**1. Introduction**

Mushrooms, due to their good nutritional attributes and richness in high-quality proteins, dietary fibers, vitamins, minerals, and phenolic compounds, are considered to be a healthy food product [1,2]. Enoki mushrooms (*Flammulina velutipes*), popularly known in di fferent countries as "golden needle", "winter", "lily" or "velvet stem" mushrooms, are widely recognized for their good nutritional value and desirable taste attributes [3,4]. Several compounds including carbohydrates, protein, lipids, glycoproteins, phenols, and sesquiterpenes have been isolated from di fferent parts of this mushroom [5]. Enoki mushrooms have also been known to exhibit good antioxidant, anti-inflammatory, immunomodulatory, anti-cancer and cholesterol-lowering activities [2,6]. The cultivated variety of this

mushroom has a pure white bean sprout, a velvety stem, and a tiny snowy-white cap, whereas the wild variety may be orange to brown with a larger, shiny cap [7]. The stem base and other parts of the mushroom are removed during harvesting and these leftovers either go to landfills or are used as compost [8].

There is a growing interest in the application of plant-based waste materials as functional food ingredients in meat products, as they are a rich source of dietary fiber and several other bioactive compounds like vitamins, minerals, and polyphenols [9–12]. These dietary fibers, in combination with phenolic compounds, form antioxidant dietary fibers (ADFs) [13,14] which can be used as dietary supplements to improve gastrointestinal health, or as technical ingredients to inhibit lipid oxidation in foods, thereby extending their shelf-life [15,16]. As far as enoki mushrooms are concerned, its extract is reported to have strong antioxidant potential, with a high 2, 2-dipheny-1-picrylhydrazyl (DPPH) radical scavenging activity and metal chelating ability. Being a rich source of dietary fiber, the extract may reduce triacylglycerol, total cholesterol, and low-density lipoprotein levels in the blood due to a variety of mechanisms [4].

In summary, mushroom powder extracts have numerous nutritional attributes such as low calorie-density, healthy lipid profile, high fiber, protein and phenolic contents that make them suitable for incorporation as functional food ingredients in a variety of food products [17]. Previously, powdered mushroom extracts have been used to fortify a variety of foods, including biscuits, cookies, crackers, and cakes [18]. There have also been a limited number of studies on the incorporation of various kinds of mushroom extract into meat products. For instance, oyster mushrooms (*Pleurotus ostreatus*) have been investigated as a substitute for pork meat in Thai glutinous fermented sausage [19]. Reports on the impact of dried portobello mushroom (*Agaricus bisporus*) on the quality characteristics of a dry spicy sausage, sucuk [20], texture and structure of meat emulsions [1] and physicochemical and sensory properties of cold-stored beef patties [21], e ffects of oyster mushroom (*Pleurotus sajor-caju*) on the color, texture, cooking characteristics, and fiber content of chicken patties [22] are also available. Besides, enoki MSW has been used as a potential substitute for antibiotics in organic egg production by chickens [8] and to enhance the growth and health status of broiler chickens [3]. To the authors' knowledge, no study has previously been carried out on the utilization of enoki MSW as a functional food additive in processed meat products. The current study was designed to analyze the dietary fiber content and antioxidant properties of enoki MSW powder, and then to evaluate its potential as a functional ingredient in goa<sup>t</sup> meat nuggets.

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

#### *2.1. Materials and Reagents*

Mushroom stem waste was collected from the harvesting and processing area of an enoki mushroom facility, cleaned properly for extraneous dirt, if any, and then dried in an oven (Static Oven, Instrumentation India, Kolkata) at 50 ◦C for 8 h. The dried MSW sample was then ground using a grinder (Kenstar, Mumbai, India) into powder (0.01 mm), which was then used as a functional ingredient in the goa<sup>t</sup> meat product formulation. The proximate composition and antioxidant activity of this powder was also quantified. Goat meat (leg part) was procured from a supermarket and then kept in a freezer (−18 ◦C) until further processing. Chemical reagents such as methanol, sodium carbonate, 2-thiobarbituric acid (TBA), trichloroacetic acid (TCA), α-amylase, protease, amyloglucosidase, Folin-Ciocalteu (F-C) reagents, and gallic acid were procured from Sigma-Aldrich (Mumbai, India). Other chemicals and reagents were of analytical grade (SRL, Mumbai, India).

