*2.7. Statistical Analysis*

Measurements of all the parameters were performed in duplicate and this study was conducted thrice. The data collected from this study were analyzed by a SPSS software (version 20.0, Armonk, NY, USA). The normal distribution and variance homogeneity were previously assessed (Shapiro-Wilk). In the case of the lipid oxidation study, the data were analyzed using two-way ANOVA with treatments (control, T2, T4 and T6) and storage time (0, 3, 6, 9 days) as the main effects (4 × 4 factorial design). To find out the impact of MSW on various parameters, Duncan's multiple range tests were applied. The values are presented here as the mean and standard error, and significance differences were identified at the 95% confidence level (P < 0.05).

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

#### *3.1. Proximate Composition and Dietary Fiber Content of Mushroom Stem Waste*

The proximate composition and dietary fiber content of enoki MSW powder is presented in Table 2. The MSW powder had high moisture (12.9%), protein (13.5%) and ash (8.2%), but a relatively lower fat content (1.5%). Available reports indicate that dried mushrooms contain relatively high moisture levels, depending on the species and other factors [31], more than 25% protein, less than 3% crude fat, and around 50% total carbohydrate [32]. Further, the ash content in mushrooms typically ranges between 5%–12% of dry matter. The composition of enoki MSW analyzed by other researchers [3,8,33] varied between 12.75% and 18.42% for crude protein, fat from 1.5% to 2.94% and ash between 6.33% and 11.6%, which is well within the level found in our study (Table 2). In this regard, variation in proximate composition could be due to differences in harvesting methods and stages of maturity of mushroom, soil types and environmental factors [34].

In this study, the SDF and IDF contents of the enoki MSW were found to be 17.3% and 15.1%, respectively (Table 2). Many researchers have reported the SDF and IDF content of mushrooms ranging between 22.4–31.2% and 4.2–9.2% of dry weight, respectively [35]. The TDF (32.3%) content determined in our study is fairly similar to that reported for raw enoki mushrooms (29.3%) [36]. In a comparison of different kinds of mushrooms, Yang, Lin, & Mau [37] found a higher dietary fiber content in enoki mushrooms than in shiitake or oyster mushrooms. The ability of enoki mushrooms to lower cholesterol

and blood pressure levels may be partly attributed to their relatively high dietary fiber content [4]. Moreover, these dietary fibers may help in the formulation of low-calorie, low-fat and high-fiber meat products, due to their ability to form gel networks that hold water and modulate texture.

**Table 2.** Proximate composition (mean values ± SE) and antioxidant activity of enoki mushroom stem waste powder/extract.


DPPH: 2, 2-diphenyl-1- picrylhydrazyl; FRAP: ferric reducing antioxidant power; GAE: gallic acid equivalents; *n* = 6.

#### *3.2. Antioxidant Activity of Mushroom Stem Waste Extract*

The total phenolics content of the enoki mushroom stem extract was 6.26 mg GAE/g dry weight, which was determined using gallic acid as a standard (Table 2). The antioxidant potential of this extract was assessed using a number of assays: the DPPH assay was used to measure the free radical scavenging ability [38]; the FRAP assay was used to measure the reducing power (Fe<sup>3</sup>+ to Fe2<sup>+</sup>); and, the iron-binding assay was used to measure the ability to chelate transition metal irons [39]. The enoki mushroom stem extract was found to have strong antioxidative potential: 84.2% DPPH scavenging; 60.1% reducing power; and 41.3% of ferrous ion chelating ability (Table 2).

In fact, phenolic compounds that are found naturally in mushrooms have antioxidant activity due to their hydrogen-donating and singlet oxygen-quenching properties [40,41]. The antioxidant properties of the enoki mushroom extract can be attributed to a number of antioxidant constituents, including p-coumaric acid, ellagic acid [42], gallic acid, pyrogallol, chlorogenic acid, caffeic acid, ferulic acid, and quercetin [40]. The phenolics content of mushrooms has been shown to be positively correlated with the results of the DPPH assay and other antioxidant assays [4]. Previous studies have shown that enoki mushrooms have higher phenolics contents, ferric reducing powers, and ferrous chelating activities than other mushrooms [43]. Taken together, these studies sugges<sup>t</sup> that enoki mushroom extracts are a good source of natural antioxidants.

#### *3.3. Physicochemical Properties and Proximate Composition of Fortified Goat Meat Nuggets*

The pH of the meat emulsion without MSW powder (control) was the lowest among all the treatments (Table 3). The addition of the mushroom powder significantly (*p* < 0.05) increased the pH. The meat emulsion containing 6.0% MSW had the highest pH value (6.44). Our results are in agreemen<sup>t</sup> with the findings of Bao, Ushio, & Ohshima [44], who earlier reported that the addition of enoki mushroom extracts to beef and fish slightly increased the pH of their products, although the increase was statistically non-significant. The increase in pH of products could be due to the abundance of basic amino acids in comparison to acidic amino acids with addition of enoki mushroom powder [45], as well as the natural buffering capacity of the mushroom proteins [46].


