Potential of Pleurotus ostreatus as a novel protein source in rice-millet-based gluten-free muffins

Abstract

The study used oyster mushroom powder (OMP) as a nutrient source to improve the nutritional value of gluten-free (GF) muffins. OMP was added at 5, 10, 15, and 20% in rice-pearl millet-based gluten-free formulation for preparing muffins. The addition of OMP significantly (p < 0.05) improved the nutritional value of muffins. The protein content of the control muffin was 7.12% which increased to 9.41% and 10.19%, with 10% and 15% addition of OMP, respectively. Further, adding mushroom powder at a 10% level or more almost doubled the amount of fibre content in the muffins. The addition of 10% and 15% OMP also increased the magnesium and potassium contents to 19.20% and 27.20%, which were 42.91% and 57.48% higher compared to the control muffin, respectively. Replacement of rice flour with OMP increased the hardness and chewiness of gluten-free muffins. At higher levels (15% and 20%), OMP had also significant changes in the colour attributes. Interestingly, these changes in textural and colour parameters as a result of OMP addition had no adverse effect on the sensory attributes of muffins. OMP-enriched muffins were in the highly acceptable range (>7.0), with the 15% OMP-enriched gluten-free muffins having the highest overall acceptability scores among the evaluated samples. Furthermore, the technology was demonstrated among gluten-intolerant persons and small-scale gluten-free product entrepreneurs. More than 90% of the respondents liked the OMP-enriched muffins compared to the control sample. Moreover, gluten-free processors also appreciated this simple food technology to enhance the nutritional value of existing gluten-free diets.

Keywords:

• Gluten-free muffins
• oyster mushroom powder
• pearl millet
• rice
• protein
• texture

1. Introduction

Celiac disease is a common disorder affecting nearly 1% of the world population. Celiac disease is a common disorder affecting nearly 1% of the world population wherein affected individuals exhibit immune reaction to eating gluten, the protein that is mainly present in wheat, rye, and barley. This immune response damages the lining of the small intestine leading to malabsorption of nutrients, and a range of symptoms including diarrhea, abdominal, fatigue, weight loss, bloating, anemia, and can lead to serious complications (N. Sharma et al., 2020).

A gluten-free diet (GFD) is the only option and treatment available for this disease (Ciacci et al., 2015) which occurs due to the consumption of gluten protein mainly present in wheat, rye, and barley (N. Sharma et al., 2020). Various formulations for gluten-free diets are available in the market; the mainly used cereals for making gluten-free diets are rice and maize in combinations with millets and pseudocereals. However, the nutritional creditability of a GFD has been questioned over and over again. Nowadays, consumers are aware of and demand healthy options in every segment of food processing. The protein quality is one of the main issues which we have to deal with GFD. Rice, maize, and millets contain an appreciable amount of protein; however, the biological value of plant protein except for a few exceptions are low compared to animal proteins.

Mushrooms are edible fungi and consumed as a vegetable which makes them an excellent option for supplementation in diets having low protein content. Mushroom proteins are considered excellent proteins owing to their complete essential amino acid profile (Li et al., 2017). Edible mushrooms have the potential to be used as a functional ingredient in a food product to improve protein quality and increase fibre and mineral content along with improved functional properties (González et al., 2020). In addition to protein content (18 to 35%), mushrooms are low in fat and calories, and high in minerals and dietary fibers with polysaccharides like β-glucans (Bach et al., 2017).

In India, button mushroom has a significant share (73%) in the commercial mushroom market; oyster mushroom production comes second (with 16% share), and the remaining 11% are accounted for by paddy straw and milky mushrooms (V. Sharma et al., 2017). Perishability of mushrooms resulted in post-harvest losses and limits their utilization; shelf-life of oyster mushroom is 1 to 3 days at ambient temperature therefore, food processing and preservation techniques are needed to extend their shelf-life (Xiao et al., 2011). For the long-term preservation of oyster mushrooms, convective drying is one of the economical methods (Apati et al., 2010). Previous studies reported the use of oyster mushroom powder as a functional ingredient in enhancing the nutritional and functional value the processed food; the addition of 6% OMP in bread significantly increased the protein content to control bread (Majeed et al., 2017); the addition of mushroom powder (5%) in noodles significantly increased their protein, fibre, calcium, and potassium compared to branded noodles available in the market (Parvin et al., 2020).

Hot air drying in a cabinet dryer is an economical method for the preservation of oyster mushrooms, and dried mushrooms could be converted into powder and used as a functional ingredient in food products.

Bakery foods such as bread, biscuit, and muffins are popular and have a reach all over the world. They owe their popularity to varied taste, longer shelf life, and low cost. Therefore, they could be used as an excellent carrier for macro and micronutrient fortification (Salehi, 2019). Baked products like muffins are traditionally prepared from wheat flour, which contains a good amount of mineral content (Marco & Rosell, 2008). On the other hand, rice and maize which are commonly used in GFD are poor in terms of protein quality and mineral content specially iron (Melini & Melini, 2019). Therefore, the objective of this study was to investigate the potential of oyster mushroom powder in rice-pear-millet-based gluten-free muffins on the nutritional, textural, colour, and sensory quality of gluten-free muffin.