#### *2.2. Chemical Composition and Extract Preparation of Mushroom Stem Waste*

For chemical composition such as moisture, protein, fat, and ash content, duplicate MSW samples were analyzed based on the methods of the Association of O fficial Analytical Chemists [23]. The enzymatic-gravimetric process was used for dietary fiber estimation [24]. Briefly, di fferent enzymes

such as heat-stable α-amylase, protease and amyloglucosidase were used for sequential enzymatic digestion of MSW samples after its proper dispersion in phosphate bu ffer solution. Then, after filtration of the insoluble dietary fiber (IDF), warm distilled water was used to wash the residue. Ethanol (95%) was used for the precipitation of soluble dietary fiber (SDF) from the combined solution of filtrate and washings. The weight was noted after proper drying of residue in an oven (Static Oven, Instrumentation India, Kolkata) at 50 ◦C. These IDF and SDF residues later were also analyzed for protein and ash content. Both IDF and SDF fractions were combined for total dietary fiber (TDF) calculation.

For the preparation of MSW extract, water was used as a solvent [9]. Briefly, 20 g of MSW powder was accurately weighed into a conical flask. To this, 1000 mL of solvent was added and the whole content was held at room temperature (29 ± 1 ◦C) for 10 h, stirring frequently with a glass rod. The mixture was shaken at a constant rate (500 rpm) using a shaker, vortexed at high speed for 10 min, and finally centrifuged (REMI NEYA 8, Kolkata, India) at 5000× rpm for 10 min. The content of extract was then passed through a 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 aqueous extracts obtained from repeated extractions were analyzed for total phenolic content (TPC), DPPH radical scavenging activity, ferrous ion chelating ability and reducing power assays. The e fficacy of the extract was determined based on the dry weight of the mushroom powder.

#### *2.3. Antioxidant Activity of Mushroom Stem Waste*

#### 2.3.1. Total Phenolics Content

The total phenolics content (TPC) of MSW was estimated using the Folin-Ciocalteu method. Briefly, 0.1 mL aqueous extract was properly mixed with 0.75 μL of F-C reagen<sup>t</sup> and then a final volume of the above mix was increased ten-times using deionized water. Then, a sodium carbonate solution (750 μL) was added in each test tubes after 5 min and these tubs were incubated (in the dark) for 90 min at room temperature. The absorbance of test samples at 725 nm was taken using a spectrophotometer (Thermo Scientific, Wilmington, NC, USA) against a blank. Di fferent concentrations of gallic acid were used for preparation of a standard curve and the TPC was calculated as gallic acid equivalents (GAE) in mg/g dry weight basis of MSW.

#### 2.3.2. DPPH Radical Scavenging Activity

The method developed by Shimada et al. [25] was used for the measurement of DPPH radical scavenging activity. Briefly, 4 mL of methanol was added to a 1 mL extract from the MSW powder in a test tube and then 1 mL of 0.2 μM DPPH methanol solution was added and mixed. Samples were then incubated for 30 min and later the absorbance at 517 nm was measured using a spectrophotometer (Thermo Scientific, Wilmington, NC, USA). The scavenging activity was calculated by the following formula:

$$\text{Scavenging activity}(\%) = \left\lfloor 1 - \left(\frac{\text{absorbase of extract}}{\text{absorance of control}}\right) \times 100\% \right\rfloor \tag{1}$$

#### 2.3.3. Ferrous Ion Chelating Ability

The ferrous ion chelating ability of the MSW extract was measured using the procedure outlined by Dinis et al. [26]. Briefly, 3.7 mL methanol and 0.1 mL of 2 μM FeCl2, MSW extract (0.1 mL) were mixed properly and then held for 30 s before adding 0.1 mL of 5 mM ferrozine solution. The mixture was then kept for 10 min at room temperature for incubation purposes. Finally, the absorbance using a spectrophotometer (Thermo Scientific, Wilmington, NC, USA) was recorded at 562 nm. The formula for calculating the ferrous ion chelating ability is as follows:

$$\text{Checking ability}(\%) = \left[ 1 - \left( \frac{\text{absorance of extract}}{\text{absorance of control}} \right) \times 100\% \right] \tag{2}$$

