Lipid oxidation and auto-oxidation play a major role in quality deterioration, reduced shelf-life and a decline in the nutritive value of muscle foods. Oxidation can also cause other detrimental effects, such as loss of essential fatty acids, flavor and discoloration, leading to changes in organoleptic attributes [1
] and the formation of genotoxic and cytotoxic compounds [2
]. Lipid oxidation can be influenced by several factors, such as the degree of lipid unsaturation, muscle type, animal diet and the additives used during processing, cooking and storage. Additionally, reduced pH of muscle food enhances lipid oxidation because of intensified autoxidation of hemoglobin [3
]. To control quality deterioration in muscle foods due to lipid oxidation, synthetic antioxidants, such as butylated hydroxytoluene (BHT) and butylated hydroxy anisole (BHA), are extensively used [4
Recently, the use of synthetic antioxidants has been viewed negatively due to toxicity, adverse effects on human health and food safety [6
]. Therefore, research on safe and effective natural antioxidants from natural sources, such as phenolic compounds, is being explored to control lipid oxidation. Presently, natural antioxidants present in foods and other biological materials have attracted considerable attention because of their safety and potential nutritional and therapeutic values [7
]. Phenolic extract from plants, as well as many phenolic compounds have been successfully demonstrated as natural antioxidants in retarding lipid oxidation in different foods [9
]. The antioxidant effects of various plant extracts, as well as individual phenolic compounds in different muscle food systems have been evaluated and tested by various workers [1
Litchi (Litchi chinensis
Sonn.) is a tropical and subtropical fruit native to South East Asian countries and now widely cultivated throughout the world [13
]. India is the second largest producer of litchi in the world after China. Litchi or lychee, a fruit with a rough brown pericarp surrounding a white flesh (aril), is popular for its delicious taste and possible health benefits [14
]. Litchi fruit pericarp (LFP), which accounts for approximately 15% by weight of the whole fresh fruit, is usually discarded as waste during processing. Recent studies have reported that litchi pericarp contains significant amounts of polyphenols, flavonoids, anthocyanins and polysaccharides [15
]. The major phenolics in LFP tissues were identified as epicatechin, procyanidin B4 and procyanidin B2 [13
]. These phenolics from LFP have good antioxidant activity, anti-inflammatory, anti-carcinogenic and immune-modulatory properties [14
]. However, to our knowledge, no studies have been conducted regarding the antioxidant potential of LFP extract in muscle foods. Herein, we report the first study of the use of LFP extract in a meat product formulation. The objectives of this study were to investigate the antioxidant potential of LFP, an abundant and under-utilized natural resource, and to evaluate its effectiveness in retarding lipid oxidation of cooked meat products.
2. Materials and Methods
2.1. Preparation of Litchi Fruit Pericarp Extract
Fresh and mature litchi fruit was purchased from the local market. The rough brown outer covers (pericarps) were carefully removed from fruits, cleaned with water and dried in an oven at 50 °C. After drying, a fine powder of pericarp was made using a home grinder. Ten grams of litchi pericarp powder were added in 100 mL boiled distilled water and left for 1 h followed by filtration through Whatman No. 1 filter paper to obtain a water extract of litchi fruit pericarp.
2.2. Sheep Meat Nugget Preparation
Sheep meat nuggets were prepared as per the procedure outlined by Das et al.
]. Briefly, sheep meat from leg and loin cuts was collected from the experimental slaughterhouse and kept under frozen storage at −18 °C until further processing. Before processing, meat was thawed and cut into small cubes and minced (Tallers Ramon Model P-22, Barcelona, Spain). For meat emulsion preparation, salt, sugar, phosphate and nitrite were thoroughly mixed to the pre-weighed quantity of minced sheep meat in a bowl chopper (Seydelmann K20 Ras, Stuttgart, Germany), and ice flakes were added during chopping to maintain a lower temperature (8 ± 2 °C). Condiments, dry spice mix, fine wheat flour and LFP extract (0%, 1% and 1.5%) were added, and chopping was continued until uniform mixing of all ingredients. About 500 g of emulsion were placed in a mold and steam cooked for 40 min to prepare cooked meat blocks. Blocks were sliced and cut into small nuggets. Sheep meat nuggets were aerobically packed in low-density polyethylene (LDPE) pouches and kept at refrigerated temperature (4 ± 1 °C) for further analysis. The formulation for control and treated nuggets with LFP extracts and BHT (100 ppm) is present in Table 1
. The whole experiment was replicated thrice.
2.3. Analysis of Litchi Fruit Pericarp Extract
2.3.1. Analysis of Total Phenolics Content
Total phenolics content in litchi fruit pericarp extract and BHT were determined by the Folin-Ciocalteu (F-C) method [20
]. Zero-point-seven-five microliters (0.75 µL) of Folin-Ciocalteu reagent were added in 100 µL of different dilutions of extract, and the final volume was made ten times with distilled water (7.65 mL). After 5 min, 0.75 mL of a sodium carbonate solution (7.5%) were added to each tube. The tubes were incubated for 90 min at room temperature in the dark, and absorbance (U-28000 Spectrophotometer, Hitachi, Tokyo, Japan) was measured against a blank at 725 nm. A standard curve was plotted using different concentrations of gallic acid, and the amount of total phenolics was calculated as gallic acid equivalents (GAE) in mg/g of dried LFP powder.
