GROWTH, YIELD AND ANTIOXIDANT ACTIVITY OF GREY OYSTER MUSHROOM (PLEUROTUS PULMONARIUS) GROWN IN SAWDUST SUBSTRATE WITH THE SUPPLEMENTATION OF ALKALINE MATERIALS
M. P. M.F. Radzi1, M. Azizah2, T. Maininah3 and A. Sumaiyah1*
1Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia; 2Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia; 3University Agriculture Park, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
Corresponding author’s e-mail: sumaiyah@upm.edu.my
ABSTRACT
The study aims to evaluate the effect of different alkaline materials’ supplementations on grey oyster mushroom (Pleurotus pulmonarius) to assess mycelial growth, initiation of primordial, yield and antioxidant activity. The sawdust substrate for mushroom cultivation was subjected to three different treatments, including lime, zeolite and gypsum, each designed in a completely randomised block with 10 replications. The prescribing of these alkaline materials significantly influences mushroom cultivation. The combinations of gypsum showed the lowest number of days taken for mycelial growth (30.7 ± 4.12 days) and the emergence of primordia (7.7 ± 4.55 days). The mushroom fruiting body collected from each flush showed that zeolite treatment produced the most acceptable yield (62.36 ± 9.67 g) compared to the other treatments. A maximum inhibitory effect against 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals, expressed as IC50 value, was emanated by zeolite treatment from the third flush (63.48 ± 28.804 µg/ml). The total phenolic content (TPC) showed that zeolite exerted the highest phenolics in the fourth flush (98.96 ± 10.07 µg GAE/mg). The application of zeolite and gypsum as additives in mushroom breeding is highly recommended for the rapid growth, increase in yield and antioxidant properties of grey oyster mushroom.
Keywords:Pleurotus pulmonarius, Mycelial growth, Biological efficiency (%), 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, Total Phenolic Content (TPC)
https://doi.org/10.36899/JAPS.2021.6.0372
Published online March 31, 2021
INTRODUCTION
The mushroom industry in Malaysia is one of the trades that continuously contribute to the national economic growth; mushroom was recognised as a potential commodity that can generate higher incomes for the country. Out of 17 commercial varieties, oyster mushroom shows an upsurge in demand, receiving extensive market globally. Grey oyster mushroom, Pleurotus pulmonarius (Fr.) Quél (1827), also known as P. sajor-caju (Fr.) Singer (Li & Yao, 2005), belonging to Pleurotaceae, Agaricomycetes, Agaricales is among the most popular edible mushrooms that garner worldwide interest from culinary courses to scientific studies in mycology, biotechnology, nutrition, medicine, immunomodulatory and pharmacology. This highly nutritious food has been widely cultivated for commercial purposes and is an inexpensive source of protein, especially for the poor.
Pleurotus spp. are presently appreciated for their chemical constituents that possess many health benefits and have been considered as functional foods. The biological activities were mainly determined from their composition in a wide range of phenolic compounds, polysaccharides, amino acids, terpenes and sterols. In the past, P. pulmonarius were tested into different extracts: methanol-dichloromethane extract, water fraction, hot water, aqueous extract and hexane fraction. The tests successfully revealed potent antioxidant activity against Low-Density Lipoprotein (LDL) oxidation and Human Aortic Endothelial Cell (HAEC) damage from H2O2-induced oxidative stress due to the content of ergothioneine that possesses a protective effect (Abidin et al., 2016). Smiderle et al. (2008) also reported that the bioactive compound (1®3)(1®6)-linked β-glucan isolated from P. pulmonarius exhibited anti-inflammatory and analgesic properties on a rodent model. Out of the edible mushrooms, the oyster mushroom is recognised for its potentialities in antinociceptive, antitumour, antioxidant and immunological activities (Li et al., 2017).
