2.1 Experimental material

We used potato dextrose agar (PDA) (PanReac AppliChem, Spain) media to culture the two different strains of oyster mushroom such as, P₁ (V₁) P. ostreatus (white oyster) and P₃ (V₂) P. sajor-caju (phoenix oyster). Medicinal and Mushroom lab, Institute of Horticultural Sciences at University of Agriculture, Faisalabad was used to conduct the whole experiment. The material preserved at 4 °C for further use.

2.2 Preparation of spawn

The spawn preparation is completed by using animal waste, wheat grains mix with Calcium sulfate (2% CaSO₄) and Calcium carbonate (2% CaCO₃) in 500 mL flask. Initially, we boiled the wheat grains till it reaches in to soften stage after that mixed with rest of material. Then it left for autoclave at 121 °C for the time duration of 20 min, cooled it overnight and potato dextrose agar (PDA) was used for the inoculated material of fresh mycelium. At the 17th of inoculation to get the a fairly homogenous collection of colonized grains the spawn was perturbed well and re-incubated to check the mycelium growth immersed on the grains for 8 days.

2.3 Substrate preparation and polythene bag fill-up

Substrate (cotton waste) was permeated in water and maintained pH by adding 2% percent lime in it. The water-soaked substrate was conglomerated and wrapped with polythene for the time duration of 4–5 days allowing substrate to undergo fermentation. Then substrate was left to remove the excessive amount of water by evaporation on the floor. Cotton waste substrate was treated with various levels of thiourea at 2 mM, 4 mM, 6 mM, 8 mM and 10 mM per liter. The experiment was conducted in three replication with CRD design. Thiourea in different concentration was added into substrate and mixed it thoroughly and left the substrate for one day under sun drying. At the end, the polypropylene bags of size (15 × 25) cm were filled with the substrate, mouths were loosely tied with rubber bands keeping bag weight of 800 g (Estrada and Royse, 2007).

2.4 Growth parameters

The inoculated bags were kept under light at 24 ± 2 °C and 80% relative humidity for mycelium colonization. After colonization, bags were moved in cropping room at the temperature18 ± 2 °C and relative humidity 80–90% to enhance fructification. Different growth-related parameters i.e., days to initiation of spawn running, days to completion of mycelium growth (100%), days to completion of flushes (1st, 2nd and 3^rdflush) and days for pinhead formation, yield, biological efficiency (BE) were measured.

2.5 Estimation of sugar contents and TSS

The sugar contents (Total sugars, non-reducing sugar and reducing) and TSS calculated in mushroom extract following the method explained by (Hortiwitz, 1960).

2.6 Determination of macro nutrients and other minerals contents

Digestion of grinding mushroom samples was done by using methods described by (AOAC, 1990) for analysis of macro-nutrients and other minerals. Nitrogen (N) was estimated by using kjeldhal’s method while and Phosphorous (P) was determined by using vonadomolybdate spectrophotometric method described by (Afiukwa et al., 2013). Calcium (Ca) and Magnesium (Mg) were determined by EDTA complexion-metric titration method described by (Udoh and Ogunwale, 1986). Sodium (Na) and Potassium (K) were quantified by flame photometric method described in AOAC (1990), whereas manganese (Mn), lead (Pb) and zinc (Zn) were determined by Atomic Absorption Spectroscopy (AAS) (Afiukwa et al., 2013).

2.7 Ascorbic acid (mg/100 mL)

The concentration of ascorbic acid in mg/100 mL was calculated by analysis (Ruck, 1961). Whereas, it’s quantitative measurement was done by 2,6-dichloro-indo-phenol dye method (AOAC, 2002).

2.8 Estimation of proximate

The crude protein contents in mushroom were calculated by the macro-Kjeldahl method. Carbohydrates, proteins, fat and ash contents were measured by using AOAC procedures (AOAC, 1995).

2.9 FTIR-spectroscopy

The variations in the molecular study of two different oyster mushroom strains due to different application of thiourea were estimated by the grounded sample pellet with FTIR spectroscopy (Agilent 680, University of Engineering and Technology Lahore, Faisalabad campus, Pakistan). The samples of Oyster mushrooms were cleaned and kept under air for drying. Dried sample (1 mg) of fruiting body was milled with 100 mg KBr, pressed into capsule form and spectrum was noted in the range of 650–4000 cm⁻¹.

