*2.10. Statistical Analysis*

The present study was repeated three times and, in each replication, measurements of all parameters were done in duplicate. The analysis was carried out using statistical package for the social sciences (SPSS) software (version 20.0, IBM SPSS, Armonk, NY, United States) and for storage study, data was analysed using two-way analysis of variance (ANOVA) with interaction using SPSS software where treatment (control, T1 and T2) and storage time (0, 5, 10, 15 and 20) were considered as factors. The experiment was carried out in 3 × 5 factorial design according to a completely randomised design. The obtained data were subjected to variance analysis, and Duncan's multiple range test was used for comparing the means to find out the effect of antioxidant dietary fibres on various parameters and the differences were considered at α = 0.05 level. The values were presented as mean along with standard error (mean ± standard error).

#### **3. Results and Discussion**

#### *3.1. Chemical Composition, Phenolic Content and Antioxidant Activity of MF Extract*

The chemical composition and dietary fibre contents of the MF extract are shown in Table 2. The protein, ash and lipid content of MF were 17.87 ± 0.28%, 7.87 ± 0.45% and 2.95 ± 0.07%, respectively. In general, the chemical composition depends on the edible part of a plant being analysed. Our findings are in disagreement with the data reported by other authors in MF extract [35,36]. This fact could be attributed to the soil type, cultivars, stage of maturity of flowers and influence of the climatic or weather conditions in the region [37]. As presented in Table 2, MF extract had 36.14 ± 0.77% TDF, 3.90 ± 0.14% SDF and 32.24 ± 0.82% IDF contents. The content of total dietary fibre in MF extract is similar to that reported by Sánchez-Machado et al. [36]. The presence of grea<sup>t</sup> amount of IDF and SDF in the MF extract indicate that MF is a promising source of dietary fibre with a very good physiological effect; better than some cereals like wheat bran (2.9% SDF; 41.1% IDF), and oat bran (3.6% SDF; 20.2% IDF) as reported by Grigelmo-Miguel et al. [38].


**Table 2.** Proximate composition, total phenolic content (TPC), IC50 (μg/mL) and phenolic compounds of Moringa flower (mean values ± error standard of six samples).

AE = Aqueous extract; AEH = Aqueous ethanolic extract, ND = Not detected, IC = Inhibitory concentration.

In the present study, the aqueous ethanolic extract of MF presented the higher total phenolic compounds (TPC) (19.49 ± 1.35 mg GAE/g powder) followed by water extract (18.34 ± 1.16 mg GAE/g powder), although nonsignificant (Table 2). The higher TPC might be attributed to different degree

of polarity of the solvents used for the extraction of polyphenolic compounds, and thus could have contributed significantly to the antioxidant and free radical scavenging activity. Similar observation was also demonstrated by Tekle et al. [39], who reported that the total polyphenolic content of MF extract was relatively higher in ethanolic extract compared to its water counterpart.

The phenolic compounds in MF extracts were identified and quantified as shown in Table 2. Among the phenolics, ferulic acid was present in high concentration and other compounds, including flavonoids (quercetin) were also present. In fact, antioxidant activities of plants mainly depend on the amount of phenolic acids (gallic acid, feluric acid and ca ffeic acid) and flavonoids (catechin, myricetin and quercetin). Several studies indicate that flavonoids are the main contributor for plant's antioxidant activity [40,41]. However, di fferent phenolics exhibit di fferent antioxidant potential, and other compounds may influence the antioxidant activities [42]. The strong antioxidant activity exhibited by MF extracts could be due to abundance of ferulic acid and quercetin.

According to DPPH scavenging activity, our results showed that aqueous ethanol extract of MF contained higher radical scavenging activity than aqueous extract (Figure 1). Both extracts demonstrated purple bleaching reaction at increased concentrations, showing the presence of compounds responsible as free radical scavengers thus reducing the initial DPPH concentration.

**Figure 1.** DPPH radical scavenging activity of MF extracts. AE = Aqueous extract; AEH = Aqueous ethanolic extract; BHT = Butylated hydroxytoluene.