To date, Pleurotus spp. have been cultivated on various types of substrates through biotechnological processes that acquire selective lignocellulosic material from any accessible source of agricultural wastes. Some examples of agricultural waste are cotton waste, rice straw, and wheat straw (Yang et al., 2013; Sardar et al., 2017), sawdust (Obodai et al.,, 2003), banana leaves (Reddy et al., 2003), sugarcane bagasse (Hasan et al., 2015), elephant grass, coat cross and coffee husks (Corrêa et al., 2016). Besides, the supplementation on mushroom planting bed is crucial for a high yield and excellent quality of mushroom. Added materials, including wheat bran, rice bran, cotton seed hulls and perilla stalks, have been reported to improve mushroom’s marketable qualities (Yang et al., 2013; Li et al., 2017). With several research revolving around the increase in the yield of mushroom production, the selectivity of different substrates with other supplementary materials warranted a major concern.
The mushroom industry finds the solution to enhancing mushroom at the expense of yield by cultivating mushroom on a substrate prepared by composting due to cut-off cost and relatively reducing the rate of infection of other competitors (Vieira & Andrade, 2016). For the time being, the technique of composting is well established only for particular species that have been popularly applied in producing Agaricus bisporus (Martos etal., 2017; Lishma & Das, 2017; Gea et al., 2012). Similar to most mushroom substrates, compost used to have supplementation as a casing layer covering at its surface. The addition of a casing layer is useful to address the environmental conditions that support optimum mushroom growth (Colauto et al., 2011). These materials should be non-toxic and have less nutritional value to avoid any negative consequences that might inhibit the initiation of mushroom growth. Amid their application, casing materials, as emphasised by several studies, exert a range of neutral to alkaline pH, calcium source, a potent cationic exchange capacity, low magnesium content and low toxic trace compound (Pardo et al., 2010). Among those additives favoured in alkaline pre-treatment are lime, zeolite, bentonite, alkaline bauxite (Li etal., 2012; Kim, Lee, & Kim, 2016; Awasthi etal., 2016), biochar (Czekała etal., 2016), gypsum (Febrisiantosa, Ravindran, & Choi, 2018), wood fly ash, lime, phosphor-gypsum, polyethylene glycol and jaggery (Gabhane etal., 2012).
Alkaline materials benefit most agricultural activities, but since the study of their application in a mushroom substrate is still limited, the effect of alkaline materials on mushroom has not been resolved yet and the distinguished functional role of its uses is still obscure. One study found that the application of different calcium sources of bentonite and gypsum increases the potential of early mushroom fructification (Zied et al., 2012). Therefore, the implementation of alkaline materials in a substrate medium are really important to be investigated for its outstanding effects on mushroom cultivation. The potential of using this agile method for various crops is as desirable as many had claimed, yet there is a lack of evidence of its benefits and values that apply in mushroom cultivation. Herein, the study aimed to evaluate the different alkaline materials, including agricultural lime, zeolite and gypsum, to produce a high yield and optimum antioxidant status of grey oyster mushroom.
MATERIALS AND METHODS
Mushroom strains and spawn preparation: Pleurotuspulmonarius’ pure culture was obtained from the University Agriculture Park, Universiti Putra Malaysia (UPM). The subculture of Pleurotuspulmonarius was prepared by inoculating (2 × 2 mm) mycelial agar on Potato Dextrose Agar (PDA) media and incubated at 25oC until a fully grown culture was obtained.
Wheat grain was used as a spawn substrate for the cultivation of Pleurotus pulmonarius. Wheat grains were washed and boiled in hot water for 30 min. The cooked grains were filled in a polypropylene bag and sterilised in an autoclave at 121oC for 1 hr. The sterilised grains were inoculated with one disc (7 mm diameter) of mycelial agar and incubated until full colonisation of spawn.