2. Material and methods

2.1. Preparation of flours

Non-aromatic rice, pearl millet, and oyster mushroom were purchased from the local market of Bathinda in Punjab, India. Rice and pearl millet were soaked overnight, fan-dried, cleaned, and milled into flour using a laboratory stone grinder. Prepared flour was sieved through 50 mesh screen and stored in polyethene bags for later use. Other ingredients for making muffins (eggs, sugar, butter, baking powder, salt, and xanthan gum) were also purchased from the local market.

2.2. Preparation of oyster mushroom powder

Fresh oyster mushrooms were graded and washed with water and, damaged parts were discarded. Mushrooms were cut into smaller pieces (5 to 6 cm slices) and treated with 2% brine solution for 5–6 min, and then blanched at 80ºC for 2–3 min. Afterward, mushroom pieces were immersed in sodium bisulphate (0.5%) and citric acid (0.25%) solution for 15 min, and then placed in trays at the rate of 1.25 kg/sqm. Drying was carried out in a hot air cabinet drier at 45ºC for the first 5–6 h, and then at 50ºC until the completion of drying (moisture content 11%). Finally, dried mushrooms were ground into a fine powder using a cemotec mill and sifted through a 50-mesh sieve.

2.3. Proximate analysis of raw materials and muffins

Proximate analysis of rice flour (RF), pearl millet (PMF) flour, and oyster mushroom powder (OMP) for moisture, crude protein, crude fat, crude fibre, and ash followed the approved methods of the AACC (2000). Carbohydrates were determined as 100- % proximate composition. On the other hand, muffin samples were first dried in a hot air oven and then defatted using the soxhlet apparatus before doing the proximate analysis as described above.

2.4. Mineral analysis of raw material and muffins

For mineral analysis of flours and blends, 1 g of sample was taken and digested using 3:1 HNO₃ and HClO₄solution. After digestion, samples were diluted with 50 mL deionized water followed by filtration using Whatman number 1 filter paper. The minerals (Minerals namely iron, zinc, magnesium, potassium, and calcium were measured by inductively coupled plasmamass spectrometry (ICP-MS) (X-Series2; ThermoFisher Scientific), and mineral content was expressed in mgkg⁻¹ (B. Kaur et al., 2018).

2.5. Formulation and muffin preparation

A composite 70:30 rice flour-pearl millet blend for muffin making was used as the control in this study based on preliminary trial for sensory attributes and physical quality (colour, texture, and specific volume). Muffins were prepared with (5, 10, 15, and 20%, v/v) or without OMP in the RF-PMF flour blend. The levels of OMP supplementation were based on literature review and initial trials. To summarize, the flour blend formulations used in the study are described below:

Control: 70% RF 70% + 30% PMF

F1: 65% RF + 30% PMF+5% OMP

F2: 60% RF + 30% PMF+10% OMP

F3: 55% RF + 30% PMF+15% OMP

F4: 50% RF + 30% PMF+20% OMP.

For each 100-g flour blend formulation, the muffins were prepared along with butter (50 g), sugar (90 g), 4–5 eggs, salt (0.5 g), xanthan gum (0.6 g) and essence (2–3 drops). For muffin making, sugar and fat were creamed in the kitchen aid mixer at high speed for 4 min. Next, eggs and a few drops of essence were added to the creamed mixture and mixed again for 2 min. Then, the sieved composite flour containing baking powder and salt was added to the creamed mixture and mixed slowly to achieve the desired consistency of the batter. The prepared batter was poured into greased muffin trays in equal proportion (50 g in each mould) and baked at 180ºC for 25 min. The muffins were cooled and packed for characterization and further analysis.

2.6. Quality characteristics of muffins

The physical quality of muffins was measured in terms of bake loss, crust, and crumb moisture, muffin weight, volume, specific volume, colour, and texture. All the parameters were measured after the cooling of muffins (2–3 h after baking). Muffin weight was measured using a weighing balance, and volume was determined by rapeseed displacement as per the AACC method. Specific volume was calculated as a ratio of volume and weight (AACC, 2000). The moisture content of the muffin was determined by oven drying for 2 h at 100ºC. Bake loss was calculated using the formula of Heo et al. (2019):

$\mathrm{B}\mathrm{a}\mathrm{k}\mathrm{e}\mathrm{L}\mathrm{o}\mathrm{s}\mathrm{s}\left(\mathrm{g}/100\mathrm{g}\right)=\frac{\mathrm{B}\mathrm{a}\mathrm{t}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\left(\mathrm{g}\right)-\mathrm{M}\mathrm{u}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\left(\mathrm{g}\right)}{\mathrm{B}\mathrm{a}\mathrm{t}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\left(\mathrm{g}\right)}X100$

2.7. Colour characteristics of muffins

Muffins crumb and crust colour was measured using a Minolta CR-300 Chroma Meter (Konica Minolta, Osaka, Japan) in terms of “L*”, “a*”, “b*” colour scales (Nanke et al., 1999). A white and black standard was used to calibrate the instrument. The L* value indicates the lightness of the product; the positive a* value indicates the redness, and the negative a* shows greenness. A positive b* value is for yellowness, while a negative b* denotes the blueness of the tested product.

2.8. Textural profile of muffins

The texture profile of gluten-free muffins with and without OMP was evaluated using the instrument “Stable Micro System Texture Analyzer Mode” l (TA-H di England) using settings Test-TPA, Probe-75 mm Cylindrical, Pre-test speed-1 mm/s, Test speed-1 mm/s, Post-test speed-1 mm, Force- 10 kg. The muffin samples were subjected to a double compression test to observe the crumb’s hardness, springiness, cohesiveness, gumminess, and chewiness as indicators for the texture profile analysis.