Figure 1 clearly illustrates that the radical scavenging activity of Moringa flowers in both extracts was comparable to butylated hydroxytoluene (BHT) at concentrations from 0 to 250 μg/mL. The levels of inhibition of DPPH radical by the AE, AEH and BHT were 68.52, 72.22 and 76.64%, respectively, and in a concentration-dependent manner. The BHT used as positive control displayed similar level of inhibition at 250 μg/mL, which was nonsignificantly di fferent in comparison to AE. A high correlation exists between DPPH radical scavenging potential and TPC of plant extracts. A study conducted by Siddhuraju et al. [43] reported that at a dosage ranging from 0.2 to 0.6 mg of acetone extract, *M. oleifera* (pericarp of immature drumstick and flower) and *S. grandiflora* (flower and leaf) showed higher free

radical scavenging activity (8.45–65.03%). Sreelatha and Padma [44] noticed that methanol extract of *M. oleifera* leaves significantly reduced DPPH radicals, though lower than our observed results. These variations could be due to di fference in the polarity of solvents and geographical location of the plants [44].

In terms of IC50, the lowest value was shown by the positive control, BHT (118 μg/mL), followed by AEH (121.42 μg/mL) and AE (126.20 μg/mL) of MF extracts (Table 2). Alhakmani et al. [45] reported that DPPH radical scavenging activity of *M. oleifera* flower extract was compared with standard ascorbic acid. Although standard antioxidant had higher scavenging activity at all tested concentrations, the flower extract still showed good free radical scavenging activity. This radical scavenging activity of extracts might be related to the nature of phenolics, thus contributing to their electron transfer/hydrogen donating ability [22,43].

Regarding FRAP assay, MF extract presented good activity on a dose dependent manner. As the aqueous ethanol-extracted MF extract exhibited an overall higher activity at the highest concentrations compared to water extract MF (Figure 2). This outcome showed that it was more e fficient in extracting antioxidants from plant materials. In this regard, Tekle et al. [46] also found that ethanolic extract of M. oleifera leaf and flower exhibiting higher ferric reducing antioxidant activity (*p* < 0.05) compared to the synthetic antioxidant, BHT.

**Figure 2.** Ferric reducing antioxidant power of MF extracts. AE = Aqueous extract; AEH = Aqueous ethanolic extract.

#### *3.2. E*ff*ect of MF Extract on Physico-Chemical Properties of Meat Nuggets*

Table 3 shows the influence of the MF extract on the physicochemical properties of meat nuggets. There was a significant (*p* < 0.05) decrease in emulsion pH with incorporation of MF extract at both levels used in this study. The highest pH value was observed in control (6.33) followed by T1 (6.25) and T2 (6.22) batches, although nonsignificant (*p* > 0.05). The decrease in pH of meat emulsion might be due to acidic pH value (5.44) of MF extract. Our findings agree with the results of Devatkal et al. [47], who noticed that kinnow rind extract significantly decreased the pH values of cooked goa<sup>t</sup> meat patties due to its acidic pH. In a similar study, Habib et al. [48] also found a decrease in pH on the incorporation of pomegranate rind powder at three different levels. However, Das et al. [49] noticed that the use of *M. oleifera* leaves extract did not modify the pH of raw and cooked goa<sup>t</sup> meat patties, whereas Hazra et al. [50] observed that the addition of *M. oleifera* leaves extract increased (*p* < 0.05) the pH values of cooked ground buffalo meat.

**Table 3.** Effect of Moringa flower on physico-chemical parameters of chicken nuggets (mean values ± error standard of six samples).


Treatments: Control = no additive; T1 = 1.0% Moringa flower extract and T2 = 2.0% Moringa flower extract. a–c Mean values in the same row not followed by a common letter differ significantly. Sig. Significance; ns: not significant; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.

