2.1 Research material

Two commercial strains of oyster mushroom that is white tree oyster (V₁P₁) and Phoenix oyster (V₂P₃) were cultured on potato dextrose agar (PDA) (PanReac AppliChem, Spain) media at Medicinal and Mushroom lab, Institute of Horticultural Sciences, University of Agriculture, Faisalabad.

2.2 Spawn preparation

The spawn was prepared by mixing boiled wheat grains with animal waste manure 2% CaCO₃ and 2% CaSO₄ as described by (Khan et al., 2019). Firstly, wheat grains were boiled until softness and then mixed with other constituents. The substrate was autoclaved at 121 °C for 20 min followed by overnight cooling and inoculation with mycelium on PDA. At the 17th day of inoculation, the spawn was shaken for even distribution of wheat grains and re-incubated for eight days until grains were fully impregnated with mycelium.

2.3 Preparation of substrate and bag filling

Cotton waste was used as a substrate. It was soaked in water and pH was maintained by adding two percent lime. After that substrate was wrapped with polythene and placed in open for 5 days to allow fermentation. Excessive water from substrate, evaporated by spread on floor. The substrate was amalgamated with different level of humic acid solutions of 0 (Control), 2, 4, 6, 8, 10 mM/L designated as T₁, T₂, T₃, T₄, T₅, and T₆ respectively. The trial was conducted in triplicated CRD design. Each replication had 12 bags (each bag has 800 g of substrate), one for each combination of variety and treatment (Rodriguez Estrada and Royse, 2007).

2.4 Agronomic features

In the presence of light, the bags were incubated at 24 ± 2 °C and 80% R.H. for mycelia growth. After the completion of colonization, bags were shifted in cropping room at the temperature of 18 ± 2 °C with 80–90% R.H. in order to increase the fructification. Different agronomic features were measured such as (i) time to mycelium growth initiation, (ii) time to completion of 1st, 2nd and 3rd flushes, (iii) time to pinhead formation, (iv) total yield and (v) BE.

2.5 Determination of sugar contents

Reducing, non-reducing and total sugars along with total soluble solids (Brix) were estimated in mushroom extract as described by (Hortwitz, 1960).

2.6 Nitrogen, phosphorus, potassium, and ascorbic acid contents

Fruiting bodies of oyster mushrooms were oven dried at 60 °C for 48 h and grinded to pass through 1 mm sieve. One gram of grinded sample was digested in HNO₃ (0.6 mol/L). Finally, this prepared solution was used for the determination potassium by flame photometer, phosphorus by spectrophotometer and nitrogen by kjeldhal’s method (Mapya, 1998). Ascorbic acid was measured quantitively with titration method using 2, 6- dichloro-indo-phenol dye Tillmans reagent (Tillman’s method).

2.7 Estimation of minerals contents

One gram of fruiting body powder was burnt for 15 h at 560 °C in a muffle furnace (Atila et al., 2017). After that, ash was digested with HNO₃ (0.6 mol/L). Mn, Cu, Zn and Fe measured by atomic absorption spectrometer (230ATS), while Ca, Mg and Na measured by flame photometer (Mapya, 1998).

2.8 Estimation of proximate

Protein, fat, ash and total carbohydrates were estimated as described by AOAC (1995). The crude fiber was measured with the procedure that was recommended by Ranganna (1986).

2.9 Molecular structure

Structural changes at molecular level in both strains of oyster mushroom were studied through Fourier-transform infrared spectroscopy (FTIR) (Agilent 680, Department of Chemistry, University of Engineering and Technology Lahore, Faisalabad, Pakistan). 1 mg of grounded sample was mixed in 100 mg KBr and compressed to form tablets. Spectra were recorded from 650 to 4000 per cm (Muhammad et al., 2019).

2.10 Statistical analysis

Experiment was conducted in completely randomized design (CRD). Data was subjected to analysis of variance (one way). The data were analyzed with the software of Statistix 8.1. Significance of differences among treatments means were tested using LSD test α = 0.05 (Steel and Torrie, 1980).

