as a vehicle to transport omega-3 fatty acids to the body. Burgers are low-priced, juicy, delicious food, multipurpose applications, which makes it a common meal choice.^[8] Burgers are usually prepared from beef, chicken, and production of fish burgers is significantly lower than the first two types. While using omega-3 fatty acids in burgers, it is technically challenging for the food scientists to deal with issue of lipid oxidation. Optimum shelf life of burgers is three months. Omega-3 fatty acids are polyunsaturated fatty acids (PUFA), which are extremely susceptible to lipid oxidation. In oxidized form, omega-3 fatty acids lose their therapeutic properties, lipid oxidation of PUFA leads to the production of highly objectionable flavors and potentially toxic oxidation products such as aldehydes, ketones, alcohols, and so on.^[9] Intake of oxidized/rancid fats is even worse than consuming bad fats (saturated/trans fats). To prevent lipid oxidation of omega-3 fatty acids, addition of antioxidants in food substrate is necessary however, the perceived carcinogenicity of certain antioxidants such as butylate hydroxy anisole, butylated hydroxy quinone and tertiary butylated hydroxy quinone has directed the food scientists to use natural antioxidants. Large number of natural antioxidants have been discovered and researchers are trying to discover potential sources of antioxidants.^[10,11] MKO is highly resistant to lipid oxidation, among all dietary sources of lipids, it showed the highest induction period, it is a great source of phenolic compounds.^[12] Mangiferin, chlorogenic acid, caffeic acid, quercetin, and carotenoids were the major phenolic compounds in MKO.^[13] Application of mango kernel oil in sunflower, cookies, butter oil and cheddar cheese significantly improved the oxidative stability.^[14,15] Antioxidant activity of MKO for the oxidative stabilization of fish oil in burgers is not previously studied. Therefore, this study was aimed to explore the effect of MKO and fish oil blends on fatty acids composition, antioxidant properties, lipid oxidation, induction period and sensory properties of fish burgers by means of innovative methods.

Materials and methods

Raw materials

The fish Rohu (Labeo rohita) was collected from market and ground through a perforate plate (5 mm). Oil from mango kernel (Chaunsa variety) was extracted through a screw press at 25–30°C, only cold pressed oil (without any added antioxidant) was used in this experiment. Food-grade fish oil (refined, bleached, deodorized without antioxidant) was used in this investigation, both oils have less than one-month age. For performing laboratory testing, chemicals were supplied by Sigma–Aldrich (St. Louis, MO).

Analysis of fish meat, burgers, MKO and fish oil

Moisture, crude protein, ash content, and crude fat in fish meat and burgers were determined by the standard protocols.^[16] MKO and fish oil were tested for free fatty acids, melting point, saponification value refractive index moisture, iodine value, unsaponifiable matter, peroxide value was determined using the standard procedures.^[17]

Experimental plan

Five different types of fish burgers were produced in triplicate, for each type, composition of spices mixture was as salt 2%, modified starch 1%, and spice mixture 3% (onion powder 25%, garlic powder 10%, ginger powder 10%, red chili powder 30%, ground black pepper 10%, and white cumin powder 5%). For first type of burgers, 80% ground fish meat was blended with 10% fish oil, 0% MKO, 4% water, and 6% spices mixture (control). For second type of burgers, 80% ground fish meat was blended with 7.5% fish oil and 2.5% MKO, 4% water, and 6% spices mixture (T₁). For third type of fish burgers, 80% fish meat was blended with 5% fish oil, 5% MKO, 4% water, and 6% spices mixture (T₂). For the production of fourth type of burgers, 80% ground fish meat was blended with 2.5% fish oil and 7.5% MKO, 4% water, and 6% spices mixture (T₃). For the production of fifth type of burgers, 80% ground fish meat was blended with 10% MKO, 0% percent fish oil, 4% water, and 6% spices mixture (T₄). Mixture was transformed to burgers (5 mm thickness and 90 g weight) by burger maker, all types of burgers were packaged in polyethylene bags and stored at −16 to −18°C for 90 days and analysis were conducted at 0, 45, and 90 days.

Oil extraction from burgers

Oil was extracted from burgers using cold extraction method briefly, 3 parts of HPLC grade methanol was mixed with chloroform and oil was extracted by Folch method, excess solvent was evaporated using rotary evaporator at 40°C.

Total antioxidant capacity (TAC)

Reagent solution was prepared by mixing 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate, sample 0.1 ml (immediately after production and frozen storage), and 1 ml reagent solution were mixed and heated to 995^oC/90 min. Absorbance was recorded at 695 nm using ascorbic acid as standard^[18].