Substrate preparation: The medium substrates were analysed to compare the different treatments of alkaline materials (lime, zeolite and gypsum) as a supplement. The different substrates were prepared in the ratio of 100%: 10%: 1%, consisting of rubber sawdust, rice bran and alkaline materials, respectively. The water content of the mixed raw materials was adjusted to 75%. The mixtures were filled to 600 g in polypropylene bags and compressed densely. In each treatment, 10 replicates of polypropylene bags were used for different compositions of substrates. For all treatments, each bag was measured for pH level in order to compare the changes in the condition of the substrate (Table 1).The substrates were sterilised in a steamer at 121oC for 8 hr. The sterilised substrates were inoculated with Pleurotuspulmonarius at an amount of 5–8 g of grain spawn. The inoculated culture bags were then transferred to a mushroom house and incubated for 10–12 weeks before the harvesting stage. The mushroom house was maintained at 25–30°C and 60% relative humidity and kept in the dark until mycelia completely covered the bags. In the harvesting period, the environment was set to 25–30oC, 85–95% and 100–200 lux of light intensity. Mushroom growth and yield were observed in the early incubation period until mushroom was produced.
Table 1. The characteristic of pH level between different substrates.
Substrate Formula with Alkaline Treatment
|
pH Level
|
100% SD + 10% RB + 1% Lime
|
7.13ab
|
100% SD + 10% RB + 1% Zeolite
|
7.21a
|
100% SD + 10% RB + 1% Gypsum
|
7.05b
|
Note: SD, Sawdust; RB, Rice Bran.
The different connotations show a significant difference at P ≤ 0.05 using Tukey’s test.
Cultivation test of Pleurotuspulmonarius: To test the growth performance and yield of Pleurotus pulmonarius cultivated in different treatments, selective parameters were applied, such as spawn running, initiation of primordial, yield of the fruiting body and biological efficiency. Spawn run was measured based on the number of days taken to complete the mycelial growth of the cultured bags during incubation. Through the determination of primordia’s initiation, the cultured bags were observed for the time taken for primordial to form after the removal of the cap. The yield of mushroom was recorded based on the fresh weight of the fruiting body from the first flush to the fourth flush. The term ‘flush’ refers to the harvesting period, with the interval between flushes normally being 7–10 days. In this experiment, four flushes of fruiting bodies were harvested and recorded over the cultivation cycle. The weight of the fruiting body was converted into biological efficiency by calculating the ratio of the weight of fresh mushroom to the dry weight of substrates. All of the aforementioned parameters were evaluated in the experiment of 10 replicates for each different treatment in order to discriminate mushroom production performance. The grown fruiting bodies were freshly harvested from each bag and compared for their effectiveness according to the analysed parameters. The fresh mushrooms collected were subsequently screened for antioxidant activity.
Methanolic extract preparation of Pleurotuspulmonarius fruit body: Mushroom samples from different treatments were prepared for methanolic extracts in accordance with Jeena et al. (2014). The extracts were set into screening of five different concentrations (1, 0.5, 0.25, 0.125, 0.0625 mg/ml) for further analysis.
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity: The free radical scavenging capacity of Pleurotus pulmonarius was determined according to Brand-Williams et al. (1995) with several modifications. The percentage of DPPH radicals scavenged by the samples was calculated using Prieto’s DPPH microplate assay (Prieto, 2012). A solution of 0.06 mM of 2,2-diphenyl-1-picrylhydrazyl was prepared using methanol, sealed with aluminium foil and kept in the fridge. The tested samples (1-0.0625 mg/ml) were dissolved in 100 µl of methanol and mixed with 100 µl of DPPH solution in a 96-well microplate. The mixture was kept in complete darkness and incubated at room temperature for 30 min. The absorption was measured at 517 nm using a microplate reader (Multiskan FC, Thermo, Waltham, MA, USA). Each tested sample was analysed in four replicates. The standard antioxidant of ascorbic acid was used as a positive control. The radical scavenging activity was calculated as a percentage of DPPH discoloration using the following formula:
Inhibition of DPPH radical (%) = [(A0 − As)/A0] x 100
Where Acontrol = Absorbance of control (blank sample),
Asample = Absorbance of samples after 30 minutes
The half-inhibitory concentration (IC50) was calculated from the graph of radical scavenging activity percentage against the extract concentration.