2.9. Pasting property of flour blends

The pasting characteristics of gluten-free flour with and without OMP were evaluated using a Rapid Visco-Analyser starch master R & D pack V 3.0 (Newport Scientific Narrabeen, Australia) following the method of AACC (2000). For analyzing RVA profile of composite flour, a 3-g sample was taken and mixed with distilled water (25 mL). The temperature was initially kept at 50ºC for 1 min and increased thereafter to 95ºC for 4.45 min. The sample was kept at 95ºC for 2.5 min followed by cooling at 50°C for 4 min; at last sample was kept at 50ºC for 1.5 min. RVA profile of samples was calculated in terms of pasting temperature (ºC), and time (minutes); viscosities at different temperature and time frame as peak, trough, final, breakdown, and setback viscosity (cp).

2.10. Sensory evaluation of muffins

Control and OMP-enriched gluten-free muffins were assessed by 20 panelists selected randomly from the staff members of the Regional Research Station, Bathinda, India, ages ranging between 25 and 55 years, for appearance, colour, texture, aroma, and overall acceptability using the 9-point hedonic scale.

2.11. Dissemination of technology

The acceptability of technology was measured in terms of responses from the targeted processor and end-users (gluten intolerant person). For this, a training programme conducted on “value addition of cereal and pulses” for gluten-intolerant entrepreneurs and farmers engaged in food processing. Under this training, gluten-free muffins were prepared as optimized in this study, and after the training, the responses of the participants were recorded via a questionnaire. To avoid any bias, the response was taken anonymously from 31 participants: 60% of the participants were “gluten-intolerant”, 31% of the participants were food processor associated with gluten-free flour formulations along with cereal and pulse milling, and 9% of the participants had an immediate family member having gluten intolerance.

2.12. Statistical analysis

All data were analyzed using ANOVA at a p ≤ 0.05 significance level using SPSS 19.0 statistical software. The results were expressed as the mean ± S.D. of at least three replications.

3. Results and Discussion

3.1. Pasting profile of flours

Pasting properties are conducted to study the starch behaviour of flours; the addition of low starch material affects the pasting characteristics of flours and their ability to form a batter with desired consistency. The batter viscosity is an important factor affecting quality of muffins and cakes (Olawuyi & Lee, 2019). Results of pasting profile of control and OMP-supplemented gluten-free flour formulations indicated a significant (p < 0.05) change in the pasting profile of the rice-millet composite flour after the addition of OMP (Table 1). These results revealed that the addition of OMP significantly (p < 0.05) reduced the peak, trough, breakdown, final, and setback viscosities of the gluten-free flours. The differences in the starch and protein composition of flours could affect paste viscosity and properties (Batey & Curtin, 2000). Peak viscosity is defined as the ability of starch to swell before leading to loss of physical integrity (Ragaee & Abdel-Aal, 2006). Peak viscosities of gluten-free flour blends decreased from 2040 to 1560 cp. As the OMP level progressed in the blend and rice flour proportion decreased it led to decrease in carbohydrate content as starch is the main carbohydrate in rice flour; dilution of starch content decreased the peak viscosities of oyster mushroom enriched flours; higher the starch content, higher the peak viscosity (Kumar & Khatkar, 2017). Furthermore, high protein content of OMP also had a lowering effect on peak viscosity of mushroom enriched flour formulation than control. In a previous study conducted on pasting profile of composite flour of soy flour, maize starch, and sweet potato flour suggested a decrease in peak, breakdown, and setback viscosities as a result of interaction of soybean fat and protein with starch of potato flour and maize (Julianti et al., 2017). Earlier study involved high protein content flour observed a decrease in peak viscosities; supplementation of cowpea protein isolates in rice for making gluten-free muffins reduced flour paste (peak, breakdown and final) viscosities (Shevkaniet al., 2015). This explains the highest peak viscosity of control flour blend and lowest for flour formulation contained 20% OMP.

Table 1. Pasting properties of flour blends

Samples	*PV	*TV	*BV	*FV	*SV	Peak time (minutes)	Pasting temperature (ºC)
C	2040 ± 15.21^e	1705 ± 14.98^e	1339 ± 11.78^d	2486 ± 21.78^e	2320 ± 19.57^e	8.18 ± 0.18^a	68.93 ± 1.59^a
F1	1930 ± 22.78^d	1610 ± 16.89^d	1128 ± 14.58^c	2185 ± 19.78^d	2120 ± 16.89^d	8.23 ± 0.12^a	70.49 ± 1.78^a
F2	1810 ± 18.56^c	1515 ± 21.58^c	918 ± 9.78^a	1946 ± 12.89^c	1946 ± 21.67^c	8.49 ± 0.13^a	76.29 ± 2.34^b
F3	1685 ± 18.45^b	1420 ± 22.78^b	908 ± 11.67^a	1714 ± 11.78^a	1714 ± 17.49^b	8.87 ± 0.18^a	81.59 ± 1.38^c
F4	1560 ± 13.78^a	1330 ± 15.48^a	997 ± 9.67^b	1890 ± 12.48^b	1482 ± 11.59^a	9.21 ± 0.17^bc	80.38 ± 1.21^c

Data are expressed as means ± SD (n = 4). Values with the same letter in the same row are not significantly different at P < 0.05; *PV=Peak Viscosity, TV: Trough Viscosity, BV: Breakdown Viscosity, FV: Final Viscosity and SV: Setback Viscosity