1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity

DPPH solution (20 mg/l) was prepared in methanol, sample (0.75 ml, immediately after production and frozen storage) was mixed with 1.5 ml DPPH solution and reacted in the dark for 30 min. Absorbance was recorded on spectrophotometer at 517 nm and results were reported in percentage.^[19]

Antioxidant activity in linoleic acid

Sample (1 ml) was dissolved in 1.5 ml of 0.1 M phosphate buffer (pH 7.0) and mixed with 1 ml (50 mM linoleic acid in ethanol), incubated for 30 min at 40°C. Test portion (50 µl) was mixed with 2.3 ml (75% ethanol & 50 µl of 30% NH₄SCN and 5 µl of 20 mM FeCl₂ in concentrated HCl and measured on a spectrophotometer at 500 nm.^[20]

Fatty acid profile

Extracted fat (50 mg) was taken in test tube, followed by the addition 2 ml each n-hexane and 0.5N sodium methoxide (prepared in HPLC methanol). Samples were vortex at 2000 rpm for 2 min, stay time of 15 min was given, upper layer was extracted, transferred to GC-Vials and 1 µL was injected (split ratio 50:50) through auto liquid sampler in a GC-MS (7890-B, Agilent Technologies) in a fused silica capillary column SP-2560 (100 m × 0.25 mm, Supelco) using helium as a carrier gas at the follow rate of 2 ml/min, while the follow rates of hydrogen and oxygen were 4 ml and 40 ml/min, respectively. Temperature of inlet and flame ionization detector was set at 200°C and 250°C. FAME-37 standards were used to identify and quantify the individual fatty acids.^[21]

HPLC characterization of phenolic compounds of MKO and fish oil

Sample (0.2 g) was dissolved in n-hexane and 4 µl was injected to HPLC system (Agilent-1100) equipped with 4 × 2-mm id C18 ODS column and DAD (G1315-A). Aqueous acetic acid (2%) and 0.5% acetic acid in acetonitrile constituted the mobile phase with a flow rate at 0.6 ml/min at 370 nm. Five standards each of mangiferin, chlorogenic acid, caffeic acid, catechin and quercetin were used to quantify the phenolic compounds of MKO and fish oil using calibration curve.^[22]

Measurement of lipid oxidation during storage and after cooking

Free fatty acids (FFA), peroxide value (POV) in frozen storage and cooked burgers were determined at 0, 45 and 90 days.^[18] Induction of period of fish burgers were determined on Professional Rancimat (Model 892) equipped with eight reaction vessels and moisture free oxygen generator. Extracted fat sample (2.5 g) was loaded in each reaction vessel, temperature and oxygen flow was set at 120°C and 20 liter/hr and operated with Stab Net Software. Break point in the curve was considered as an induction period.

Sensory evaluation

Sensory evaluation of cooked fish burgers was performed at 0, 45 and 90 days of storage, for descriptive sensory analysis, panel of ten trained judges was verified, selected and trained according to the standard method [53]. For the standardization of pre-drafted vocabulary (color, flavor, and texture), four training sessions (each two hour) were conducted. Quantitative and qualitative calibration of panel was attained by familiarization with reference materials, scale definitions. Sensory evaluation was performed in individual sensory evaluation booths in an illuminated and ventilated laboratory at 20°C on a 9-point scale. Data of sensory analysis were analyzed by FIZZ software, version 2.46B. During the sensory analysis, unsalted crisp bread and distilled water were given for the rinsing of palate.

Statistical analysis

For the execution of current research work, completely randomized design was used. All kinds of burger were prepared in triplicate and also analyzed three times, mean was the product of three replicate and three times analysis, that is, 3 × 3 = 9. Different letters on means were given on the basis of DMR analysis with the aid of 9.4 SAS software.

Results and discussion

Analytical characterization of fish oil and mango kernel oil (MKO)

FFA, melting point, saponification value refractive index@40°C, moisture, iodine value (wijs), unsaponifiable matter and POV in fish oil were 0.08%, −13.5°C, 193 mg KOH/g, 1.4788, 0.18%, 204 cg/100 g, 1.27%, and 0.24 (MeqO₂/kg). Free fatty acids, melting point, saponification value, refractive index@40°C, moisture, iodine value (Wijs method), unsaponifiable matter, POV in MKO were 0.11%, 35.4°C, 191 mg KOH/g, 1.4557, 54.8 cg/100 g, 1.72%, and 0.25 (MeqO₂/kg). Results of fish oil and MKO observed in the present study are almost similar to the literature.^[4,15]

Composition of fish burgers

Moisture, fat, protein and ash contents of tilapia fillet recorded in current investigation were 77.8%. 2.14%, 18.7%, and 1.12%, respectively (Table 1). Proximate composition of tilapia fillet is almost similar to findings of Filho.^[23] Minor amount of glycogen is also present in fish muscle, which is used for energy, carbohydrate contents of five fish varieties ranged from 1.5 to ,4.5%.^[24] Moisture, protein, lipid and ash content in raw fish was 78.26, 18.12%, 2.3% and 0.89%.^[25] Addition of either fish oil or MKO did not affect proximate composition of all types of burgers. Protein content of control and all treatment ranged from 15.14 to 15.42% (p > .05). Lipid content among control and all treatments ranged from 11.74 to 11.92% (p > .05). For each type of burger, either fish oil, MKO or their blends were added at 10% concentration, rest of the lipids were contributed by fish flesh. No significant difference was recorded among ash content of control and all the treatments. This non-variation among ash contents of fish burgers was due to same formulation, processing facility and researcher. dos Santos Fogaça & Sant’Ana^[26] produced fish burger by blending 98.8% tilapia fillet and 1.2% condiments, moisture, lipid, protein and ash content of burgers were 76.1%, 1.63%, 20.7%, and 2.33%. In present study, lower protein content was due to the lower concentration of protein source 80% tilapia fillet as compared to 98.8% tilapia fillets as compared to the study.^[20] Proximate composition of present investigation was almost similar to grilled burger produced from catfish, moisture, fat, protein, ash, and carbohydrate contents in grilled burgers were 68.3%, 6.1%, 18.9%, 2.6%, and 4.2%.^[27]

Table 1. Composition of fish burgers.