#### 2.3.4. Ferric Reducing Antioxidant Power

The ferric reducing antioxidant power (FRAP) of the MSW extract was determined based on the method described by Madane et al. [9]. Briefly, 2.5 mL of extract taken in a 10 mL test tube was added with 2.5 mL of phosphate bu ffer (0.2 M, pH 6.6) and 2.5 mL of 1% ( *w*/*v*) potassium ferricyanide. Then, 2.5 mL of 10% TCA was added after the mixture was kept for incubation at 50 ◦C for 20 min. After that, 2.5 mL of deionized water and 0.5 mL of ferric chloride (0.1% *w*/*v*) were mixed with 2.5 mL of the supernatant. Then, absorbance of samples was taken at 700 nm using a spectrophotometer (Thermo Scientific, Wilmington, NC, USA) and expressed as a percentage.

#### *2.4. Preparation of Goat Meat Nuggets*

Four formulations (control and treatments-T2, T4 and T6) of goa<sup>t</sup> meat nuggets were prepared following the standard method described by Das et al. [27]. The first batch was considered as a control (meat without MSW powder), whereas in the case of T2, T4 and T6 formulations, MSW powder at 2.0%, 4.0% and 6.0% was included, respectively, replacing an equal percent of goa<sup>t</sup> meat. Therefore, the total weight was 100 g with salt, condiments, spice mix, oil and wheat flour (Table 1).


**Table 1.** Formulation of goa<sup>t</sup> meat nuggets with different levels of mushroom stem waste (MSW) powder.

Treatments: Control = no additive; T2 = 2.0% MSW powder, T4 = 4.0% MSW powder and T6 = 6.0% MSW powder. \* Condiments: fresh garlic and onion (4:1). \*\* Dry spice mix (18 g/kg nuggets): aniseed, black pepper, capsicum, caraway seed, cardamom, cinnamon, cloves, coriander powder, cumin seed, turmeric and dried ginger (Cookme, Kolkata, India).

Before processing, the frozen goa<sup>t</sup> meat was thawed, cut into small cubes, and then minced using a meat mincer (Stadler, Mumbai, India). Meat emulsion was prepared separately for each group (control, T2, T4 and T6) by thoroughly mixing goa<sup>t</sup> meat cubes with other ingredients (salt, sugar, phosphate, and nitrite) in a bowl chopper. During chopping, ice flakes were added to prevent excessive heating. Condiments, dry spice mix, and fine wheat flour were then added and chopped continuously until all the ingredients were uniformly mixed. About 500 g of emulsion from each formulation was placed in a mold, and cooked for 40 min with a steam-cooker (Stadler, Mumbai, India). The cooked meat blocks obtained were then sliced and cut into small pieces. Nugget samples of di fferent formulations were then analyzed for various parameters (physicochemical, textural, colour) and also aerobically packed into low density polyethylene pouches and stored for up to nine days at 4 ± 1 ◦C for lipid oxidation study.

#### *2.5. Analysis of Meat Products*

#### 2.5.1. pH, Emulsion Stability and Cooking Yield

The pH of the meat emulsion and nugge<sup>t</sup> samples were measured after blending a 10 g sample with 50 mL of deionized water for a minute in a homogenizer (Omni International, Kennesaw GA, USA) and then using a digital pH meter. For emulsion stability, 25 g of emulsion was placed in a polypropylene bag and heated in a thermostatically controlled water bath for 20 min at 80 ◦C. The cooked mass was then cooled and weighed after draining out the exudate. For cooking yield, the weight of each meat block before and after cooking was recorded. The cooking yield was calculated and expressed as a percentage.

$$\text{Cooling yield } (\%) = \frac{\text{Weight of cooled meat block}}{\text{Weight of raw meat block}} \times 100\tag{3}$$