Breakdown and final viscosities ranged from 297 and 339 cp to 2190 to 2486 cp, respectively. Addition of OMP also resulted in decreased breakdown and final viscosities of composite flours. Breakdown viscosity is directly proportional to the peak viscosity (Devi & Haripriya, 2014). Lower peak viscosities means lower breakdown viscosity which in turn is related to lower starch content of OMP-enriched flours. With the addition of mushroom powder, protein and fibre particles of OMP compete with starch particles for water and caused water shortage for starch and thus starch granules had low hydration capacity. High hydration capacity of starch granules increases their swelling, viscosity, and gelatinization ability (Cornejo-Ramírez et al., 2018). The final viscosity measures the retrogradation of starch particles during cooling after starch gelatinization phase (Julianti et al., 2017; Ortega-Ojeda et al., 2004). However, the lower breakdown and final viscosities indicate the higher gelling ability of the flour formulations during cooking and cooling and therefore, provide better stability to shear stress during stirring (Lee et al., 2012). Setback viscosity indicates the ability of starch gel to form a semi-solid paste. In our study, setback viscosities decrease from 2320 to 1482cp and the decrease was corresponding to the level of OMP. This might be due to decrease in re-association or retrogradation of starch granules during cooling which has been earlier reported on flours from Indian corn varieties (Sandhu et al., 2007).

In RVA profile, the peak time indicates the cooking time and the pasting temperature is the minimum temperature needed for gelatinization of starch-based food samples. At this temperature, first measurable increase in viscosity was observed and it occurs due to the water absorption and swelling of starch molecules (Goksen Oksen & Ekiz, 2019). Addition of OMP, increased the peak time from 8.18 to 9.21 min. Furthermore, an increase in pasting temperature was also observed as the amount of OMP increased in the flour formulations. The reason for increase in peak time and pasting temperature might be due increase in fibre and protein content of the formulation and they compete with starch molecules for available water. This competition delayed the starch gelatinization time and temperature and thus an increase in peak time and pasting temperature was observed for the composite flour enriched with OMP. In an earlier study, it was observed that fortification of chickpea flour at 10, 20 and 30% levels significantly increased the pasting temperature (62 to 66.5ºC) corresponding to the level of chickpea in the formulation (Mohammed et al., 2014). Thus, lesser water is available for starch gelatinization which led to an increased pasting temperature for OMP-enriched formulations. Therefore, in this study, an increase in peak time, pasting temperature, and decreased viscosities of OMP-enriched blends indicated starch dilution and competition between OMP protein and fibre with starch for available water. On the basis of RVA profile, minor changes were done (mentioned in material and methods) in muffins making to obtain the batter with desired consistency.

3.2. Proximate composition of raw material and gluten-free muffins

Compositional analysis of raw material suggested the potential of oyster mushroom powder as a functional ingredient to improve the protein, fibre, ash, and mineral content of rice flour and pearl millet composite flour. A significant variation was found in the proximate composition of RF, PMF and OMP (Table 2). Oyster mushroom powder had the highest protein (29.3%), fibre (10.41%), and ash content (10.91%) among the analyzed samples. The earlier study observed a high nutritional value of oyster mushrooms; crude protein (36.42%), crude fat (0.38%), crude fibre (22.41%), ash (8.81%), and carbohydrates (22.5%) on a dry weight basis (Siyame et al., 2021). Mushrooms are edible fungi, and their proteins are of excellent quality having all the essential amino acids (Kayode et al., 2015).

Table 2. Proximate composition of RF, PMF, OMP, and muffins

Parameter	RF	PMF	OMP	Muffins
				Control	F1	F2	F3	F4
Moisture (%)	10.21 ± 0.32^b	8.45 ± 0.22^a	8.29 ± 0.31^a	25.78 ± 1.36^a	26.09 ± 1.56^a	27.13 ± 1.78^b	27.31 ± 1.26^b	28.78 ± 1.55^c
Protein (%)	6.34 ± 0.62^a	12.56 ± 1.52^b	29.31 ± 1.67^c	7.12 ± 0.14^a	8.17 ± 0.1^b	9.41 ± 0.11^c	10.19 ± 0.36^d	10.34 ± 0.16^d
Fat (%)	1.24 ± 0.15^b	3.98 ± 0.11^c	0.25 ± 0.13^a	20.67 ± 1.12^a	21.03 ± 1.67^a	20.89 ± 1.09^a	20.78 ± 2.14^a	21.05 ± 1.48^a
Fibre (%)	1.09±.0.22^a	2.90 ± 0.11^b	10.41 ± 1.84^c	1.12 ± 0.02^a	1.48 ± 0.05^a	2.11 ± 0.04^b	2.45 ± 0.03^b	2.87 ± 0.05^bc
Ash (%)	1.81 ± 0.23^a	2.34 ± 0.32^a	10.91 ± 1.22^b	2.01 ± 0.01^a	2.14 ± 0.02^a	2.55 ± 0.03^b	2.89 ± 0.04^c	3.05. ± 0.03^c
CHO (%)	75 ± 2.23^c	69 ± 1.89^b	42.14 ± 1.34^a	42.17 ± 1.21^a	41.091.15^a	37.15 ± 1.37^b	35.19 ± 1.64^c	35.01 ± 2.25^c
Mineral content (mg/100 g)
Iron	1.21 ± 0.11^a	5.78 ± 0.13^b	11.74 ± 0.12^c	2.16 ± 0.11^a	2.56 ± 0.17^a	3.11 ± 0.16^b	4.09 ± 0.15^c	4.25 ± 14^c
Zinc	1.49 ± 0.11^a	37.56 ± 1.14^c	7.89 ± 0.34^b	9.21 ± 1.02^a	10.12 ± 1.23^b	11.27 ± 1.15^b	12.11 ± 1.18^c	12.52 ± 1.15^c
Magnesium	109.67 ± 2.89^a	231 ± 3.89^b	294 ± 2.78^c	125 ± 2.21^a	138 ± 1.23^b	149 ± 1.19^c	159.34 ± 2.16^cd	167.48 ± 2.55^e
Potassium	270 ± 4.54^a	320 ± 5.67^b	1267 ± 3.56^c	247 ± 1.57^a	315 ± 5.16^b	353 ± 3.28^c	3.89 ± 2.17^c	456 ± 3.17^d
Calcium	11.56 ± 1.78^a	40.23 ± 1.21^b	47.21 ± 1.45^c	30 ± 0.16^a	33.57 ± 0.15^a	36.13 ± 1.05^a	38.9 ± 0.15^ab	39 ± 0.34^c