Treatments	Protein (%)	Fat (%)	Ash (%)
Control	15.22±0.13	11.88±0.16	2.13±0.02
T1	15.42±0.08	11.95±0.13	2.09±0.06
T2	15.14±0.05	11.74±0.07	2.15±0.04
T3	15.25±0.11	11.81±0.18	2.10±0.01
T4	15.29±0.03	11.92±0.09	2.11±0.03

Means mentioned in individual columns of protein, fat and ash are non-significantly different from each other (p>.05)

Control: 100% Fish Oil.

T1: 75% Fish Oil & 25% Mango Kernel Oil.

T2: 50% Fish Oil & 50% Mango Kernel Oil.

T3: 75% Fish Oil & 25% Mango Kernel Oil.

T4: 0% Fish Oil and 100% Mango Kernel Oil.

HPLC characterization of phenolic compounds of MKO and fish oil

Analytical characterization of phenolic compounds of MKO was performed on HPLC, five major phenolic compounds viz mangiferin, quercetin, catechin, chlorogenic acid and caffeic acid were found in MKO. Magnitude of mangiferin, quercetin, catechin, chlorogenic acid and caffeic acid were 1381, 87, 512, 973 mg/100 g, respectively. Schieber et al.^[22] performed analytical characterization of phenolic compounds of MKO and reported that mangiferin, quercetin, catechin and chlorogenic acid were most abundant phenolic compounds. Ferulic acid, kaempferol, catechin, quercetin and mangiferin were present in MKO.^[28] Nadeem et al.^[15] compared the total phenolic contents of MKO with commercially available edible oils, mango kernel had highest total phenolic contents than other vegetable oils. Total phenolic contents of MKO were 11.6 mg GAE/g. Takeoka and Dao^[29] reported that anthocyanin content of MKO was 415 mg/100 g, antioxidant activity of anthocyanin is scientifically established and extensively reported in literature. Wide range of flavonols and xanthone glycosides have also been reported in MKO by Schieber et al.^[22] Concentrations of phenolic compounds in MKO were higher than other parts of the kernel.^[30] Abdalla et al.^[13] studied the phenolic compounds of MKO by HPLC, coumarin, gallic acid, mangiferin, chlorogenic acid, ferulic acid and cinnamic acid were found in MKO. HPLC characterization of fish oil revealed that mangiferin, quercetin, catechin, chlorogenic acid and caffeic acid were not detected. Non-detection of phenolic compounds in fish oil can be justified by the fact that refined bleached and deodorized (RBD) version of fish oil was used in this study. RBD fish oil was produced by refining crude fish oil with 14% NaOH followed by three hot water washings (95°C), bleaching was performed at 110°C using acid activated clay and activated carbon for 30 min under vacuum (600 mm Hg) and deodorization was performed at 240°C for 3 hrs. by the injection of live steam at 3.5 kg/cm2, under high vacuum −760 mm Hg followed by cooling and polish filtration. Refining, bleaching and deodorization can virtually eliminate phenolic compounds from processed oils.^[31]