Total phenolic content determination using Folin-Ciocalteu’s method: The total phenolic content of extracts was estimated following Folin-Ciocalteu’s method developed by Bobo-García et al. (2015) with several modifications. A solution of 20µl of sample mixed with 80µl of diluted Folin–Ciocalteu’s phenol reagent (1:4) in a 96-well microplate was shaken for 1 min and left for 2 min until dissolved. The mixture was added to 75 μl of sodium carbonate solution (100 g/l), shaken at medium-continuous speed for 1 min and kept in the dark for 2 hr of incubation. Then, the absorption was measured at 765 nm using the microplate reader (Multiskan FC, Thermo, Waltham, MA, USA). Gallic acid was used to prepare the standard curve (1-0.0625 mg/ml). Results were expressed as mg of gallic acid equivalents (GAE) per gram of the extract.
Statistical analysis: The data obtained were analysed using SAS statistical software. Each of the treatment was arranged into a completely randomised design (CRD) with post-hoc Tukey’s test. The data were expressed as means ± standard deviation.
RESULTS
Growth performance in spawn running and primordial initiation: The number of days for spawn running was noticeably significant among the Pleurotuspulmonarius cultivated with different treatments (Table 2). The treatment of gypsum showed the earliest spawn run period to complete the mycelial growth (30.7 ± 4.12 days), followed by lime (48.4 ± 2.8 days) and zeolite (50.5 ± 3.5 days). In primordial initiation, the treatment of gypsum was also the fastest (7.7 ± 4.55 days) compared to that of lime and zeolite. Statistically, the treatment of gypsum showed a high significance according to Tukey’s test, which was considerably different from the treatment of lime and zeolite with a similar level of significance.
Yield of mushroom from the first flush to the fourth flush and biological efficiency (%): The total yield was obtained from the fresh weight of mushroom collected from each bag, 10 replicates for all treatments. The result in Table 3 shows that all of these treatments were considerably varied in the yield of mushroom counts from every flush, as presented in Figure 1. The treatment of zeolite accumulated the biggest yield ranging from 18.88 ± 3.22 g to 62.36 ± 9.67 g, while the lowest was found in the treatment of gypsum (11.61 ± 5.82 g) at the fourth flush. The maximum yield of mushroom was observed in the first flush of the treatment of zeolite with 62.36 ± 9.67 g. The biological efficiency of mushroom indicated that the treatment of zeolite (26.05 ± 11.18 %) was the highest compared to that of lime (18.77 ± 8.34 %) and gypsum (17.95 ± 8.21 %). There was a high significance in the treatment of zeolite according to Tukey’s test as compared to the level of significance in the treatment of lime and gypsum. On average, the efficiency of yield from every flush was simplified as the highest was obtained in the treatment of zeolite (39.07 g), followed by lime (28.15 g) and gypsum (26.92 g).
DPPH scavenging activity: All the tested samples had outcome into significant reactions to DPPH radicals as summarised in Table 4. The treatment of gypsum showed the greatest inhibition of radicals in the first and second flushes with 187.38 ± 2.97 µg/ml and 207.84 ± 11.71 µg/ml, respectively, while the treatment of zeolite was the most reactive in the third and fourth flushes with 63.48 ± 28.804 µg/ml and 152.16 ± 17.504 µg/ml, respectively. The minimum IC50 value, which was the highest antioxidant activity, was documented in the treatment of zeolite (63.48 ± 28.804 µg/ml) from the third flush of grey oyster mushroom, from as low as 31.25 µg/ml to 500 µg/ml of concentration. Collectively, the third flush of zeolite treatment was depicted as the most effective among all the four flushes in scavenging the free radicals.