Statistical analysis showed that the moisture and lipid content did not differ significantly (*p* < 0.05) between control and treated samples (Table 3). Our findings agree with the data reported by Al-Juhaimi et al. [51], who observed that the moisture content of raw patties decreased as the percentage of *Moringa oleiferi* seed flour increased, but the decline rate was found to be not significant. In addition, Hazra et al. [50] also found that the inclusion of *Moringa oleiferi* leaf extract did not modify the moisture and lipid content of cooked ground buffalo meat. The protein content of the control nugge<sup>t</sup> was 14.38%, whereas the treated nuggets (T1 and T2 groups) presented protein values of 15.27% and 16.32%, respectively. In fact, Moringa flowers have a very high crude protein content varying between 18.92 and 26.16% [35,36]. Therefore, the increase in protein percentage of chicken nuggets might be due to higher protein (17.87%) percentage of MF extract as found in this study. There was a significant difference in the percentage of ash content between control and treated nuggets. The increased ash content in treated chicken nuggets might be due to high ash content of MF (7.87%). A similar trend was reported by Al-Juhaimi et al. [51] who noticed that the ash content of raw patties increased as the percentage of *Moringa oleifera* seed flour increased in cooked ground buffalo meat.

The incorporation of MF significantly (*p* < 0.05) enhanced the emulsion stability of treated nuggets than the control (Table 3). The control and treated chicken nuggets (T1 and T2 groups) showed a 94.45%, 95.56% and 96.47% emulsion stability, respectively. The probable reason for increased emulsion stability could be due to the presence of dietary fibre in MF (36.14%). In this regard, Das et al. [18] also observed that bael pulp residue at 0.5% significantly improved (*p* < 0.05) the emulsion stability in goa<sup>t</sup> meat nuggets. Similarly, Sarıçoban et al. [52] studied the effect of different concentrations (2.5%, 5%, 7.5% and 10%) of lemon albedo (raw and dehydrated) on the functional properties of emulsions and found an increase in the emulsion capacity at 5 percent of added emulsions. A similar trend was also observed in emulsion stability values. Malav et al. [53] reported that there was an increasing trend in emulsion stability with increase in levels of red kidney bean powder. The emulsion stability and cooking yield increased on incorporation of dietary fibre extracted from rice bran at di fferent levels in meat batters [54].

On the other hand, a significant (*p* < 0.05) improvement in cooking yield was observed due to the incorporation of MF at di fferent levels (Table 3). The higher percentage of cooking yield (97.83% and 97.26%) was observed in nuggets with MF than the control (96.79%). The articles available in the literature regarding this aspect indicate that non-meat ingredients with high dietary fibre content, when used in emulsion type of meat products, improve the cooking yield. A similar trend was reported by Ham et al. [55] who noticed that increasing lotus rhizome powder levels as a source of ADF lowered the cooking loss of emulsion sausages (5.89–6.25%) significantly (*p* < 0.05) than that of control sausage (7.31%). In addition, Anderson and Berry [56] also observed that 10% fat beef patties extended with pea fibre had high cooking yield. The probable reasons for the increased cooking yield of chicken nuggets with MF extract could mainly be attributed to the presence of high amount of dietary fibre and its ability to bind more water and fat as reported by Verma and Banerjee [4]. However, this finding disagrees with the data reported by Das et al. [49] who observed that the use of *M. oleifera* leaves extract did not influence the cooking loss of goa<sup>t</sup> meat patties.

On the contrary, Hazra et al. [50] found that the inclusion of *M. oleifera* leaves extract showed a significant reduction in cooking loss of cooked ground bu ffalo meat. In this study, the expressible water content of nuggets ranged between 21.71% and 27.14% (Table 3). Although chicken nuggets with MF extract had lower expressible water, indicating higher water-holding capacity (WHC), there was no significant e ffect (*p* > 0.05) compared to control group. This result agrees with those reported by Vural et al. [57] who observed that the use of sugar beet fibre increased the water-holding capacity of frankfurter without any significant changes on sensory properties. In addition, Chang and Carpenter [58] also reported that more water was retained in frankfurters with an increase in oat bran level. Our findings clearly indicate that fibre can be used in cooked meat products to increase the WHC.