Antioxidant capacity of fish burgers

Total antioxidant capacity (TAC) mentions the antioxidant status of food systems and measures their capacity of scavenging or neutralizing the free radicals. It is a prestigious way of measuring the oxidative stress. TAC is directly proportional to the oxidative stability of foods. For the assessment of antioxidant activity of MKO, Arif et al.^[32] used TAC. Foods having higher TAC are perceived to have superior oxidative stability and vice versa.^[33] Results of TAC of MKO supplemented fish burgers is shown in Table 2. Addition of MKO considerably increased the TAC (p < .05). TAC of control, T₁, T₂, T₃, and T₄ was 18.97, 31.47, 47.69, 65.37, and 78.82%. TAC of MKO was 89.52%, which was the reason for higher TAC of MKO added fish burger samples. MKO is a great source of polyphenols, carotenoids, antioxidant capacity of MKO was 84.2%, it is highly resistant to lipid oxidation, among all dietary sources of lipids, it showed highest induction period, it is a great source of phenolic compounds, mangiferin, chlorogenic acid, caffeic acid, quercetin and carotenoids.^[34] Addition of MKO in butter oil from 2.5% to 10% concentrations significantly increased the antioxidant capacity.^[15] Mango kernel powder (12% oil content) was used in beef mince, addition of mango kernel powder considerably enhanced the antioxidant capacity.^[35] For the assessment of antioxidant status of foods, DPPH is regarded as an empirical assay. DPPH value of control, T₁, T₂, T₃, and T₄ was 14.43, 19.74, 23.72, 34.48, and 40.66%, respectively. DPPH value of MKO, chia oil, sesame oil, groundnut oil and date seed oil were 91, 74, 83, 69 and 78%.^[31] Addition of MKO in formulation of cookies raised total phenolic contents from 3.84 to 24.37 mgGAE/g and DPPH value increased from 1.2% to 17.6%.^[36] Jafari et al.^[12] introduced MKO at 1, 5 and 10% levels in tallow and studied the antioxidant characteristics of blends. Addition of MKO in tallow at all concentrations significantly raised antioxidant capacity. Addition of MKO in burgers at 25, 50, and 75% concentrations significantly raised ALA values. ALA value of control, T₁, T₂, T₃, and T₄ was 12.33, 15.92, 17.87, 22.36, and 29.36%. Fish oil is highly vulnerable to lipid oxidation and objectionable flavors are quickly developed. Therefore, for the food scientists, it is challenging to control lipid oxidation in food systems having fish oil. In this investigation, control over lipid oxidation in fish burgers was achieved by using MKO as a natural antioxidant. Findings of this investigation are highly valuable for the fish processing industries and waste utilization view points as most of the waste of mango processing facilities is not utilized. This study also paved the way and suggested another utilization or application of MKO.

Table 2. Antioxidant capacity of burgers.

Treatments	Storage days	TAC (%)	DPPH (%)	ALA (%)
Control	0 30 60 90	18.97±0.14^i 16.27±0.19^j 11.38±0.07^k 8.74±0.06^l	14.43±0.32^j 12.49±0.31^k 10.32±0.24^l 9.13±0.29^m	12.33±0.02^j 11.16±0.09^k 9.24±0.13^l 8.14±0.06^m
T1	0 30 60 90	31.47±0.24^f 29.44±0.31^f 26.38±0.16^g 23.57±0.42^h	19.74±0.13^g 18.69±0.27^g 16.36±0.15^h 14.19±0.34^i	15.92±0.29^g 15.59±0.48^g 14.24±0.08^h 13.97±0.17^i
T2	0 30 60 90	47.68±0.41^d 47.27±0.35^d 46.55±0.49^d 42.38±0.65^e	23.72±0.03^e 23.61±0.41^e 23.11±0.08^e 20.18±0.07^f	17.87±0.09^e 17.62±0.27^e 17.35±0.30^e 15.42±0.26^f
T3	0 30 60 90	65.37±0.26^c 64.88±0.39^c 64.52±0.74^c 62.37±0.57^d	34.48±0.21^c 34.22±0.06^c 34.07±0.14^c 32.28±0.06^d	22.36±0.15^c 22.15±0.05^c 21.83±0.10^c 19.76±0.16^d
T4	0 30 60 90	78.82±0.18^a 78.43±0.22^a 77.82±0.65^a 75.72±0.54^b	40.66±0.43^a 40.53±0.07^a 40.26±0.18^a 38.89±0.25^b	29.36±0.21^a 29.22±0.08^a 29.11±0.14^a 28.74±0.33^b

If a Mean is mentioned with a different letter in a column, this reveals statistically significant difference (p<.05).

TAC: Total Antioxidant Capacity.

DPPH: 2,2-diphenyl-1-picryl-hydrazyl-hydrate.

ALA: Antioxidant Activity in Linoleic Acid.