Total phenolic content (TPC): The total phenolic content was crucially comparable in tested samples among different treatments (Table 5 and Fig 2). The treatment of gypsum exerted the highest content of phenolics in the second flush with 188.44 ± 11.38 µg/ml, while the treatment of zeolite accumulated the most phenolics in the third and fourth flushes with 321.84 ± 21.79 µg/ml and 369.44 ± 101.31 µg/ml, respectively. However, all the tested samples showed no significant difference in the total phenolic content analysis in the first flush. The greatest phenolics composed in a fruiting body was found in the treatment of zeolite (369.44 ± 101.31 µg/ml) from the fourth flush. Therefore, the treatment of zeolite from the fourth flush was the most pronounced in phenolic content among the samples.
Table 2. Rate of growth of Pleurotus pulmonarius in spawn running and primordial initiation.
Treatment
|
Spawn-run (day)
|
Primordial initiation (day)
|
Lime
|
48.4 ± 2.80a
|
10.0 ± 2.67ab
|
Zeolite
|
50.5 ± 3.50a
|
12.7 ± 4.11a
|
Gypsum
|
30.7 ± 4.12b
|
7.7 ± 4.55b
|
Values are the mean ± SD (n=10).
The different letter(s) in the column indicate considerable differences among the means within each treatment at P ≤ 0.05 using Tukey’s test.
Table 3. Yield of fresh samples harvested in every flush (1st – 4th flush) and biological efficiency (%).
Treatment
|
Yield of mushroom (g)
|
BE (%)
|
1st flush
|
2nd flush
|
3rd flush
|
4th flush
|
Lime
|
43.64 ± 9.69b
|
28.15 ± 7.62b
|
26.2 ± 6.71b
|
14.62 ± 2.60ab
|
18.77 ± 8.34b
|
Zeolite
|
62.36 ± 9.67a
|
40.62 ± 5.14a
|
34.42 ± 2.51a
|
18.88 ± 3.22a
|
26.05 ± 11.18a
|
Gypsum
|
41.98 ± 6.65b
|
28.31 ± 7.05b
|
25.8 ± 3.65b
|
11.61 ± 5.82b
|
17.95 ± 8.21b
|
Values are the mean ± SD (n=10).
The different letter(s) in the column indicate considerable differences among the means within each treatment at P ≤ 0.05 using Tukey’s test.
Table 4. IC50 values of the methanol extracts from each treatment of Pleurotus pulmonarius in antioxidant activity.
Treatment
|
IC50 of DPPH radical by mushroom extracts (µg/ml)
|
1st flush
|
2nd flush
|
3rd flush
|
4th flush
|
Lime
|
333.0 ± 23.49a
|
302.42 ± 11.00b
|
332.65 ± 8.32a
|
201.27 ± 12.33a
|
Zeolite
|
309.62 ± 27.51a
|
393.45 ± 15.16a
|
63.48 ± 28.80c
|
152.16 ± 17.50b
|
Gypsum
|
187.38 ± 2.97b
|
207.84 ± 11.71c
|
283.36 ± 1.41b
|
219.32 ± 9.53a
|
Values are the mean ± SD (n=4).
The different letter(s) in the column indicate significant differences among the means within each treatment at P ≤ 0.05 using Tukey’s test.
Table 5. Phenolic content in three different treatments of mushroom samples.
Treatment
|
Total phenolic content of mushroom extract (µg GAE/mg)
|
1st flush
|
2nd flush
|
3rd flush
|
4th flush
|
Lime
|
209.51 ± 118.45a
|
163.4 ± 7.20b
|
202.03 ± 33.96b
|
292.66 ± 8.24ab
|
Zeolite
|
273.51 ± 59.57a
|
98.96 ± 10.07c
|
321.84 ± 21.79a
|
369.44 ± 101.31a
|
Gypsum
|
398.88 ± 27.42a
|
188.44 ± 11.38a
|
174.96 ± 23.38b
|
187.25 ± 3.007b
|
Total phenolic content was expressed as gallic acid equivalent (GAE) which resembled the linear equation, y = 0.0045x − 0.2306, where R2 = 0.9857.
The values are the mean ± SD (n=4). The mean differences with different letter notations were significant at P ≤ 0.05 using Tukey’s test.