3.1 Time to spawn initiation, 100% mycelium growth and pinhead formation

Analysis of variance revealed no significant differences in time to spawn initiation among both varieties of oyster mushrooms subjected to various levels of humic acid. However significant differences were observed in time taken for full mycelium growth and pinhead formation in response to different levels of humic acid. Out of six humic acid levels, least time for complete mycelium growth was given by T₆ (39.1 days) whereas max days (48.7, 48.0 days) were observed in T₁ and T₂ for white oyster mushroom. Same trend was observed for mycelial growth in Phoenix oyster mushroom i.e maximum days for mycelial growth was observed in T₁ (52.5 days) and minimum days were observed in T₆ (39.1 days). Moreover, raw cotton substrate complemented with humic acid exhibited fast mycelium growth. It is depicted that different levels of humic acid and strains of oyster mushroom significantly affected the time to pinhead formation. Minimum time (53.0 days) was shown by T₆ and maximum time (65.0 days) in T₁ by variety white oyster. However, in phoenix oyster, treatments T₆ (57.0 days) and T₁ (67.0 days) significantly affected the days taken to pinhead formation as compare with others (Table1).

3.2 Time to maturity of flushes and yield (g)

The different concentrations of humic acid exhibited significant effects on days taken to harvest ready flushes in both strains of oyster mushroom. Less time was taken by treatment T₆ and maximum was taken by T₁. In both strains of oyster mushroom treatment T₆, T₅, and T₃ showed min days for the maturity of 1st and 2nd flushes as compared to control T₁. Moreover, minimum time taken for the completion of 3rd flush by the treatments T₆ and T₄ than control T₁. The yield of mushroom obtained from cotton waste supplemented with humic acid was significantly higher in treatments T₆, T₅, and T₃ in both varieties as compared to control T₁ (Table 1).

3.3 Reducing, non-reducing and total sugars and total soluble solids (TSS)

Humic acid significantly increased total soluble solids (⁰Brix). Among different treatments of HA, max total soluble solids were noticed in treatment T₆, following T₅ and minimum TSS was recorded in T₁. The highest value of TSS was regarded for T₆ as compared to T₁ in both varieties. Maximum TSS was observed in white oyster as compared to phoenix oyster. Cotton waste enriched with HA showed the significant increase in the total sugar contents. HA significantly boosted the reducing and non-reducing sugars. Maximum reducing and non-reducing sugars were seen in treatment T₆ and min was observed in T₁ in both varieties (Table 2).

3.4 Ascorbic acid and K, P, N contents of mushroom (mg/100 g)

Humic acid significantly increased the ascorbic acid contents of fruiting body. Highest concentration of ascorbic acid was recorded in T₆ and T₅ in white oyster and phoenix oyster. Concentration of K, P and N was higher in T₆. The highest amount of potassium (266.6 mg/100 g), phosphorus (122.3 mg/100 g) and nitrogen (84.3 mg/100 g) were noticed treatment T₆ in white oyster mushroom. The highest amount of potassium (48.2 mg/100 g), phosphorus (62.3 mg/100 g) and nitrogen (78.6 mg/100 g) were noticed in phoenix oyster cultivated on cotton waste substrate supplemented with 10 mM humic acid (Table 3).

3.5 Biological efficiency (%)

Biological efficiency (BE) is the ratio of fresh edible weight of mushroom to dry substrate weight expressed in %. Humic acid significantly increased the biological efficiency in two strains of oyster mushroom. Highest biological efficiency was observed in T₆ and T₅ followed by T₁. Highest BE was recorded in phoenix oyster as compared to white oyster mushroom (Table 1).

3.6 Proximate analysis (%)

Carbohydrate concentrations of fruiting body were examined between 46% and 72%, depending upon the diverse treatments of HA in substrate. The highest amount of crude protein noted in treatment T₆ as compared to control (Fig. 1) By the application of humic acid enriched with cotton waste low level of fat (2.66%) was noticed in T₆ in variety V₂P₃ and in case of variety (V₁P₁) 16% fats were observed (Fig. 1) Maximum ash content (10.8%) was noticed in T₆ in V₂P₃, while in variety (V₁P₁), T₆ and T₅ gave the highest ash content as compared to control. The carbohydrates, fibers, crude protein, fat, ash contents of mushroom were significantly altered by different concentrations of HA (Fig. 1).