Fatty acid profile by GC-MS

GC-MS analysis of fish oil revealed that contents of C18:1, ALA, EPA, and DHA were 25.63, 1.12, 7.46, and 8.27%. Amounts of C18:0, C18:1, C18:2, ALA in MKO were 38.65, 44.51, 5.21, and 0.52%, EPA and DHA were not found in MKO (Table 3). Higher amounts of C18:0 and C16:0 suggest that MKO possesses great functional properties and it can be applied to large number of food applications and offers an excellent replacement of palm oil. Further, MKO is pale yellow in color with mild pleasant flavor and industrial processes such as refining, bleaching and deodorization are not required to produce food-grade MKO.^[14] Fatty acids composition of fish oil recorded in this investigation are almost similar to the earlier investigations.^[37,38] Fish oil and MKO were blended in three different proportions, that is, 75% fish oil and 25% MKO, 50% fish oil and 50% MKO, 25% fish oil and 75% MKO. Blending of fish oil and MKO at all three levels significantly altered the fatty acids composition. C18:0, C18:1, ALA, EPA and DHA increased as the concentration of fish oil increased. Impact of blending on fatty acids composition of oils and fats is described in literature. Blending of MKO with watermelon seed oil significantly modified the fatty acids composition, blends had lower concentrations of C18:2 and C18:3 than pure watermelon seed oil.^[39,40] At zero day, concentrations of ALA, EPA, and DHA in control were 1.13, 7.39, and 8.29%. At zero day, concentrations of ALA, EPA, and DHA in T₁ were 0.97, 6.19, 6.35%. At zero day, concentrations of ALA, EPA, and DHA in T₂ were 0.85, 3.62, and 4.19%. At zero day, concentrations of ALA, EPA, and DHA in T₃ were 0.67, 1.84, and 2.18%. Weight of fish burger in this study was 90 ± 2 g, consuming one fish burger containing 50% fish oil and 50% MKO can provide about 7.8 omega-3 fatty acids. According to the recommendations of FDA, minimum 2 g omega-3 fatty acids should be taken on daily basis to prevent life-style related disorders. In present investigation, impact of three months storage phase on fatty acids composition was determined to find a clear picture of fatty acids profile during the long-term storage so that fish processing industries can get a reasonable time to sale their value-added fish burgers. It is clear from fatty acids composition, that control burger underwent serious lipid oxidation and fatty acids composition significantly changed during three months storage. In control after three months of storage, loss of ALA, EPA and DHA was 55, 14 and 11%. In T₁ after three months of storage, loss of ALA, EPA, and DHA was 27, 7, and 15%. At T₂ and T₃ concentration, antioxidant compounds of mango kernel significantly altered the lipid oxidation and efficiently inhibited the free radical mechanism and non-significant changes were recorded between fatty acids composition of fresh and three months stored fish burgers (p > .05). In T₂ after three months of storage, loss of ALA, EPA, and DHA was 3.6, 1.98, and 1.71% only. Statistically, no significant difference was recorded between fatty acids composition of T₂ and T₃ while, T₄ was produced from 100% MKO, less than 0.2% concentrations of EPA and DHA was found in it. Phenolic compounds and other antioxidant substances of MKO inhibited lipid oxidation during the course of three months storage. Estimation of fatty acids composition provides a clearcut picture of lipid oxidation in oils and fats. Abdalla et al.^[13] studied the effect of mango kernel addition on fatty acids composition of sunflower, results suggested that MKO strongly inhibited the lipid oxidation, fatty acids composition of fresh and two months stored sunflower oil was not different from each other. Muffins were prepared by blending shortening with MKO and transition in fatty acids composition was monitored for 60 days, fatty acids composition of freshly prepared muffins were not different from 60 days old stuff.^[41]

Table 3. Effect of fish oil and mango kernel oil on fatty acid profile burgers of mg/100 g.

Fatty acid			Control		T1		T2		T3		T4
	Fish oil	Mango kernel oil	0-Day	90-Days	0-Day	90-Days	0-Day	90-Days	0-Day	90-Days	0-Day	90-Days
C14:0	5.24±0.14^a	NF	5.22±0.26^a	4.43±0.25^b	3.87±0.13^c	3.62±0.12^d	2.65±0.12^e	2.61±0.27^d	1.39±0.21^g	1.35±0.16^g	0.18±0.01^h	0.17±0.01^h
C16:0	12.76±0.10^a	7.21±0.15^c	12.43±0.34^a	10.67±0.44^b	8.37±0.20^c	7.11±0.44^c	6.37±0.26^d	6.25±0.34^d	3.24±0.46^e	3.18±0.35^e	7.18±0.67^c	7.13±0.39^c
C18:0	2.55±0.17^f	38.65±0.28^a	2.32±0.24^f	1.22±0.17^g	10.65±0.33^d	9.44±0.16^e	20.18±0.11^c	19.85±0.53^c	28.77±0.24^b	28.63±0.14^b	39.75±0.41^a	38.66±0.16^a
C18:1	25.63±0.22^f	44.51±0.18^a	25.94±0.36^f	22.85±0.51^h	30.44±0.29^d	28.59±0.54^e	35.94±0.36^c	35.47±0.87^c	39.62±0.16^b	39.49±0.05^b	44.53±0.33^a	44.21±0.28^a
C18:2	2.27±0.13	5.21±0.09^a	1.95±0.02	1.66±0.03	3.11±0.02	2.24 ±0.09	3.78±0.06^c	3.76±0.73^c	4.39±0.10^b	4.32±0.05^b	5.25±0.02^a	5.23±0.05^a
C18:3 Alpha Linolenic Acid	1.12±0.01^a	0.52±0.02^f	1.13±0.01^a	0.51±0.09^f	0.97±0.06^b	0.71±0.07^c	0.85±0.07^d	0.82±0.04^d	0.67±0.07^e	0.63±0.07^e	0.49±0.04^f	0.48±0.14^f
C20:5 Eicosapentaenoic acid	7.46±0.08^a	NF	7.39±0.10^b	6.35±0.01^c	6.19±0.02^d	5.91±0.03^e	3.62±0.13^f	3.55±0.02^f	1.84±0.02^g	1.81±0.02^g	0.17±0.01^h	0.14±0.05^h
C22:2 Docosapentaenoic acid	1.29±0.03^a	NF	1.25±0.03^b	0.78±0.02^e	0.92±0.01^c	0.82±0.01^d	0.68±0.02^f	0.66±0.01^f	0.13±0.03^g	0.12±0.01^g	0.10±0.01^g	0.11±0.02^g
C22:6 Docosahexaenoic acid	8.27±0.22^a	NF	8.29±0.16^a	7.41±0.01^b	6.35±0.01^c	5.54±0.01^d	4.19±0.01^e	4.12±0.01^e	2.18±0.07^f	2.14±0.03^f	0.15±0.01^g	0.12±0.03^g

If a Mean is mentioned with a different letter in a row, these reveals statistically significant difference (p<.05).