2.10 Statistical analysis

The data were analyzed statistically adopting ANOVA (analysis of variance) and comparing mean differences at level of significance 5% along with least significance difference (LSD) estimation (Steel and Torrie, 1980).

3.1 Initiation of spawn running, mycelium growth, pinhead formation (days)

In Table 1 analysis of variance elucidated the non-significant effects of thiourea treatments on both varieties regarding initiation of spawn running (days). The outcomes confirmed that varieties and thiourea treatments to great extent influence on completion of mycelium growth at 100%. Amongst various thiourea treatments, minimum (38 days) taken by treatment T₆ and maximum time (50 days) was measured in T₁ in variety V1 respectively. The same results were obtained for T₆ (39 days) and (46 days) in T₁ in variety (V₂). These consequences described that substrate enriched along thiourea motivated growth of mycelium as compared with treatment (T₁). Results demonstrated that varieties and thiourea treatments appreciably impact on days for initiation of pinhead formation. Between the different treatments of thiourea, minimum (65 days) expressed by T₆ and maximum time (73 days) in T₁ by variety V₁. Although in V₂, treatments T₆ (61 days) and T₁ (70 days) significantly involved the initiation of pinhead’s formation (days) contrast with other treatments.

3.2 Days for completion of flush and yield (g)

The significant effects of varieties and thiourea treatments on completion of flushes in days (Table 1) confirmed that different level of thiourea had considerably impact on accumulation of flushes (1st flush, 2nd flush and 3rd flush). Along with the various treatments of thiourea, minimum time was taken by treatment T₆ and maximum time was measured by T₁. The impact of thiourea on both varieties (V₁, V₂) demonstrates statistically different results. Mushroom yield attained from substrate (cotton waste) mixed with thiourea was considerably greater in treatments T₆, T₅ in both varieties as compared to control (Table 1).

3.3 Biological efficiency (%)

Biological efficiency is the percentage conversion of dry substrate to fresh fruit bodies. Thiourea significantly enhanced the biological efficiency of white oyster and phoenix oyster mushroom. Amid the different treatments of thiourea, maximum biological efficiency was measured in treatments (T₆ and T₅) in both varieties (Table 1).

3.4 Total soluble solids (Brix⁰)

Two different strains of oyster mushroom showed appreciable amount of TSS (Brix^o) cultivate on substrate of cotton waste mixed along different application of thiourea. Amid the treatments of thiourea, maximum TSS was recorded in treatment (T₆) and lowest TSS was noticed in (T₁). Greatest value of TSS was observed in V1 as compared V2. (Table 2).

3.5 N.P.K and ascorbic acid contents (mg/100 g)

Maximum nitrogen (84.3 mg/100 g), Phosphorus (122 mg/100 g) and potassium (164 mg/100 g) were measured in oyster mushroom cultivated on substrate augmented with thiourea at 10 mM per liter. Different concentrations of thiourea enhanced the quantity of Vitamin C in edible part of mushroom. Highest quantity of Vitamin C was noted among strains V₁, V₂ of oyster mushroom at the concentration of 10 mM, while lowest quantity of Vitamin C was obtained in control (Table 2).

3.6 Total sugars contents (%)

A positive significant trend in total sugars (reducing sugar and non-reducing sugar) in mushroom was observed at different treatments of thiourea enriched cotton waste. Treatment T₆ (10 mM/L) among all other treatments showed high percentage of all sugar contents in both strains (Table 2).

3.7 Estimation of macro and micro minerals contents in mushroom

Both macro and micro mineral (Na, Fe, Zn, Mg, Mn and Ca) contents were observed in increased concentration under different treatments of thiourea in both varieties. The largest amount of minerals contents were recorded in T₅ and T₆, for Na (10.4 mg/100 g), Fe (2.5 mg/100 g), Zn (2.4 mg/100 g), Mg (28.6 mg/100 g), Mn (0.50 mg/100 g) and Ca (50.3 mg/100 g) on dry weight basis (Table 3).

3.8 Estimation of proximate contents in mushroom

Total fat, crude protein, carbohydrates, crude fiber and ash contents were also significantly enhanced (Figs. 1–5). Maximum amount of crude protein was calculated in T₆ of variety V₂ as compared to T₁. Crude fiber ranges from 12% to 43% were estimated in fruit body of mushroom. Greatest value of carbohydrate was examined in treatment T₆ of variety V₂ as compared to T1 (control).