Fig. 1. Total yield of mushroom of each treatment collected from the 1st flush to the 4th flush based on the weight of fresh samples (g).
Fig. 2. Total phenolic content of Pleurotus pulmonarius (methanolic extract).
DISCUSSION
The grey oyster mushroom provides various raw materials to be manipulated in mushroom cultivation, which often adds value to its growth, yield, flavour and nutritional properties. The treatment of alkaline materials reacts significantly with a wide spectrum of performance in terms of mycelial growth, primordial formation, yield of production and antioxidant status. In another way, our results disregarded the morphological change of the treatment on the fruiting body, as there are no appreciable differences among distinct treatments regarding the size of pileus and stalks that have been produced.
The rate of mycelial growth is one of the measures of mushroom cultivation that considers the time taken to reach the harvesting stage. Mycelial growth is mainly influenced by the basal substrate where all those essential nutrients are available to be absorbed for the development of premature mycelium. Meanwhile, the primordial growth that occurs after the completion of spawn running is another insight into determining the productivity of mushroom cultivation. Mushroom grows at a specific pH level that provides a suitable condition to support growth from an early stage. A previous study showed that oyster mushrooms had the best growth rate at neutral pH levels (Khan et al., 2013). In the bottom line, the mushroom can only absorb the nutrients at an optimum pH level that was prescribed in the media (Arif et al., 2015).
Generally, the ratio of alkaline material used plays an important role in mushroom cultivation, especially in supporting the availability and transportation of the nutrients. However, the ratio of alkaline materials used for substrate preparation is according to the ratio standard practiced by local growers. In the current study, all these alkaline materials at 1% of treatment showed notable effects on mushroom growth over the spawn running and primordial growth. The results were in line with the previous study performed by Firdaus et al. (2015) who reported that a substrate mixture with 1% of lime took 36 to 48 days for a complete spawn running.
The variation in the duration of spawn running might be due to the presence of different calcium concentrations from various types of alkaline materials. Specifically, the essential element that encompasses magnesium, calcium, iron, copper, manganese, zinc and molybdenum is favourable for fungal growth (Jennings, 1996). Calcium is responsible for stimulating intracellular effects through chemical, electrical and physical signals. Amidst mycelial development, calcium ions were transferred to the hyphal tips and supplied to the subapical zones through passive mechanisms whereby it initiated the gradient of H+, pH, electrical and ions (Royse & Sanchez-Vazquez, 2003). The concentration of calcium in a substrate could have an adverse effect on growth, cell proliferation and sporulation.
Yield and biological efficiency create awareness among the growers in attempting to meet the demands of both the local and global markets. In general, mushroom cultivation with the supplementation of other beneficial additives improves the yield and quality of mushroom (Yang et al., 2013). For instance, many studies indicated that the variety of agricultural wastes mixed into substrates following the ratio of the mixture would be a betterment of the yield of mushroom (Alananbeh et al, 2014). The full exploitation of lignocellulosic material solely without supplementation is inadequate to provide the optimum nutrient requirement to increase the performance of mushroom production (Atila et al, 2017).
The considerable factors in attaining the high productivity of mushrooms presumably point out the composition of the nutrient and physical and chemical systems of the substrates (Hoa, Wang, & Wang, 2015). The recent result showed a significant difference in the yield of these treatments, which is lower than the yield recorded in Pleurotus ostreatus cultivation grown on rice straw (735.9–900.9 g), weed plants (154.1–195.36 g) and maize and pineapple residues (218–675 g) (Hlerema, Eiasu, & Koch, 2017; Islam & Riaz, 2017; Cayetano et al., 2018 ). A past study (Salmalian, Peyvast, & Olfati, 2016) reported that Pleurotus eryngii produced a yield between 14.71 g and 164.54 g, which was in line with the yield in the present study.
