**1. Introduction**

Muscle foods are an excellent source of high-quality protein with high biological value. Moreover, the bioavailability of micronutrients such as iron, selenium, vitamins A, B12, folic acid, sodium, potassium and magnesium of these matrices is much higher than from plant sources [1]. In spite of being nutritious, meat is deficient in dietary fibre, a complex mixture of polysaccharides, which is naturally present as a part of plant material in cereals, vegetables, fruits and nuts. Lack of adequate quantity of dietary fibres in our diet has been involved in several health disorders such as colon cancer, obesity and cardiovascular diseases [2]. On the other hand, a fibre-rich diet is lower in energy density and richer in micronutrients thus reducing several disorders [3], and thereby promotes a healthier lifestyle. However, many food and food products, including meat products, not only lack of minimum amounts of dietary fibre [4] to fulfil the recommended dietary intake, but also di ffer in the quantity and composition of fibres.

As per the American Dietetic Association, the recommended dietary fibre intake for a healthy adult should be approximately 25 to 30 g/day, being the insoluble/soluble fibre ratio 3:1. Hence, to ge<sup>t</sup> the above health benefits of dietary fibre, attempts have been made to incorporate dietary fibres from various sources in meat product formulation to promote various technological benefits and the acceptability of meat products benefitting human health [5].

In a developing country like India, industrialisation, rapid urbanisation, globalisation as well as increasing number of women workforce are factors contributing to a rapidly growing inclination towards fast and convenient meat and food products [6]. However, meat and meat products contain an important amount of unsaturated fatty acids, which are sensitive to lipid oxidation [7,8]. Further, processing such as grinding, chopping, flaking, emulsification and cooking also accelerate lipid oxidation of meat and meat products [8]. Moreover, most meat products are processed using vegetable oils in order to overcome the problems of saturated fatty acids and cholesterol associated with animal fats [9,10]. These vegetable oils, with a high degree of polyunsaturation, accelerate oxidative processes, leading to meat quality deterioration. Therefore, lipid oxidation is considered the major causes of meat quality deterioration with development of undesirable flavours and odours, thus reducing the nutritional, sensorial and functional properties of meat products as well as consumer acceptability [11,12].

The use of antioxidants is considered as an e ffective method to minimise or inhibit lipid oxidation as well as inhibit the formation of toxic oxidation products in muscle foods, thereby improving the shelf-life of products [13–15]. Although synthetic antioxidants have been widely used in the meat industry, consumer concerns over safety issues of products have renewed the interest of food industry in search of antioxidants from natural sources [16]. Thus, both dietary fibre and natural antioxidants are considered as two important dietary fractions and could be very valuable in improving meat product quality and storage stability.

The natural ingredients having dual properties, i.e., a source of dietary fibre besides having antioxidant potential are known as antioxidant dietary fibres (ADF). ADF is defined as dietary fibre concentrate containing a significant content of natural antioxidants linked to the dietary fibre (DF) matrix in a single material [17]. To counter the shortcomings (low fibre content and oxidation) associated with meat and meat products, researchers and meat processors are continuously searching for natural ingredients and materials/extracts, especially of plant origin, to use as additives. The incorporation of ADF in meat products not only increases the shelf life during storage by inhibiting lipid oxidation due to presence of phenolic antioxidants, but also improve the texture, physicochemical and sensory properties of meat products [4,18,19].

*Moringa oleifera*, commonly known as horse radish tree or drumstick tree, is one of the most widely cultivated species native to the sub-Himalayan tracts of India. Almost all parts of this plant, like fruit (pods), gum, root, seed, bark, leaf, flowers and seed oil, are used as a nutritional and nutraceutical resources for human and animal diets [20]. The leaves and flowers are good source of protein and dietary fibre with an adequate profile of amino acids and ash [21,22]. The extracts of flowers of *M. concanensis* (fresh or dried) contain a grea<sup>t</sup> amount of ascorbic acid, polyphenols, tannins and flavonoids with high DPPH scavenging activity [23]. Considering the benefits of both dietary fibre and antioxidants in a single material, the objective of this study was to assess the potential use of Moringa flower (*M. oleifera*) as ADFs or functional ingredients in meat food system to enhance the nutritional quality, storage stability and acceptability of meat products.

#### **2. Materials and Methods**

#### *2.1. Reagents and Plant Materials*

Fresh mature moringa flowers (*M. oleifera*) were collected from a local market of Kolkata, India. Flowers were cleaned thoroughly to remove extraneous dirt, dried completely in a hot air oven at 45 ± 2 ◦C, ground in a grinder (Kenstar, Mumbai, India) and sieved (#60 mesh sieves). The powder obtained was stored in an air tight container at room temperature until further use. Chicken meat (breast) was obtained from West Bengal Livestock Development Corporation, Kolkata, India and kept under frozen storage at −18 ◦C till further processing. Di fferent chemicals such as methanol, butylated hydroxytoluene (BHT), α-amylase, protease, amyloglucosidase trichloroacetic acid, sodium carbonate, 2-thiobarbituric acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), phosphate bu ffer, potassium ferricyanide, F-C reagents and gallic acid, ca ffeic acid, ferulic acid and quercertin were purchased from Sigma-Aldrich (Mumbari, India). All the chemicals used for this experiment were of analytical grade.