NF: Not Found.

Lipid oxidation

Lipids in foods may suffer from hydrolytic and oxidative deterioration. Lipids having higher concentration of unsaturated fatty acids undergo rapid oxidative deterioration, rate of oxidation in PUFA is about 100 times more than MUFA. EPA and DHA (5 and 6 double bonds) are much more susceptible to lipid oxidation, every double bond is a place for H₂ abstraction and enable to produce more than 16 and 20 hydroperoxides for EPA and DHA, respectively.^[42] In the present study, FFA and POV were used as indicators of lipid oxidation in storage phase (0, 45, 90 days) and cooked fish burger (170°C, 20 min) (Table 4). Addition of fish oil with MKO at all levels did not affect FFA content, FFA of food-grade fish oil and MKO were 0.10 and 0.08% (oleic acid) which was the reason for non-variation in FFA content of all treatments. In present study, transition in FFA during the storage phase of 90 days and cooking (175°C, 20 min) was monitored. During storage phase, FFA of control and all the treatments went on increasing slowly and steadily, determination of FFA at 45 and 90 days of storage revealed non-significant variation (p > .05). It was observed that addition of MKO in fish oil at all concentrations did not affect the generation of FFA. This can be justified as FFA are product of hydrolysis of triglyceride molecules. Hydrolysis of triglycerides may be caused by hydrolytic enzymes such as lipases, other factors include moisture, metal ions, storage duration and conditions.^[43] Fish oil has higher degree of unsaturation than MKO however, FFA generation has no connection with degree of unsaturation. Azeem et al.^[41] did not find any correlation between rise of FFA and degree of unsaturation. FFA of all types of cooked fish burgers were significantly higher than uncooked burgers, this was due to catalytic effect of temperature employed during cooking. FFA of oils and fats increased during frying.^[44] Higher FFA content may lead to the production of off-flavors in foods therefore, food industries strive to keep FFA content at the lowest possible level. According to the standard of EU, FFA content of food should be less than 0.2%, FFA content of all types of samples in current investigation were below than allowable limit. POV is the most commonly used test for the assessment of lipid oxidation in oils and fats. POV is directly connected to the storage stability, foods having lower POV can be kept for a longer period of time. Addition of mango kernel oil in fish burgers at T₂ and T₃ concentrations effectively inhibited the lipid oxidation during long term storage and cooking as well. During storage phase of (−16 to −18oC), POV of 45 days stored control, T₁, T₂, T₃, and T₄ was 1.16, 0.85, 0.35, 0.32, and 0.29 (MeqO₂/kg). During storage phase of (−16 to −18°C), POV of 90 days stored control, T₁, T₂, T₃, and T₄ was 3.42, 1.55, 0.63, 0.41, and 0.31 (MeqO₂/kg). During cooking, POV of all types of fish burgers increased but to different extents, treatments having MKO resisted the lipid oxidation during cooking and yielded lower POV as compared to control. After cooking, POV of freshly prepared control, T₁, T₂, T₃ and T₄ was 0.72, 0.68, 0.41, 0.37 and 0.35 (MeqO₂/kg). After cooking, POV of 90 days stored control, T₁, T₂, T₃, and T₄ was 4.88, 2.29, 0.82, 0.77, and 0.38 (MeqO₂/kg). Effective inhibition of lipid oxidation at T₂ and T₃ concentrations was due to the presence of naturally occurring antioxidant compounds in MKO. MKOs is a rich source of large number of phenolic compounds and antioxidant substances such as mangiferin, quercetin, catechin, chlorogenic acid and caffeic acid. According to the allowable limits of European Union, POV in foods should be less than 10 (MeqO₂/kg). Frying oil was blended with MKO, frying stability was compared butylated hydroxy toluene (BHT), oxidative stability of frying oil containing MKO was better than BHT.^[27] Bolonga-type mortadella was produced by blending beef, pork, fat and MKO, sample containing 100 ppm BHT was used as control, samples were stored at 4–5°C for 21 days. MKO effectively altered the lipid oxidation, measurement of TBA value at the end of storage phase showed that samples added with MKO had lower TBA value than control.^[45]

Table 4. Effect of mango kernel oil on lipid oxidation of fish burgers during storage and cooking.