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Fig. 1

Crude protein (%) of two strains oyster mushroom cultivated on different concentration of TU enriched cotton waste. (T₁ = 100% cotton waste, T₂ = 2 mM/L TU, T₃ = 4 mM/L TU, T₄ = 6 mM/L TU, T₅ = 8 mM/L TU, T₆ = 10 mM/L TU); V1 (P1); V2 (P3).

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Fig. 2

Crude fiber (%) of two strains oyster mushroom cultivated on different concentration of TU enriched cotton waste. (T₁ = 100% cotton waste, T₂ = 2 mM/L TU, T₃ = 4 mM/L TU, T₄ = 6 mM/L TU, T₅ = 8 mM/L TU, T₆ = 10 mM/L TU); V1 (P1); V2 (P3).

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Fig. 3

Total fat (%) of two strains oyster mushroom cultivated on different concentration of TU enriched cotton waste. (T₁ = 100% cotton waste, T₂ = 2 mM/L TU, T₃ = 4 mM/L TU, T₄ = 6 mM/L TU, T₅ = 8 mM/L TU, T₆ = 10 mM/L TU); V1 (P1); V2 (P3).

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Fig. 4

Carbohydrates (%) of two strains oyster mushroom cultivated on different concentration of TU enriched cotton waste. (T₁ = 100% cotton waste, T₂ = 2 mM/L TU, T₃ = 4 mM/L TU, T₄ = 6 mM/L TU, T₅ = 8 mM/L TU, T₆ = 10 mM/L TU); V1 (P1); V2 (P3).

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Fig. 5

Ash contents of two strains oyster mushroom cultivated on different concentration of TU enriched cotton waste. (T₁ = 100% cotton waste, T₂ = 2 mM/L TU, T₃ = 4 mM/L TU, T₄ = 6 mM/L TU, T₅ = 8 mM/L TU, T₆ = 10 mM/L TU); V1 (P1); V2 (P3).

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3.9 Features of FTIR spectra

FTIR spectrum characterized revealed complete biochemical contents of two different strains of Oyster mushroom (4000–1800 cm⁻¹). Bands spectrum assessment in variety (V₁) placed particular 3360, 2911, 2922, 2929 and 3255 cm-1, that might be allocated to C–H and O–H group. Distinguished bands located in spectrum of Pleurotus ostreatus (V₁) as compared to T₁, 3255 and 3360(C–H and O–H), 1610 (Carbonyl group), 1140 and 1026, 1023, 1019, 1015, 1013, 1375(β (1 → 3) glucan, C–O bond, cell wall, protein, polysaccharide) (Table 4). In variety (V₁) the band spectrum showed the significant change as compared to Pleurotus sajor-caju (V₂). Largest changes in spectrum bands were noted in both varieties treatment (T₆) (Table 5).

4.1 Initiation of spawn running, mycelium growth, pinhead formation (days)

The Thiourea has no effects on initiation of spawn strains of both mushrooms under feasible environment. In different species of mushroom the mycelium growth mostly relies on different factors like as accessibility of nutrients in substrate, type of substrate, rate of spawn. Mycelium growth also depends on the growth conditions such as temperature, light during incubation period, humidity, carbon dioxide and oxygen (Hassan et al., 2010). The early formation of pinhead started due to highly rich substrate and presence of macro and micro elements in it.

4.2 Days for completion of flushes, yield (g)

The number of mushroom flesh yield decreased subsequently by increasing the number of flushes presentence of nutrients in the substrate in very low quantity (Rizki and Tamai, 2011). These were in accordance to the findings of Amin et al. (2013) revealed that maize yield enhanced by the application of thiourea at different levels.

4.3 Biological efficiency (%)

Bhattacharjya et al. (2014) declared that if different agricultural waste material is amalgamated with various chemicals, the biological efficiency of oyster mushroom increases.

4.4 N,P,K and Vitamin C contents of mushroom (mg/100 g)

Nitrogen plays important role in the cultivation and growth of mushroom. Nitrogen considerably influences the growth of mycelium, spawn run, formation of pinheads, edible fruit body formation, protein concentration and cultivation of mushroom (Elhami et al., 2008). Nitrogen plays role in the composition of amino acids (production of proteins), polysaccharide, pyrimidines, vitamins and purines (chitin helps to make cell wall of mushrooms (Miles and Chang, 2004). In case of maize plant thiourea application considerably increases nitrogen (N) in the leaves; it is helpful to improve the biomass production of maize plant (Anjum et al., 2011).