**Table 3.** Effect of enoki mushroom stem waste (MSW) on the physicochemical, textural and color attributes of goa<sup>t</sup> meat nuggets (*n* = 6).

Treatments: Control = no additive; T2 = 2.0% MSW powder, T4 = 4.0% MSW powder and T6 = 6.0% MSW powder. a–c Mean values in the same row bearing different superscript differ significantly (*p* < 0.05).

There was a significant (*p* < 0.05) increase in the emulsion stability of the treated meat nuggets, whereas cooking loss (%) reduced significantly with an increase in level of MSW powder incorporation, compared to control. On the other hand, the expressible water (%) decreased with increased powder level, indicating an improvement in water holding capacity, although this change was not statistically significant (*p* > 0.05). The improved emulsion stability and reduced cooking loss were probably because of the higher TDF (32.3%) content of enoki MSW which enhanced the oil absorption and water retention properties of the meat emulsion [47]. The improvement in water binding and fat retention in meat products, upon the addition of dietary fiber from several sources, have been reported by various researchers [16,47].

The TPC of meat nuggets increased significantly (*p* < 0.05) with the increasing level of MSW powder, rising from 0.14 to 0.96 mg GAE/g dry weight of product as the MSW content increased up to 6.0%. This is in agreemen<sup>t</sup> with the findings of different researchers who reported significantly increased TPC of meat nuggets upon addition of guava powder [16] and dragon fruit peel powder [9]. The increased phenolics content in treated meat nuggets could be due to addition of powdered enoki mushroom stem extract, which is reported to possess several bioactive phenolic and polyphenolic compounds [40,42,43].

Incorporation of MSW powder did not have a significant effect on the moisture, protein, and fat contents of the meat nuggets, but it led to a significant (*p* < 0.05) increase in the ash and dietary fiber contents (Table 3). These effects can be attributed to the presence of relatively high levels of minerals and dietary fibers in the mushrooms. It has been reported that this kind of mushroom is not only rich in potassium and phosphorus [43,48], but also contains several other minerals in minor amounts such as sulfur, sodium, copper, iron, and zinc [31]. In addition, the powdered enoki mushroom extract contained higher levels of TDF (32%), which would contribute to the fiber content of the final meat product.

#### *3.4. Textural and Color Attributes of Meat Nuggets*

The effects of incorporating powdered enoki MSW on the textural and color attributes of the meat nuggets is presented in Table 3. Although various textural parameters like hardness, springiness, cohesiveness, and gumminess of the nuggets decreased slightly, the reduction was not statistically significant. However, the chewiness of treated nuggets decreased significantly (*p* < 0.05) with an increased level of mushroom powder addition. Our results are in tandem with the findings of Choe et al. [49] who added enoki mushroom extracts to emulsion-type sausages and obtained similar results. Other researchers have shown that replacing chicken meat with 25% or 50% oyster mushroom (*P. sajor-caju*) reduced the hardness and improved the textural parameters of chicken patties [22]. The addition of portobello mushroom powder has been shown to increase the hardness and cohesiveness of model meat emulsions up to 3%, but then decrease their textural attributes at higher addition levels [1]. The textural properties of cooked meat products containing plant materials are related to the gelation of the myofibrillar proteins from the meat [50], as well as the biopolymer networks formed by the dietary fibers from the plants [47]. The addition of dietary fiber may have influenced the gelation of the meat proteins, thereby decreasing the gel strength and leading to a softer texture [14,15,47].

The color of any fresh or processed food product plays an important role in influencing the decision of consumers [51]. It is, therefore, imperative that any functional ingredient added to improve the nutritional properties of a meat product does not cause undesirable changes in its appearance. Increasing the amount of powdered enoki mushroom extract in nugge<sup>t</sup> formulations increased the lightness (L\*) and reduced redness (a\*), but did not change their yellowness (b\*) values (Table 3). No statistical differences (*p* > 0.05) in the lightness and redness of the meat nuggets containing 2.0% and 4.0% mushroom extract were observed compared to the control group. However, there was a significant increase in lightness and reduction in redness (*p* < 0.05) in nuggets samples prepared with 6.0% MSW. This might be a result of the dilution of the meat protein due to the addition of mushroom powder as a percentage of the meat and the white color of mushroom powder. Moreover, there may have occurred an increase in the degree of light scattering by the particles in the mushroom powder, which caused the meat product to become lighter. Similar results were reported by Choe et al. [49] in enoki mushroom powder added to emulsion-type sausages. In another study, it was reported that adding portobello mushroom extracts to a meat emulsion led to a decrease in L\* value and an increase in a\* value [1], which suggests that the effects may be system dependent.