Treatments	Days	FFA% during storage and cooking		POV (MeqO2/kg) during storage and cooking
		During storage at –16 to −18°C	After cooking at 175°C/20 min	During storage at −16 to −18°C	After cooking at 175°C/20 min
Control	0 45 90	0.08±0.02^g 0.11±0.03^e 0.14±0.02^d	0.14±0.02^d 0.18±0.01^b 0.19±0.02^b	0.24±0.05^k 1.16±0.02^e 3.42±0.01^b	0.72±0.04^g 2.34±0.10^c 4.88±0.13^a
T1	0 45 90	0.09±0.01^g 0.11±0.02^e 0.15±0.01^c	0.13±0.01^d 0.18±0.03^b 0.19±0.01^b	0.25±0.02^k 0.85±0.04^f 1.55±0.01^d	0.68±0.03^g 1.19±0.06^e 2.29±0.09^c
T2	0 45 90	0.11±0.02^f 0.12±0.01^e 0.16±0.02^c	0.14±0.01^d 0.16±0.02^c 0.19±0.03^b	0.22±0.01^k 0.35±0.02^j 0.63±0.04^h	0.41±0.02^j 0.65±0.03^h 0.82±0.07^f
T3	0 45 90	0.10±0.01^f 0.12±0.02^e 0.15±0.03^c	0.12±0.02^e 0.15±0.01^c 0.19±0.01^b	0.24±0.02^h 0.32±0.01^k 0.41±0.03^j	0.37±0.04^j 0.61±0.02^h 0.77±0.08^g
T4	0 45 90	0.09±0.01^f 0.12±0.03^e 0.16±0.01^c	0.12±0.02^c 0.16±0.01^c 0.22±0.03^a	0.26±0.03^k 0.29±0.01^k 0.31±0.04^k	0.35±0.01^j 0.52±0.03^i 0.38±0.05^j

Within two columns of a same parameter, if means are mentioned with a different letter, it shows statistically significant difference (p<.05).

FFA: Free Fatty Acids.

POV: Peroxide Value (MeqO2/kg).

Induction period

For the estimation of antioxidant activity of natural and synthetic antioxidants, measurement of induction period provides a reliable information regarding their antioxidant activity. Further, induction period is highly suitable to determine the oxidative stability of a food matrix in a short duration. Conventional oxidation techniques require several months to know the oxidative stability of food matrix therefore, accelerated oxidation technique is preferred over conventional methods to save precious time for product development and formulation alterations.^[46] For the assessment of oxidative stability and anticipating the expected keeping quality of vanaspati formulated from MKO and palm oil blends, induction period was used.^[32] For the anticipation of expected shelf life of olein and super olein fraction of date seed oil, induction period was measured on Professional Rancimat.^[47] For the enumeration of oxidative stability of olein and super olein fractions of flaxseed oil, Azad et al.^[48] used Rancimat method and strong correlation was established between POV and induction period (R₂ = 0.9875). In this investigation, induction period of fish oil, MKO, T₁, T₂, T₃, and T₄ was measured on Professional Rancimat 892. Induction period of fish oil, MKO, T₁, T₂, T₃, and T₄ was 2.45, 64.28, 7.33, 13.42, 18.39, and 62.84 hrs (p < .05) (Figure 1). MKO is a rich source of large number of phenolic compounds and antioxidant substances such as mangiferin, quercetin, catechin, chlorogenic acid and caffeic acid. Anthocyanin content of MKO was 415 mg/100 g, antioxidant activity of anthocyanin is scientifically established. Wide range of flavonols and xanthone glycosides has been discovered in MKO by Schieber et al.^[22] Significantly higher induction period of fish burger can be connected to the presence of above-mentioned phenolic compounds. Induction period of MKO, soybean oil, sunflower oil, cottonseed oil, chia oil, watermelons oil, and butter oil were 85.2, 2.41, 2.35, 3.82, 4.1, and 10.56 hrs, respectively.^[49] Abdalla et al.^[13] determined induction period of large number of commercially available cooking oils, among all the tested samples MKO showed the highest induction period. Among all edible oils, MKO possesses the highest induction period.^[50] Form the results of induction period, it can be assumed that MKO can be used for the oxidative stabilization of value-added meat products however, this should be studied in detail.

Figure 1. Rancimat oxidative stability index of fish oil and MKO blend in burgers with different antioxidant activity of natural and synthetic antioxidants treatments.

Sensory characteristics

Addition of MKO in fish burgers at all levels did not affect color score of all treatments (Table 5). Color score of control and other treatments ranged from 8.0 to 8.5 (p > .05). During three months frozen storage (−16 to −18°C) of control and experimental burgers, no surface discoloration was recorded. Addition of MKO at all concentrations did not have any impact on flavor score. Flavor score of control and T₁ decreased during frozen storage of 90 days and cooking process Flavor score of 90 days stored control, T₁, T₂, T₃, and T₄ was 7.1, 7.0, 7.9, 8.0, and 8.0 (p < .05). Decline in flavor score of control and T₁ was not due to the addition of MKO in the formulation of fish burgers rather it was due to lipid oxidation of PUFA present in fish oil. Some of the panelist criticized control and T₁ for having oxidized flavor. Addition of MKO at all levels significantly improved the texture of fish burgers. Evaluation of texture score at 0, 45, and 90 days stored fish burgers showed that texture score of control was significantly lower than all the treatments. Few panelists write in their comments that control had softer texture and lower crispiness as compared to all other treatments. Fatty acids composition of mango kernel showed that it had 55% saturated and 45% unsaturated fatty acids with a melting point of 35.5°C. Control was prepared from 100% fish oil which has higher levels of PUFA with sub-zero melting point. Improvement in texture score of treatments can be connected to higher solid fat index, more number of saturated fatty acids and higher melting point. Color flavor and texture score of 90 days frozen stored T₂ was 91, 90, and 86% of total score. MKO and fish oil can be used at 50:50% concentration to formulate fish burgers of acceptable sensory properties. Baugreet et al.^[9] produced beef burgers from vegetable oils, sensory evaluation indicted that addition of vegetable oils did not affect sensory attributes.