Addition of MF significantly (*p* < 0.001) increased the total dietary fibre (TDF) and total phenolics content (TPC) in chicken nuggets (Table 3). The TDF content was the highest in nuggets with 2% MF extract (2.03%) followed by T1 group (1.39%), while the lowest values were found for control samples (0.76%). This outcome agrees with the data reported by Verna et al. [6] who noticed that the incorporation of guava powder as ADF significantly improved the TDF content of meat nuggets. Similarly, TPC content was significantly higher in treated nuggets (0.789 and 1.121 mg GAE/g for T1 and T2 groups, respectively) than control samples (0.059 mg GAE/g). Such a high dietary fibre and TPC level in treated chicken nuggets might be due to use of MF extract, which had very high phenolic content (36.14 mg/g dry powder) and good source of dietary fibre (36.14%). This result agrees with the data observed by Das et al. [49] who showed that TPC of cooked goa<sup>t</sup> meat patties with *M. oleifera* leaves extract was significantly (*p* < 0.05) higher compared to control group.

In addition, Das et al. [18] also found significantly increased (*p* < 0.05) TPC by incorporating bael pulp residue as ADF in meat products. Moreover, Das et al. [34] reported that sheep meat nuggets incorporated with 1% and 1.5% litchi fruit pericarp extract had significantly higher TPC than control nugget.

The incorporation of MF extract did not influence (*p* > 0.05) the textural parameters (hardness, cohesiveness, gumminess and chewiness) of the product, although slightly lower hardness values were found in T2 group, indicating softer chicken nuggets compared to control (Table 3). A similar trend was found by Verma et al. [6] who observed that hardness, adhesiveness, cohesiveness, gumminess and chewiness values were not significantly a ffected (*p* > 0.05) by the addition of guava powder. Similarly, Choi et al. [54] studied the addition of rice bran fibre on the textural properties of heat-induced gel, and found that not only hardness, but springiness, cohesiveness, gumminess and chewiness were

also lower in samples with added rice bran fibre relative compared to control treatment. Moreover, Ham et al. [55] reported that textural properties, notably hardness, cohesiveness, and gumminess of cooked sausages were unaffected (*p* > 0.05) even when formulated with different levels of lotus rhizome powder. Other textural properties recorded in this study were nonsignificantly different (*p* > 0.05) between control and treatment chicken nuggets, though all the values decreased with increasing levels of MF. Wan Rosli et al. [39] while studying the textural properties of chicken patties formulated with different levels (0, 25% or 50%) of grey oyster mushroom, as a source of fibre and fat replacer, found lower cohesiveness, gumminess and chewiness values compared to control.

#### *3.3. E*ff*ect of MF on pH, TBARS Values and Total Plate Count of Chicken Nuggets during the Storage Time*

The pH values of the control and treated chicken nuggets, which were aerobically packaged and stored under refrigerated conditions, were evaluated at five-day intervals up to 20th day and are presented in Table 4. Statistical analysis displayed significant difference (*p* < 0.05) between storage days and treatments. As storage period progressed, a significant (*p* < 0.05) increase in pH values was observed in both the control and treated nuggets. The pH values increased from 6.30 ± 0.02 to 6.50 ± 0.01, from 6.27 ± 0.01 to 6.36 ± 0.01 and from 6.26 ± 0.01to 6.37 ± 0.01 from day 0 to day 20 for control, T1 and T2, respectively. The increase in pH during the storage period of meat product might be because of accumulation of metabolites due to the growth of Gram-negative bacteria like Pseudomonas, Moraxella, Acinetobacter, etc. [59]. Das et al. [60] also observed an increase in pH of ground and cooked meat added with curry leaf (*Murraya koenigii*) during the storage time. However, the increase in pH during the refrigerated period was significantly (*p* < 0.05) less in treated samples compared to control treatment, which might be due to the inhibitory effects of MF extract on oxidation of protein and lipid and some antimicrobial effects of the plant powder [61].