#### *2.2. Preparation of Moringa Flower Extract*

Moringa flower (MF) was extracted using either aqueous (AE) or aqueous ethanol (AEH) as solvent (60:40, *v*/*v*). Briefly, for preparation of the extracts, 2 g of each MF was accurately weighed into separate conical flasks. To this, 100 mL of solvent were added and the whole content was held at room temperature (27 ± 1 ◦C) for 10 h, stirring frequently with a glass rod. The mixture was shaken at constant rate (500 rpm) using a shaker, vortexed at high speed for 10 min, and finally centrifuged (REMI NEYA 8, Kolkata, India) at 5000x rpm for 10 min. The content of each extract was then passed through Whatman filter paper No. 1 (HiMedia ®, Mumbai, India). The resulting extract was kept in a container and stored at 2 ◦C for further studies. The extracts (aqueous or aqueous ethanol) obtained from di fferent solvent were analysed for total phenolic contents (TPC), 1, 1 diphenyl-2-picrylhydrazil (DPPH) radicals scavenging activity and ferric reducing antioxidant power (FRAP) assays. The e fficacy of the extracts was determined based on the weight of respective dry powders.

#### *2.3. Analysis of Polyphenols and Antioxidant Capacity*

The total phenolics content of the extracts was assessed using the Folin–Ciocalteu (F-C) method previously described by Singleton et al. [24], while in cooked nuggets the procedure described by Escarpa & González [25] with slight modifications was used. The results were expressed as mg gallic acid equivalents (GAE) /g of dry matter.

HPLC-DAD phenolic composition of MF extracts was carried out according the method of Zeb [26], using an Agilent 1260 Infinity HPLC system equipped with a quaternary pump, degasser, autosampler and diode-array (DAD) detector. An Agilent rapid resolution Zorbax Eclipse plus C18 (4.6 × 100 mm, 3.5 μm) column was used for the separation, using a gradient elution at 0.6 mL/min with gradient program (0–20 min, 95–75% A; 20–40 min, 75–50% A; 50–20%, 40–50 min A; 50–60 min, 20% A) with 1% formic acid in water as solvent A and methanol as solvent B. The chromatograms were obtained at 280 nm.

On the other hand, the method proposed by Fargere et al. [27] was used to determine the free radical scavenging activity (FRSA) using DPPH assay. Free radical scavenging activity (FRSA) was calculated using the following formula.

$$FRSA(\%) = \frac{(absor\,bance\,\,control - absorbonce\,\,sample\,\,sample)}{absor\,bance\,\,control} \times 100\tag{1}$$

The Ferric reducing antioxidant power (FRAP) assay was assessed following the procedure previously established by Oyaizu [28].

#### *2.4. Determination of Dietary Fibre Content*

The dietary fibre composition was analysed according to the enzymatic–gravimetric method [29]. A phosphate buffer was used to disperse the sample and a sequential enzymatic digestion using α-amylase, protease and amyloglucosidase was carried out. The total dietary fibre (TDF) was calculated as the sum of insoluble dietary fibre (IDF) and soluble dietary fibre (SDF) obtained from the enzymatic digestion.

#### *2.5. Preparation of Chicken Nuggets*

Approximately 8 kg of meat sample (chicken breast meat, breast trimmings and chicken skin), obtained from West Bengal Livestock Development Corporation (West Bengal, India), was minced twice (minced through a 6 mm grinding plate followed by 4 mm plate) in a meat mincer. After mincing, meat samples were divided into three (3) different batches. The composition of ingredients for each batch was based on the standard formulation (Table 1). The first batch was considered as control (meat without any ADF), whereas MF at 1% and 2% level were used as ADF for chicken nuggets in formulations (T1 and T2) replacing equal per cent of breast trimmings.

**Table 1.** Formulation of chicken nuggets with different levels of Moringa flower (MF) as antioxidant dietary fibre.


Treatments: Control = no additive; T1 = 1.0% Moringa flower (MF) extract and T2 = 2.0% Moringa flower extract. \* Condiments: garlic and onion (4:1). ADF = Antioxidant dietary fibre. \*\* Dry spice mix (18 g/kg nuggets)—aniseed, black pepper, capsicum, caraway seed, cardamom, cinnamon, cloves, coriander powder, cumin seed, turmeric and dried ginger.

All batches of minced meat samples were prepared separately in a bowl chopper. In order to prepare meat emulsion, salt, phosphate and nitrite were added and mixed to the meat using a bowl chopper. Ice flakes were added to keep temperature at ~8 ± 2 ◦C. Afterwards, condiments, dry spice mix, and fine wheat flour were added, and chopping was continued till uniform mixing of all the ingredients. The emulsion prepared was steam-cooked (100 ◦C) for 40 min to ge<sup>t</sup> cooked chicken meat nugget. The blocks were sliced uniformly to obtain small cubes (size) of chicken nuggets. The formulated nuggets (C, T1 and T2) were aerobically packaged in low density polyethylene (LDPE) pouches and kept under refrigerated conditions (4 ± 1 ◦C) to evaluate different physicochemical parameters, including storage stability (0, 5, 10, 15 and 20 days) and sensory attributes. The whole experiment was conducted three times and samples were analysed in duplicate.