Table 5. Sensory Evaluation of burgers produced from fish oil and mango kernel oil blends.

Treatments	Days	Color during storage at −16 to −18°C	Color after cooking at 175°C/20 min	Flavor during storage at −16 to −18°C	Flavor after cooking at 175°C/20 min	Texture during storage at −16 to −18°C	Texture after cooking at 175°C/20 min
Control	0 45 90	8.4±0.10^a 8.5±0.13^a 8.1±0.02^a	8.2±0.04^a 8.1±0.07^a 8.0±0.09^a	7.5±0.13^b 7.4±0.15^b 7.1±0.03^b	7.2±0.09^b 7.1±0.03^b 6.3±0.02^d	7.1±0.03^b 7.3±0.06^b 7.2±0.03^b	7.2±0.05^b 7.0±0.07^b 7.1±0.12^b
T1	0 45 90	8.1±0.05^a 8.2±0.02^a 8.0±0.05^a	8.1±0.03^a 8.0±0.09^a 7.9±0.08^a	8.4±0.02^b 7.3±0.03^b 7.0±0.06^b	8.1±0.05^b 6.7±0.02^c 6.4±0.07^d	8.2±0.14^a 8.3±0.19^a 8.1±0.08^a	8.1±0.06^a 8.2±0.16^a 8.0±0.15^a
T2	0 45 90	8.2±0.06^a 8.4±0.09^a 8.2±0.06^a	8.3±0.06^a 8.2±0.03^a 8.1±0.04^a	8.1±0.06^a 8.0±0.09^a 7.9±0.04^a	8.0±0.02^a 7.9±0.06^a 8.0±0.12^a	8.3±0.17^a 8.3±0.13^a 8.1±0.04^a	8.1±0.19^a 8.2±0.13^a 8.2±0.08^a
T3	0 45 90	8.3±0.04^a 8.1±0.12^a 8.0±0.12^a	8.4±0.11^a 8.2±0.10^a 8.1±0.02^a	7.9±0.11^a 8.1±0.14^a 8.0±0.12^a	8.2±0.14^a 8.0±0.17^a 8.1±0.13^a	8.2±0.12^a 8.0±0.09^a 8.1±0.05^a	8.3±0.07^a 8.2±0.09^a 8.3±0.06^a
T4	0 45 90	8.1±0.03^a 8.0±0.07^a 8.2±0.05^a	8.2±0.13^a 8.1±0.09^a 8.0±0.15^a	8.1±0.06^a 8.3±0.02^a 8.0±0.05^a	8.3±0.14^a 8.4±0.09^a 8.2±0.14^a	7.9±0.05^a 8.1±0.04^a 8.0±0.07^a	8.1±0.17^a 8.3±0.15^a 8.2±0.22^a

Within two columns of a same parameter, if means are mentioned with a different letter, it shows statistically significant difference (p<.05).

Conclusion

Addition of mango kernel oil from 25% to 75% levels did not affect compositional properties of fish burgers rather it improved antioxidant activity of fish burgers. Mango kernel oil effectively inhibited lipid oxidation, fatty acid profile of 90 days stored T₂ was almost similar to freshly prepared burgers. Peroxide value of 90 days stored T₂ and control was 1.55 and 3.42 (MeqO2/kg). Induction period of control and T₂ was 2.45 and 13.42 hrs. Color, flavor and texture score of T₂ was better than control. These results suggest that Mango kernel oil can be successful used in the formulation of fish burgers.

Author contributions

Conceptualization, M.N. and M.I.; methodology, S.S.; software, F.A.; validation, M.N., M.I., C.G.A., and M.A.B.; formal analysis, M.H.U.R and C.G.A.; data curation, M.N.; writing – original draft preparation, S.S.; visualization, M.H.U.R. and C.G.A.; supervision, M.N. and C.G.A.

Consent for publication

All the authors consent to the publication

Acknowledgments

The authors are highly obliged to the Library Department, University of Veterinary and Animal Sciences (UVAS), Government College University Faisalabad (GCUF), and IT Department, Higher Education Commission (HEC, Islamabad) for access to journals, books and valuable database. The authors are also thankful to Kampala International University, Kampala, Uganda for its collaborative support.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Additional data will be made available on request

Additional information

Funding

This research work was funded by Higher Education Commission (HEC) (Project No. HEC/NRPU/2022/6978)