**Table 4.** Effect of Moringa flower on pH, thiobarbituric acid reactive substances (TBARS) and microbial count (log cfu/g) of chicken nuggets during the storage time (mean values ± error standard of six samples).

Treatments: Control = no additive; T1 = 1.0% Moringa flower extract and T2 = 2.0% Moringa flower extract. a–e Mean values in the same row not followed by a common letter differ significantly among storage times. x–y Mean values in the same column not followed by a common letter differ significantly among treatments. Sig. Significance; ns: not significant; \*\*\* *p* < 0.001.

The mean ± SE values of total plate count of aerobically packaged control and treated chicken nuggets (T1 and T2) during refrigerated storage up to 20 days is presented in Table 4. During storage, a significant increase (*p* < 0.05) in microbial count was observed in control and treated products (T1

and T2) at each interval of storage period except on 0 day, where the counts were comparable. Total plate count increased from 2.74 ± 0.06 to 6.46 ± 0.04 log10 cfu/g, from 2.64 ± 0.09 to 4.66 ± 0.10 log10 cfu/g and from 2.71 ± 0.04 to 4.51 ± 0.05 log10 cfu/g, from day 0 to day 20 for control, T1 and T2 groups, respectively. An increase of total plate count of chicken sausage during refrigerated storage was noticed by Sallam et al. [62]. However, at the end of storage time, the total plate count of treated chicken nuggets was significantly lower compared to the control group (6.46 vs. 4.66 and 4.51; *p* < 0.001 for control, T1 and T2 groups, respectively). This result might be due to its richness in polyphenolic compounds [45] exerting antimicrobial [61] effects.

It has been well documented by some researchers that polyphenols from MF extract have microbial activities against a number of pathogenic bacteria [61]. The effectiveness of MF extract in lowering the total plate count of chicken nuggets are in agreemen<sup>t</sup> with the previous findings reported by Das et al. [18] who observed that incorporation of bael pulp residue as ADF was very much effective (*p* < 0.05) in controlling the microbial counts in goa<sup>t</sup> meat nuggets throughout the 20 days of storage period.

The TBARS values of aerobically packaged chicken nuggets studied at regular intervals up to 20 days under refrigerated storage conditions is presented in Table 4. The TBARS values of chicken nuggets, irrespective of treatments, increased significantly (*p* < 0.05) from 0.37 ± 0.01 to 1.94 ± 0.05 mg MDA/kg, from 0.36 ± 0.01 to 0.84 ± 0.02 mg MDA/kg and from 0.36 ± 0.01 to 0.81 ± 0.01 mg MDA/kg, from day 0 to day 20 for control, T1 and T2 treatments, respectively. This increase in TBARS value throughout the storage period might be due to the lipid oxidation and production of volatile metabolites in presence of oxygen during aerobic storage [63–66]. Although TBARS value of all treated groups increased, but the rate of increase was comparatively slower in case of treated nuggets indicating more oxidative stability due to the presence ADF from MF extract. As observed on day 15, treated nuggets were within the spoilage limit, whereas during the same period, control nuggets (1.38) crossed the acceptable limit of 1 mg MDA/kg. The polyphenolic compounds present in the MF extract may be the reason for the strong antioxidant ability as reported by different researchers [22,47].

While comparing the treated groups, it was found that chicken nuggets in T2 group retarded the oxidation process more efficiently by maintaining TBARS values below the unacceptable range during 20 days storage. A similar result was found by Das et al. [50], who showed that TBARS values of cooked goa<sup>t</sup> meat patties with *M. oleifera* leaves extract was 47% less than control group. Sáyago-Ayerdi et al. [67] also reported similar findings using grape antioxidant dietary fibre in chicken hamburgers. In addition, this outcome agrees with those reported by other authors [9,10,68–71] who observed that the addition of natural antioxidant decrease the TBARS values of meat products.