Data are expressed as means ± SD (n = 4). Values with the same letter in the same row are not significantly different at P < 0.05. Raw material (Rice flour, Pearl millet flour, and Oyster mushroom powder) were statically compared; muffins samples were compared with each other.

Pearl millet is one of the widely grown millet. Its use in value-added products would further increase the area under its cultivation. However, pearl millet protein (12.56 %) and fibre (2.90%) were in better proportion than rice. Earlier studies reported protein content of non-aromatic rice varied between 6% and 7%, ash content 0.35–0.65%, and fibre content 0.60–0.65% (Verma & Srivastav, 2017). Earlier investigation on comparative analysis of pearl millet suggested PMF had 17.4% protein, 6.3% fat, 2.8% fibre and 2.2% ash content (Sawaya et al., 1984). In another study, it was observed that pearl millet had 11.21–12.43% moisture, 2.05–2.72% ash, 5.14–5.96% fat, 10.97–11.65% protein, and 2.07–2.63% crude fibre and 66.49–68.85% carbohydrates (Kulthe et al., 2016).

Rice flour showed a poor mineral profile compared to the other raw materials (Table 2). Earlier studies reported the low mineral profile of rice (Chiş et al., 2020; Verma & Srivastav, 2017). In our study, we observed that pearl millet contained 5.78 mg/100 g of iron, 37.56 mg/100 g of zinc, 231 mg/100 g of magnesium, and 320 mg/100 g of potassium. A previous study already reported that pearl millet was a rich source of minerals, ranging from 40.07 to 42.67 mg/100 g calcium, 255.67–327.82 mg/100 g phosphorus and 5.08–8.12 mg/100 g iron (Kulthe et al., 2016). Oyster mushroom powder contained 11.74 mg/100 g of iron. Zinc was found to be 7.89 mg/100 g, phosphorus content was 1267 mg/100 g, and calcium was found to be 47.21 mg/100 g on a dry weight basis. Variation was found among researchers regarding the mineral content of mushroom powder; it might be due to variation in the substrate and strain used for the production of mushrooms. However, all agreed upon mushroom being a rich source of iron, zinc, phosphorus, and potassium and low in sodium (Dávila et al., 2020; Mallikarjuna et al., 2013; Salami et al., 2017).

The proximate composition of muffins suggested a significant increase in the protein, fibre, and ash content of OMP-enriched muffins (Table 2). The protein content of the control muffin was 7.12% which increased to 9.41 and 10.19%, with 10 and 15% addition of OMP. The incorporation of OMP further improved the fibre and ash content of the muffins; the fibre content of muffins had 10% OMP was 2.11% which increased to 2.45% at a 15% level of OMP. The results showed that adding OMP almost doubled the fibre content of muffins at higher levels of OMP. Ash content is directly related to mineral content; as already discussed, the oyster mushroom powder was a rich source of minerals (Table 2); it significantly enhanced the ash content of mushroom-enriched muffins. Muffins enriched with 10% and 15% OMP had 26.86% and 43.78% higher ash content than the control muffins. The mineral profile of muffins revealed a significant increase in iron, zinc, magnesium, and potassium due to mushroom addition in the muffin formulation. The control muffin’s iron content (2.16 mg/100 g) was increased to 3.11 and 4.09 mg/100 g with the addition of 10 and 15% of OMP, respectively. Magnesium and potassium contents were significantly higher in OMP fortified muffins than in control muffins. With the addition of 10 and 15%, OMP magnesium and potassium contents were increased to 19.2 and 27.20%, 42.91 and 57.48% compared to the control muffin, respectively. In a study, maize flour was fortified with oyster mushroom powder at 30, 40 and 50%, significantly improving the protein, fibre, ash, and mineral content in porridges (Siyame et al., 2021). Oyster mushroom fortified noodles had higher protein, ash, and fibre content compared to control noodles (Wahyono et al., 2018); muffins prepared with dehulled de-skin groundnut meal and wheat flour had 9.77% protein, 1.64% minerals, 3.82 mg/100 g iron and 135 mg/100 g calcium content; significantly higher than the wheat muffin (Mridula et al., 2021).