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Fig. 1

Protein (%) (1); Ash content (%) (2); Fat (%) (3); Crude Fiber (%) (4); Carbohydrates (%) (5) of two strains of Oyster mushroom cultivated on different concentration of humic acid enriched with cotton waste. (T₁ = Control (100% cotton waste only), T₂ = 2 mM/L humic acid, T₃ = 4 mM/L humic acid, T₄ = 6 mM/L humic acid, T₅ = 8 mM/L humic acid, T₆ = 10 mM/L); V₁ (P₁) = White oyster mushroom; V₂ (P₃) = Phoenix oyster mushroom.

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3.7 Mineral contents in mushroom (mg/kg)

Cotton waste enriched with various level of HA significantly affected Zn, Cu, Mg, Mn, Na, Fe and Ca in fruiting body of mushroom (Table 4) Different concentrations of HA on cotton waste substrate increased the mineral contents of fruiting body in both varieties of oyster mushroom. The highest value for mineral contents was observed in T₅ and T_6, including Zn (28.0 mg/kg), Cu (74.0 mg/kg), Mg (203.4 mg/kg), Mn (28.1 mg/kg), Na (77.6 mg/kg) Fe (550.8 mg/kg) and Ca (362.0 mg/kg) (Table 4) on dry weight basis.

3.8 Fourier transform infrared (FTIR) spectroscopy

FTIR spectroscopy is vastly technique that gives the detailed information about organic compounds and functional group present in the mushrooms, that can be observed by wave number of bands. Different FTIR absorption peaks distinguished variation in nutritional contents of white and phoenix oyster mushroom in response to various levels of HA. Considerable variation was observed in absorption spectrum of both oyster mushroom varieties for important nutritional components i.e. carbohydrates, fatty acids, proteins, alkanes, alkynes and hydroxyl group. The FTIR spectral area of both verities of oyster mushroom demonstrated characteristics features in two regions. The first region range, between (4000 – 1800 cm⁻¹). Important spectral peaks in white oyster (V1P1) were 3302, 3289, 2920, 2918, 2853, 2151, 2119 cm-1, that may be allocated to O–H, CH₃, - CH₂ lipids, C≡C and C–H group. The second region, between (1500–750 cm⁻¹) is consisted of carbonyl group and C⚌C double bond. Differentiated bands were observed in spectrum of white oyster s(V1P1) as compared to control, 3289 and 3202 (O–H and C–H), 1617 (protein), 1146 and 1015 cm⁻¹ (C-O bond, β (1 → 3) glucan, cell wall, polysaccharide) (Table 5). Highest peaks value in spectra observed in (V₁P₁T₆ and V₂P₃T₆) (Table 5); Supplementary figures S2 (A-L).

4.1 Time to spawn initiation, 100% mycelium growth and pinhead formation

There was no significant effect of different level of HA on days to spawn growth initiation. According to Baysal et al. (2003) humic acid is the source of nitrogen, both high and low nitrogen contents reduce the growth of mycelia growth. The high C/N ratio in the substrate helps to increase the mycelium growth (Hoa et al., 2015). Mushroom growth mainly depends upon the different factors like nature of substrate, spawn rate, fertility of substrates, distribution of spawn and abiotic factors like humidity, light intensity, temperature, level of oxygen and carbon dioxide and moisture percentage during incubation period (Hassan et al., 2010). Fertile substrate enriched with macro and micro nutrients causes early initiation of pinhead and reduces the time to harvestable produce (Singh et al., 2011). Our results declared that HA treatment took less for pinheads’ formation as compared to control.

4.2 Time to maturity of flushes and yield (g)

The total yield obtained by first flush is higher than subsequent flushes. In the second and third flush, mushroom quality is lower as compared to the first flush. Increasing in the number of flushes decreases the yield acquired from substrate due to the less nutrient’s accessibility in the substrate (Rizki and Tamai, 2011). In tomato, humic acid is reported to enhance vegetative growth, fruit yield and quality (Kazemi, 2014). Our results correlate with Prakash et al. (2010) reveal that supplementation of substrate with HA increases the yield in white oyster mushroom.