#### *3.4. E*ff*ect of MF on Instrumental Colour Stability during the Storage Time*

The incorporation of MF extract significantly changed the colour value of treated chicken nuggets compared to control during storage time (Table 5). A significant increase in the lightness values, in treated chicken nugge<sup>t</sup> (T1 and T2 groups) was observed on 0 day in comparison to control. However, the reduction in lightness value of treated chicken nuggets was noticed after 10 days of storage period which may be attributed to the dilution of meat pigment in other meat products due to the presence of non-meat ingredients.


**Table 5.** Effect of Moringa flower on instrumental colour of chicken nuggets during the storage time (mean values ± error standard of six samples). Control = no additive; T1 = 1.0% Moringa flower extract; T2 = 2.0% Moringa flower extract.

a–e Mean values in the same row not followed by a common letter differ significantly among storage times. x–y Mean values in the same column not followed by a common letter differ significantly among treatments. Sig. Significance; ns: not significant; \*\*\* *p* < 0.001.

#### *3.5. E*ff*ect of MF on Sensory Attributes of Chicken Nuggets during the Storage Time*

Although both the control and treated chicken nuggets were comparable for all the above sensory attributes up to the 5th day of storage, the control nuggets received lower sensory scores thereafter (Table 6). This finding agrees with data reported by Das et al. [49] who showed that the addition of *M. oleifera* leaves extract had no effect on the sensory attributes. In addition, Muthukumar et al. [72] noticed that the of *M. oleifera* leaves extract did not modify colour, odour, flavour or texture, and all the products were equally acceptable as evidenced by the overall acceptability scores. The sensory attributes for treated nuggets (T1 and T2 groups), even though decreased with increasing storage time, were acceptable up to the 15th day of storage. On the other hand, the sensory scores for appearance of control, T1 and T2 treatments decreased from 6.77 ± 0.15 to 5.62 ± 0.19, from 7.02 ± 0.010 to 6.44 ± 0.10 and from 6.72 ± 0.15 to 6.38 ± 0.10, respectively. There was a significant decrease (*p* < 0.05) in appearance attribute of treated nuggets from day 15 onwards; whereas, in control samples, the values were even lower on 10th day of storage time.

This fact may be due to protective effect of MF extract preventing the fading of colour of the chicken nuggets. Control group received significantly lower flavour score on day 10 and development of rancid odour was noticed on 15th day of storage time, which might be the influencing factor to reduce the flavour and acceptability scores and was, therefore, rejected by the panellists. On the other hand, MF extract containing ADF which might have acted as stabilising agen<sup>t</sup> for retaining the flavour by inhibiting lipid oxidation in treated chicken nuggets. There was hardly any remarkable variation, and the texture of nuggets with MF was comparable to control (*p* > 0.05) up to 10th day. Sensory texture was not done in case of control from 15th day onwards, as rancid odour was detected. Likewise, a significant decrease (*p* < 0.05) in juiciness score was observed in both control and treated (T1 and T2) products from 10th day onwards. However, chicken nuggets containing MF extract were found to be more juicer than the control group, which could be attributed to the increased moisture retention of the product during cooking.


**Table 6.** Effect of Moringa flower on sensory attributes of chicken nuggets during the storage time (mean values ± error standard of six samples). Control = no additive; T1 = 1.0% Moringa flower extract; T2 = 2.0% Moringa flower extract.

ND = Not determined. a–e Mean values in the same row not followed by a common letter differ significantly among storage times. x–y Mean values in the same column not followed by a common letter differ significantly among treatments. Sig. Significance; ns: not significant; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.

As far as overall acceptability is concerned, the products also followed the same pattern that was observed for other sensory attributes. The control sample received a lower overall acceptability score, although nonsignificant (*p* < 0.05), on day 5 than treated samples, and was found to have rancid odour on day 15. On the other hand, overall acceptability scores obtained from T1 and T2 groups remained stable and were acceptable even on 15th day of storage. This could be due to incorporation of MF extract which might have extended the shelf-life. It is well documented by many researchers that meat products incorporated with natural antioxidants have higher flavour and overall acceptability scores during storage owing to the colour and flavour stabilising effect of them by inhibiting lipid and protein oxidation [73–79].