3.3. Physical quality characteristics of gluten-free muffins

The evaluation of physical quality attributes of control and OMP fortified gluten-free muffins suggested significant variations among samples with the addition of OMP (Table 3). The control muffin sample had the lowest moisture content (25.23%). The addition of OMP significantly (P < 0.05) increased the moisture content ranging from 25.23 to 27.67% for the sample containing 10% OMP and further increased to 29.89% for muffins having 20% OMP. The increase in moisture content of muffins corresponded to the increase in OMP in the formulation. This is due to the high fibre content of mushroom powder (Table 2), which increased the muffin’s water-binding capacity (Tan et al., 2016) resulting in increased moisture content compared to the control sample. Bake loss was slightly increased with an increase in OMP; however, the increase was non-significant except for muffins with 20% OMP. Bake loss is essential from an economic point of view, and a higher loss in bake value results in more product weight (Marchetti et al., 2018). Change in muffin weight was non-significant, up to 15% of mushroom powder; further increases in OMP (20%) led to a significant weight increase (47.71 g) and bake loss (12.75%) for muffins. This increase in weight is related to water absorption and the water-holding capacity of the oyster mushroom powder, owing to its higher protein and fibre content. A significant (P < 0.05) decrease in the specific volume of OMP fortified muffins was observed. The decrease in the muffin volume was more as the level of OMP increased in muffins. The addition of fruits and vegetables is associated with decreased volume of cakes and muffins (Gómez & Martinez, 2018). The fortification of fibre-rich foods often decreases the volume of cakes and muffins (Heo et al., 2019). It was reported earlier that mushroom and carrot addition decreased muffin volume along with an increased weight of muffins with a subsequent increase in hardness of enriched muffins (Olawuyi & Lee, 2019).

Table 3. Physical quality attributes of control and OMP-supplemented gluten-free muffins

Samples	Crumb Moisture (%)	Crust Moisture (%)	Bake Loss (%)	Weight (g)	Volume (cc)	Specific volume (cc/g)
C	25.23 ± 0.51^a	9.34 ± 0.51^a	10.21 ± 0.34^b	45.21 ± 1.65^a	125.82 ± 1.35^b	2.78 ± 0.12^b
F1	27.67 ± 0.29^b	10.23 ± 0.51^a	10.99 ± 0.50^b	45.92 ± 2.17^a	125.48 ± 3.35^b	2.73 ± 0.04^b
F2	27.12 ± 0.34^b	11.14 ± 0.51^ab	11.22 ± 0.57^b	46.05 ± 2.12^a	126.81 ± 2.75^b	2.75 ± 0.05^b
F3	26.34 ± 0.11^a	11.89 ± 0.51^b	11.26 ± 0.43^b	46.16 ± 1.57^a	126.03 ± 2.89^b	2.72 ± 0.05^b
F4	28.89 ± 0.21^c	12.67 ± 0.51^c	12.75 ± 0.34^a	47.71 ± 2.01^b	123.34 ± 2.45^a	2.58 ± 0.06^a

Data are expressed as means ± SD (n = 4). Values with the same letter in the same row are not significantly different at P < 0.05.

Colour is a critical quality parameter that influences the consumer preference for the product. The crust and crumb colour characteristics L*, a*, b* of the control and OMP-fortified gluten-free muffins had significant (p < 0.05) variations (Table 4). Although the mushroom powder was off-white in colour (Figure 1), slight colour variation of control and supplemented muffins occurred as the mushroom powder was light in colour. In visual appearance, there was not any marked difference in the colour of control and mushroom-fortified muffins (Figure 2). However, the instrumental analysis of colour revealed a significant difference between control and muffins that contained a higher percentage of OMP (15 and 20%). The addition of OMP at 15 and 20% levels significantly reduced the product’s lightness from lower L* values of muffins containing 15 and 20% OMP in the formulation. Further, colour attributes were analyzed differently for crust and crumb of muffins and a significant (P < 0.05) variation was found in the L* a* and b* values of the crust and crumb of muffins. The crust colour attributes of mushroom supplemented muffins were darker than the crumb. An increase in darkness was depicted from significantly lower L*values of mushroom supplemented muffins compared to control muffins. The crust colour is mainly attributed to the Maillard reaction or caramelization that occurs at high temperatures (baking temperature 160–180ºC) between protein-sugar and sugar components of ingredients, respectively (Tamanna & Mahmood, 2015). However, in the case of crumb colour, lower core crumb temperature does not favour Maillard reactions as it does for crust colour (Gómez & Martinez, 2018; Marchetti et al., 2018). This explained the darker colour of the crust of muffins compared to the crumb.

Figure 1. Flours used for gluten free muffing making prepared from Non-aromatic rice and pearl millet which were soaked overnight, fan-dried, cleaned and milled using a stone grinder. Treated and cut oyster mushroom were dried in a hot air cabinet drier (45ºC for the first 5-6 h followed by 50ºC till 11% moisture content achieved) and grinded into a fine powder using a cemotec mill. All the flours/ powder have been sieved through 50mesh screen. A. Rice flour; B. Pearl millet flour; C. Oyster mushroom powder.