Phosphorus is a major component which helps and promote plant growth. It is also helpful in different processes such as production of nucleic acid, energy generation, respiration, glycolysis, membrane stability and synthesis, activation and inactivation of enzyme, carbohydrate metabolism (Vance et al., 2003). Phosphorus is responsible for good trait and better yield of mushrooms (Beyer and Muthersbaugh, 1996). The exogenous application of thiourea plays a major contribution in cellular ion homeostasis and then increases the intake of phosphorus (P). Our result correlates with Kaya et al. (2015) thiourea profitably increases the phosphorus concentration in maize plant.

Potassium plays the very important role in the different mechanisms that are involves in the development (biosynthesis) and discrimination of cap and gills, enzyme activity, upholding carbohydrate metabolism, ionic balance (Griffin, 1996). The exogenous application of thiourea plays important role in cellular ion homeostasis and then increases the uptake of potassium (K). Our result correlates with Kaya et al. (2015) thiourea beneficially increases the potassium concentration in maize plant.

Ascorbic acid plays important role in plants, it is present generally in plants as the excessively existing micro-molecule substance. Ascorbic acid (Vitamin C) plays the important role in different physiological processes like as co-factor of some enzymes, cell division, cell elongation, plant defense against oxidization, plant development, plant growth and senescence (Horemans et al., 2000). Thiourea increases the concentration of ascorbic acid, our result similar to Babu and Yadav (2002) by the foliar spray of thiourea at full bloom stage increases the ascorbic acid contents in peaches.

4.5 Total soluble solids (Brix⁰)

Total soluble solids are the solid contents which are present in the substance. And, sugar is one of them and others such as, vitamins and minerals etc. The different solid contents contain substances called as total soluble solids. Total soluble solids of apple and some other fruits conifers the most important quality indicators which is related to the composition and texture (Peck et al., 2006). Thiourea helpful to increases the TSS in mushroom. Foliar application of thiourea significantly increases the TSS (total soluble solids) in maize plant (Amin et al., 2013).

4.6 Total sugars, reducing sugar and non-reducing sugar in mushroom (%)

Total soluble sugars contain the sucrose, sorbitol and glucose. These total sugars present in fruit tissues that are responsible for the fruit sweetness, quality and taste of fruits. In addition, sucrose balances the osmotic pressure, maintains the cell membrane structure, triggers the anti-oxidization system to eradicate free radicals and adjusts metabolic pathways (Nishizawa et al., 2008). Our study findings elucidated that total sugar (%) increased by increasing the concentration of thiourea. Earlier study by Jana and Das (2014) showed increasing the concentration of thiourea total sugar (%) also increased in Asian pear. Current study shows that by the application of thiourea reducing sugars increases. Foliar application and seed treatment with thiourea boost the reducing sugars in mung bean (Mathur et al., 2006). Thiourea increases the non-reducing sugar (%). Application of thiourea after fruit setting increases the non-reducing sugar (%) in peaches (Meitei et al., 2013).

4.7 Estimation of macro and micro minerals contents in mushroom

By foliar application of thiourea the uptake of mineral nutrients such as Ca, Mg, Mn, Zn, Na and Fe were higher because thiourea contribute in cellular ionic movements. Foliar application of thiourea increases the Ca contents in the maize leaves. Exogenous application of thiourea increases the Na concentration due to increase in the biomass production of root and shoot (Kaya et al., 2013). Singh and Paliwal (2017) noticed that application of thiourea through foliar application and seed soaking @ 500 ppm increased the zinc content in okra fruit.

4.8 Estimation of proximate contents in mushroom

Mahmoud and Ahmed (2018) discussed that by application of thiourea enhance the carbohydrates, ash, protein, fat and fiber contents in wheat grains.

4.9 Features of Fourier transform infrared spectroscopy

Different compounds with various functional groups was detected with Fourier transform infrared spectroscopy. Mushroom contains abundant amount of carbohydrates, protein, micro and macro-elements with low fats contents. Various bands and spectrum, formed through FTIR might well-ornate compositional analysis based on structural groups of compounds. The consequence of FTIR was its capacity for more precise classification of, proteins, sugars, nucleic acid, starch, lipids and macrostructure of functional groups (O’Gorman et al., 2010). Earlier studies demarcated chemical classification for Agaricus bisporus, many kinds of Amanita and truffles using bands and structural groups through FTIR spectroscopy. Major purpose of current study was to validate whether several treatments of TU influence the nutritional contents, molecular structure of two different strains of mushrooms by FTIR spectroscopy method.