#### *3.5. Sensory Characteristics of Meat Nuggets*

The sensory parameters of goa<sup>t</sup> meat nuggets containing different levels of powdered enoki mushroom extract are presented in Table 4. There was no significant difference (*p* > 0.05) in the individual sensory attributes of the meat nuggets regardless of the level of mushroom waste used. However, the appearance, flavor, and overall acceptability of nuggets decreased slightly with the addition of 6.0% MSW powder. Unlike other mushroom species, enoki mushroom is reported to have a very mild and delicate taste [52]. This is desirable and may be a beneficial attribute for many food applications, as it does not strongly alter or mask the expected sensory attributes of the original product. Few reports available in this regard sugges<sup>t</sup> that enoki mushrooms contain high levels of free amino acids, which are associated with umami or monosodium glutamate-like, sweet, and bitter tastes that are often perceived favorably by the consumers [37]. The white color of the mushrooms may also be beneficial because it does not alter the overall hue of the final meat product, but may slightly decrease its lightness.

In this regard, researchers have shown that supplementation of pork patties with the ground, white jelly mushrooms at 10%, 20%, and 30% by weight did not affect the liking of appearance, color, flavor, or texture [53], but the acceptability was better at a 10% level. In another study, Myrdal Miller et al. [54] reported that the addition of ground white button mushrooms to ground meat did not have a major impact on their perceived quality attributes. Taken together, these results sugges<sup>t</sup> that mushrooms can be used as a healthy substitute for meat products without adversely impacting their desirable appearance or flavor.

**Table 4.** Effects of enoki mushroom stem waste (MSW) powder on the sensory characteristics of goa<sup>t</sup> meat nuggets (*n* = 30).


Treatments: Control = no additive; T2 = 2.0% MSW powder, T4 = 4.0% MSW powder and T6 = 6.0% MSW powder.

#### *3.6. Lipid Oxidation of Meat Nuggets During Storage*

The impact of the powdered MSW on the oxidative stability of the meat nuggets was determined by measuring the primary (PV) and secondary (TBARS) lipid oxidation products over time and is presented in Figures 1 and 2. The treated meat nuggets (T2, T4, and T6) had significantly (*p* < 0.05) lower peroxide and TBARS values than the control group, although there was no significant difference (*p* > 0.05) between the levels of powder addition.

**Figure 1.** Effect of mushroom stem waste (MSW) powder on peroxide values of goa<sup>t</sup> meat nuggets during storage.

Hydroperoxides are primary products of lipid oxidation, hence PV are important to know the extent of initial lipid oxidation in meat samples. The results depicted in Figure 1 indicated that the initial PV (0.64 meqO2/kg) of control nuggets increased to 1.21 meqO2/kg after nine days of storage, which was significantly higher (*p* < 0.05) compared to treated nuggets. Similarly, results suggested that the control samples underwent noticeable lipid oxidation during the first six days of refrigerated storage and reached maximum PV at the end of the primary auto-oxidation. After six days of storage, the hydroperoxides formed might have gone through the decomposition to form secondary lipid oxidation products [55]. Although oxidation in the control was more intense compared to the treated samples, a decline was observed on day six. This indicates that, after the induction period, the decomposition rate of the hydroperoxides was faster than the production rate [56].

The control nugge<sup>t</sup> had an initial TBARS value of 0.32 and it reached 0.85 mg MDA/kg on the ninth day of storage study, whereas TBARS value in treated nuggets with 2–6% MW increased from 0.32–0.58 mg MDA/kg (Figure 2). There was an increase in TBARS values during storage irrespective of treatment but at a slower rate in treated nuggets compared to the control, indicating the e ffectiveness of MSW in inhibiting lipid oxidation during the storage. MSW besides supplementing the dietary fiber to goa<sup>t</sup> meat nuggets was found to retard lipid peroxidation in the product during refrigerated storage. Although the secondary reaction products showed an upward trend in all the samples as storage days progressed, a considerably slower rate was observed in the samples containing the mushroom extracts. These results sugges<sup>t</sup> that the enoki mushroom extracts were e ffective antioxidants that were able to retard lipid peroxidation in treated goa<sup>t</sup> meat nuggets during refrigerated storage study for up to nine days.

**Figure 2.** Effect of mushroom stem waste (MSW) powder on TBARS values of goa<sup>t</sup> meat nuggets during storage.

In general, mushrooms have been shown to contain a variety of di fferent antioxidant substances that make them e ffective at inhibiting lipid oxidation [4,7,31,57]. In an earlier section, we showed that the enoki mushroom extracts had strong reducing power, high scavenging activity, and good iron-binding ability (Table 2). These antioxidant e ffects may be attributed to the relatively high phenolic, dietary fiber, ergothioneine, vitamin C, and nucleotides content [7]. Antioxidant e ffects have also been reported when winter mushroom extract is added to beef and fish products [44] and emulsion-type sausages [49]. Enoki mushroom extracts have also been shown to prevent discoloration and lipid oxidation in fish and melanosis in crustaceans during postmortem storage [44,58].