Figure 2. Visual appearance of crust (above) and crumb (below) of gluten free muffins prepared from different proportions of rice, millet and wheat flour. C: control RF (rice flour): PMF (pearl millet flour): OMP (oyster mushroom powder (70:30:0), Fl: RF: PMF: OMP (65:30:5), F2: RF: PMF: OMP (60:30:10), F3: RF: PMF: OMP (55:30:15), F4: RF: PMF: OMP (50:30:20)

Table 4. Color and textural attributes of control and OMP-supplemented gluten-free muffins

Samples	Color attributes						Textural attributes
	Crust			Crumb
	L*	a*	b*	L*	a*	b*	Hardness (N)	Cohesiveness	Springiness	Gumminess (N)	Chewiness (N)
C	52 ± 1.37^d	2.89 ± 0.21^a	32.89 ± 0.81^d	72 ± 1.29^e	1.78 ± 0.15^a	41.47 ± 1.35^d	19.57 ± 0.81^a	0.47 ± 0.019^a	0.58 ± 0.034^a	8.5 ± 0.06^c	3.42 ± 0.012^a
F1	48 ± 1.25^c	3.89 ± 0.11^a	31.67 ± 1.21^d	68 ± 1.15^d	2.69 ± 0.13^b	38.78 ± 1.55^c	21.56 ± 0.47^b	0.48 ± 0.29^a	0.65 ± 0.027^a	8.9 ± 0.05^c	3.81 ± 0.050^a
F2	45 ± 1.17^c	5.18 ± 0.18^c	34.67 ± 0.81^c	63 ± 1.25^c	4.98 ± 0.32^c	39.45 ± 1.37^c	22.67 ± 0.71^b	0.58 ± 0.32^a	0.61 ± 0.008^a	8.5 ± 0.03^c	3.98 ± 0.022^b
F3	41 ± 1.76^b	4.78 ± 0.25^c	29.56 ± 0.78^b	55 ± 1.28^b	4.89 ± 0.21^c	35.34 ± 1.23^b	23.89 ± 0.65^bc	0.59 ± 0.43^a	0.62 ± 0.009^a	7.4 ± 0.05^b	4.18 ± 0.015^b
F4	37 ± 1.55^a	6.21 ± 0.29^cd	25.78 ± 0.83^a	51 ± 1.35^a	4.78 ± 0.29^c	32.78 ± 1.15^a	24.02 ± 0.55^c	0.48 ± 0.23^a	0.62 ± 0.021^a	6.5 ± 0.05^a	4.62 ± 0.014^bc

Data are expressed as means ± SD (n = 6). Values with the same letter in the same row are not significantly different at P < 0.05.

3.4. Textural quality characteristics of gluten-free muffins

With increasing levels, OMP-enriched muffins had an increasing trend in hardness and chewiness compared to control muffins (Table 4). The control sample had the lowest chewiness value (3.42 N), and muffins with 20% OMP had the highest (4.62 N). Chewiness increased gradually as the amount of OMP increased in the muffin formulation. An increase in chewiness might result from an increase in fibrous material upon the incorporation of OMP. It was observed that hardness and chewiness had a positive correlation (0.92); an increase in hardness resulted in increased chewiness. As reported in Table 2, OMP had high protein and fibre content, which contributed to a more compact structure and less expansion during baking which led to an increase in hardness and chewiness of OMP supplemented muffins. In a previous study on muffins prepared from rice and legumes (cowpea and mungbean), an increase in hardness with an increase in the level of legumes in the muffin formulation was observed (Jeong & Chung, 2018); cakes prepared with rice and legumes had a higher hardness value than the control rice muffin (Gularte et al., 2012); muffins enriched with cocoa fibre had more chewiness than the control muffin (Martínez-Cervera et al., 2011)

The addition of OMP did not affect the gumminess of muffins up to 10% level; however, at 15 and 20% levels of OMP, a significant decrease is found in the gumminess value of muffins. This might be due to the high fibre content of OMP, which decreased the gumminess of the muffins. It was reported earlier that fibre disrupts the protein matrix and reduces cereals’ gelling strength, leading to a lower gumminess value proportional to the fibre content (Cui & Roberts, 2009).

Cohesiveness is the ability of the food to stick together, and it is a good quality characteristic of baked goods in terms of textural attributes. The addition of OMP did not affect the cohesiveness of muffins. It is a desirable textural quality as a more cohesive product is better tolerant to stresses during packaging and distribution in the food supply chain. The springiness of muffins showed a similar trend as cohesiveness, no change in springiness was desirable as it measures the recovery extent after the second compression, and it is directly related to the freshness of cakes and muffins (Grigelmo-Miguel et al., 1999) which was constant at up to 15% of OMP in the formulations. This might be the reason for the non-significant variation in the springiness of the muffins.

3.5. Sensory attributes of muffins

Acceptance in terms of sensory attributes is an essential quality in the development of novel food products. It depicts the consumer acceptance or rejection of the developed product. The addition of mushroom power had a significant (P < 0.05) effect on the sensory attributes of muffins (Figure 3). However, all muffins were in the sound, acceptable range (score above 7.0). In addition, the appearance and colour of OMP-enriched gluten-free muffins were more preferred by panelists (?) compared to those of control muffins.