4.3 Reducing, non-reducing and total sugars and total soluble solid

In present study, humic acid significantly increased the TSS of fruiting body (Kazemi, 2014). The fruit quality, sweetness and taste are bounded to sugar contents like glucose, sucrose and sorbitol in fruit. These factors determined the fruit quality and its market value. Moreover, sucrose plays important role in activation anti-oxidization system, regulation of osmotic pressure, cell membrane stabilization and other metabolic pathways (Nishizawa et al., 2008). Increasing humic acid concentration significantly increases the total, reducing and non-reducing sugars in cucumber plant (Unlu et al., 2011). We observed similar increasing trend in our study.

4.4 Nitrogen, phosphorus, potassium and ascorbic acid contents of mushroom

Different levels of HA notably affected mycelia growth rate spawn growth, pinhead’s and fruiting body formation, protein contents and flush yield (Elhami et al., 2008). These results correlate that nitrogen content of lettuce plant increase in response to HA application (Haghighi et al., 2010).

For the completion of biochemical reaction within cells of mushrooms phosphorus works as a co-factor (Khan et al., 2007). Quality of mushroom depends upon the phosphorus availability (Beyer and Muthersbaugh, 1996). The HA considered to increase the uptake of nutrients like P, Ca, and Mg making it more mobile and available to plant root system (Wang et al., 1997).

Potassium plays the important role in different mechanism such as growth, carbohydrates metabolism, ionic balance, enzyme activity and cap and gills discrimination (Griffin, 1996). Humic acid considered to increase the K uptake. The HA increases the N and K concentrations in the roots of tomatoes (Turkmen et al., 2004). Our results are in accordance with Kazemi (2014) who studied and found the increase in TSS and Vitamin C in tomato plant as a result of HA application.

4.5 Biological efficiency (%)

Bhattacharjya et al. (2014) reported that BE of mushroom increases if substrate is supplemented with different chemicals. Similar results were observed by Kirbag and Akyuz (2008) regarding an increase in BE of oyster mushroom at various biological structures of substrate along with supplementation of different chemicals.

4.6 Proximate analysis (%)

Karakurt et al. (2009) investigated that different applications of HA influenced total yield and carbohydrate contents of pepper. Organic fertilizer like humic acid increases the crude protein in pumpkin seed (Jariene et al., 2007). The HA is rich source of nitrogen contents which helps to decrease the fat contents in pumpkin seed. Our results correlate with Jariene et al. (2007) stating that supplementation of substrate with HA increases the crude fiber, decrease fat in pumpkin seed. Our results of ash content are similar to Prakash et al. (2010).

4.7 Mineral contents in mushroom (mg/kg)

Atiyeh et al. (2002) demonstrated that nutrints uptake in tomato plants is significantly improved through HA application. Lime soil treatment with HA enhances absorption of Zn, Cu and Mn in maize crop (Hakan et al., 2010). Nikbakht et al. (2008) revealed that applying the humic compound in cut gerbera flowers increases the uptake of Ca. The HA application improved absorption of minerals (N, P, K, Mg, Fe and Ca) in cucumber and gerbera plants (Behzad, 2014; Nikbakht et al., 2008). We also noted similar increasing trends in our study’s results.

4.8 FTIR characterization

The presence of several functional groups of different biochemical compounds was noticed by FTIR spectroscopy (Supplementary Figs. S2 (A-L). Oyster mushrooms contained high nutritional profile like as, proteins, carbohydrates, macro and microelements with less fat. Absorption bands and spectrum formed by FTIR could properly explained phytochemical analysis relied on functional groups (Muhammad et al., 2019; Ibrahim et al., 2019: Ghramh et al., 2019b). Alcohols, alkyne, and ketone were detected by FTIR spectroscopy (Ghramh et al., 2019a). Moreover, FTIR spectra are proved to be suitable means for estimation of tiny structures in interactions between metallic nanoparticles and biomolecules (Ghramh et al., 2019c). The importance of FTIR is capability of particular characterization of starch, sugars, fats, proteins, nucleic acid and other functional groups (O'Gorman et al., 2010). Some previous work described chemical compositions for various species of Amanita, king oyster, truffles and Agaricus bisporus by FTIR spectra (Zhao et al., 2006; Khan et al., 2019; Muhammad et al., 2019). The present study illustrated that various concentrations of HA highly influenced the nutritional quality of both strains of oyster mushroom.