Total phenolics content in cooked sheep meat nuggets from control, BHT and extract-incorporated formulations was analyzed by using the Folin-Ciocalteu assay [21
] with slight modifications. Briefly, five grams of nugget were homogenized with 25 mL of 70% acetone and kept overnight for extraction at refrigeration temperature. Suitable aliquots of extracts were taken in a test tube, and the volume was made to 0.5 mL with distilled water followed by the addition of 0.25 mL F-C (1 N) reagent and 1.25 mL sodium carbonate solution (20%). The tubes were vortex mixed, and the absorbance was recorded at 725 nm after 40 min.
2.3.2. Radical Scavenging Activity Using the DPPH Assay
The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was performed according to the method of Fargere et al.
]. An aliquot of the various concentrations of the LFP extract and BHT was mixed with 3 mL of DPPH in methanol (final concentration of 250 μM), and the mixture was vortexed vigorously. Tubes were then incubated at room temperature for 30 min in the dark, and the absorbance was taken at 517 nm. Radical scavenging activity (RSA) was calculated by the following equation: RSA% = (AbsorbanceControl
) × 100.
2.3.3. Ferric Reducing Antioxidant Power Assay
The ferric reducing antioxidant power of BHT and LFP extracts was determined according to the method of Oyaizu [23
]. Different concentrations of the LFP extracts and 100 ppm BHT were mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% (w
) potassium ferricyanide. Mixtures were incubated for 20 min at 50 °C followed by the addition of 2.5 mL of 10% trichloroacetic acid and then centrifuged at 700× g
for 10 min. The supernatant (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of ferric chloride (0.1% w
). The absorbance was measured at 700 nm (U-28000 spectrophotometer, Hitachi, Japan). An increase in the absorbance of the reaction mixture indicated the reducing power of the sample.
2.4. Analysis of Cooked Sheep Meat Nuggets
2.4.1. pH and Cooking Yield of Nuggets
The pH of the cooked nuggets was determined by blending 10 g of sample with 50 mL of distilled water for a minute in a homogenizer (model PT-MR-2100, Kinematica AG, Luzern, Switzerland). The pH values were measured using a standardized electrode attached to a digital pH meter (Systronics, Ahmedabad, India). The cooking yield of nuggets was determined by recording the weight of each meat block before and after cooking. The yield was calculated and expressed in percentage as: weight of cooked meat block/weight of raw meat block ×100.
2.4.2. Sensory Evaluation of Sheep Meat Nuggets
A 10-member experienced panel evaluated sheep meat nuggets using an 8-point descriptive scale, where 8 denoted extremely desirable and 1 denoted extremely poor. Panelists were provided information about the nature of the experiments without disclosing the identity of samples and were asked to evaluate the samples for appearance, flavor, juiciness, texture and overall acceptability. Samples were warmed using a microwave oven for 1 min and served randomly to the panelists. The panelists were provided filtered water to rinse their mouth between samples
2.4.3. Lipid Peroxidation of Nuggets during Storage
Lipid peroxidation of nuggets was recorded by measuring thiobarbituric acid-reactive substances (TBARS) at an interval of 3 days during refrigerated storage. The TBARS number (mg malonaldehyde/kg) of nuggets was estimated using the extraction method outlined by Witte et al.
] with slight modifications, as the slurry was centrifuged at 3000× g
for 10 min (Biofuge Primo R, Heraeus, Hanau, Germany) instead of filtration through Whatman No. 42.
2.5. Statistical Analysis
This study was replicated thrice, and in each replication, measurements of all parameters were done in duplicate. One-way ANOVA was conducted using SPSS software (Version 20.0, IBM Corp, Armonk, NY, USA) for the calculation of different mean values (pH, cooking yield, total phenolics, DPPH and sensory attributes), whereas lipid peroxidation was analyzed using two-way ANOVA with treatment and storage time as the main effects. Statistical significance was identified at the 95% confidence level (p < 0.05).
The present findings indicate that litchi fruit pericarp powder is a good source of phenolic compounds having strong free radical scavenging activity and reducing power. The incorporation of pericarp extract in sheep meat nuggets did not have any adverse effect on pH, cooking yield and sensory attributes. Litchi pericarp extract at 1.5% significantly increased the phenolic content in sheep nuggets compared to other treatments and was effective at inhibiting the lipid peroxidation of cooked nuggets similar to the synthetic antioxidant BHT (100 ppm) over a period of 12 days. Being a promising natural antioxidant, litchi fruit pericarp extract could, therefore, be used effectively to improve the product quality, stability and safety of different meat and meat products.