Figure 3. Sensory profile of gluten free muffins enriched with different proportions of oyster mushroom powder, C-control: RF (rice flour): PMF (pearl millet flour): OMP (oyster mushroom powder) (70:30:0), Fl: RF: PMF: OMP (65:30:5), F2:RF:PMF: OMP (60:30:10), F3: RF: PMF: OMP (55:30:15), F4: RF: PMF: OMP (50:30:20).The evaluation has been done on 9-point hedonic scale based on for appearance, colour, texture, aroma, and overall acceptability

Moreover, adding mushroom powder improved the flavour of the muffins, depicted by higher aroma and flavour scores for 10 and 15% levels of OMP enriched muffins, among other samples. Mushroom-enriched muffins had a better mouthfeel than control muffins. Further increase (20%) of OMP level in the blend resulted in compromised flavour and aroma, depicted by low scores for these sensory parameters. Overall, the sensory study concluded that the addition of OMP up to 15% level had sensory scores comparable to the control or even better in some aspects. It showed that the addition of functional ingredients at an optimum level does not alter the sensory attributes of food; fibre-rich orange bagasse product addition had no effect on sensory scores of bakery products (Romero et al., 2011); green banana flour in gluten-free muffins improved the sensory attributes of muffins compared to the control sample (K. B. Kaur et al., 2018). A previous study reported that sensory scores of muffin incorporated with cowpea was comparable to the control muffin (Jeong & Chung, 2018). Gluten-free muffins that contained quality maize protein had a sensory score as good as control (8.03) muffins (Bala et al., 2019). Enrichment of mushroom and carrot pomace improved the sensory quality of muffins more than controlled rice muffins (Olawuyi & Lee, 2019).

3.6. Response of gluten-intolerant persons and small-scale gluten-free product processors toward the technology

Based on the response to the questionnaire from the gluten-intolerant persons and local processors involved in gluten-free products, it was observed that 95% of the respondents were not aware of the poor protein quality of commercially available gluten-free products (Table 5). More than 92% of the respondent liked the taste and flavour of oyster mushroom enriched muffins over control muffins. Moreover, entrepreneurs involved in gluten-free products strongly rated the technology for ease of adoption to develop a gluten-free product with an enhanced nutritional profile. This technology not only improved the protein quality of gluten-free muffins but would also be beneficial for reducing the post-harvest losses for oyster mushrooms. Most of the entrepreneurs trained are members of well-organized self-help groups (SHG); thus, based on the response received, this easy-to-adopt and cost-effective technique will be highly adopted and helpful for small-scale gluten-free processors.

Table 5. Response of gluten intolerant persons and local gluten-free product processors towards the OMP-enriched gluten-free muffins making technique

Attributes regarding muffin quality and technique	Responses
Knowledge of low nutritional quality of gluten free diets compared to wheat counterparts	Not aware (81%)	Aware (19%)	-
Knowledge of preserving oyster mushroom as a dried powder	Not Aware (70.6%)	Aware (30.4%)	-
Mushroom powder fortification as a protein source	Not aware (94.3%)	Aware (5.7%)
Ease of adoption	Strongly agree (86.8%)	Neither agree nor disagree (7.3%)	Disagree (5.9%)
Cost effective	Strongly agree (91.20%)	Neither agree nor disagree (7.6%)	Disagree (1.2%)
Organoleptic qualities*	Strongly agree (98.2%)	Neither agree nor disagree (1.8%)	-
Overall quality of the product	Strongly agree (89.8%)	Neither liked nor disliked (10.1%)	-
Willing to pay more for fortified gluten free diets with enhanced nutrition	Yes (94.10%)	May be or May be not (3.4%)	No (2.5%)
Overall rating of this technology for reducing post-harvest losses for mushroom and improving quality of gluten free formulation	Strongly agree (97.40%)	Neither agree nor Disagree (2.6%)	-

*Organoleptic qualities include colour, taste, and aroma

_#Based on information collected from 31 respondents who imparted training on “gluten free formulations”

4. Conclusion

It can be concluded from the present study that OMP is a highly nutritious functional ingredient especially rich in proteins and fibre. Mushroom incorporation resulted in composite flours with improved ash, fibre, and mineral content. Thus, when gluten-free muffins were prepared using RF-PMF flour blend supplemented with up to 15% OMP, the baked products registered increased crude protein, ash, and zinc contents. OMP also rendered the gluten-free muffins more delicious, as indicated by the higher acceptability of muffins enriched with up to 15% OMP. This study suggested that OMP can be used in the dietaries of the population to improve their nutritional status, and it can be used as an economical alternative for non-vegetarian food to acquire good quality proteins. This would lead to an increase in demand for oyster mushroom species, and hence their commercial cultivation by more farmers. This indirectly would also help in improving the farmers’ economic and nutritional standards.

Acknowledgments

The authors acknowledge the support from Punjab Agricultural University, Ludhiana for the support to conduct this research. The authors offer gratitude to Dr Satnam Singh, PAU, RRS, Faridkot for amending the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Notes on contributors

Gurpreet Kaur Dhillon

Gurpreet Kaur Dhillion (Ph.d in Post harvest technology) is currently serving as Food technologist at PAU, Regional Research station, Bathinda- India. Her main research interests are on post harvest management of fruits, and vegetables, value addition of cereals and production of low glycaemic index (GI) bakery products. Besides conducting research she is involved in imparting trainings on value addition of fruits and vegetables, pre and post harvest management practices to the framers, farm women and entrepreneurs

Amardeep Kour

Amardeep Kour (Ph.d in Fruit Science) is working as Horticulturist at PAU, Regional Research Station, Bathinda- India. She is working on high-value horticultural crops like avocado, dragon fruit, almond and strawberry

Bong M. Salazar

Bong M. Salazar works as an Assistant Professor at the Institute of Crop Science, College of Agriculture and Food Science, University of the Philippines Los Baños in the Philippines. His teaching and research activities focus on the production, nutrition, and ecophysiology of perennial crops like coffee, cacao, mango, and apple