With regard to the biological efficiency percentage, a range of 17.95 to 26.05% was observed for the harvested mushroom between treatments. According to a previous study conducted by Myronycheva et al., (2017), the biological efficiency obtained from P. pulmonarius produced from the mixture of wheat straw and sunflower husks at the ratio 2:3 ranged from 9.8 to 62% compared to the current study that recorded lower in biological efficiency. The result was less likely to contradict the study of Khan etal. (2017) that showed that Pleurotus ostreatus grown on a cotton waste substrate with wheat bran produced the maximum biological efficiency of 46.33%, whereas the lowest was only 30% in the cotton waste without supplementation. Another research also reported the contrast in result in terms of biological efficiency of Pleurotus sajor-caju on cotton waste with 81%, while the least was 48.25% in the sugarcane bagasse (Sardar etal., 2017).
The treatment of different alkaline materials on grey oyster mushrooms resulted in crucial effects on the yield, where the treatment of zeolite outweighed the other treatments. The performance corroborates the earlier findings since the application of zeolite on crops elevated the crop production with an outstanding yield that involved Aloe vera (397.17 g), potato (396.35 g), sunflower (5828 kg/ha), canola (2674.1 kg/ha) and tomato (16.972 kg/m2) (Savvas et al., 2004; Bybordi & Ebrahimian, 2013; Gholamhoseini et al., 2013; Ghannad, Ashraf, & Alipour, 2014; Hazrati etal., 2017). It has been discovered that zeolite is a subtle cationic interchanger with microporous structures, enabling it to adsorb molecules at relatively low pressure (Sozana et al., 2003). The exchange capacity of zeolite depends on its nature, which only allows selective minerals and nutrients to pass through despite its ability to adsorb a wide range of molecules. Nevertheless, Ok etal. (2003) reported that zeolite promoted enormous potentialities as it can improve water holding capacity, solubility and nutrient retention in soil. These studies indicated that zeolite is potent for its application in mushroom breeding since it may correlate to the yield thanks to the catalysation of nutrient transportation.
The scavenging activity against 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radicals measures the antioxidant capacity attributed to the plant-, fungi- or microbial- released bioactive compounds. In general, the DPPH assay is carried out in accordance with the principle that active compounds, as the free radical scavenger, donate hydrogen ions to the radicals to manifest the protective effects from oxidative damage. As the antioxidant donates a proton to these radicals, the absorption of samples decreased. The measurement to determine the radical scavenging activity was based on the reduction of the absorbance of the samples (Srivastava et al., 2006). Herein, the results of the assay were derived as an inhibitory concentration of extracts onto 50% of radicals (IC50 value).
All the different treatments demonstrated a wide spectrum of the scavenging activity in IC50 where the lowest value was recorded in the treatment of zeolite at the third flush of the harvested mushroom. This result was also supported by a study conducted by (González-Palma et al., 2016) that displayed that the IC50 of Pleurotus ostreatus fruiting body reached the minimum value of 26.99 mg GAE/L. A few past studies show corresponding results with the current findings with Pleurotus spp. (43.21–52.03 µg/ml), Pleurotus eryngii (48.53 mg/ml) and Pleurotus dryinus (24.71 mg/ml) (Obodai et al.,2014; Wong et al., 2013; Keles, Koca, & Genccelep, 2011). Our finding supported the previous study performed by Shahbaz et al. (2019), where soil remediation for wheat production in nickel (Ni) polluted soil, through the employment of biochar and zeolite, and alleviated the Ni oxidative stress while increasing the activities of antioxidant in wheat. The addition of zeolite to the soil had improved the antioxidant defence machinery of sunflower, which highly scavenged the reactive oxygen species as previously reported (Shahbaz et al., 2018).
The different types of alkaline materials applied to Pleurotus pulmonarius may have a different composition, which resulted in a wide range of IC50. The possible combination of environmental materials, such as composite additives, while prescribing the use of zeolite could be beneficial for crop management, hence promoting growth, postharvest quality and antioxidant enzyme activity in the long run of crop production (Shi et al., 2016).
The phenolic compound is a secondary metabolite that possesses antioxidant properties and in which over 10,000 compound species were identified as being most abundantly extracted from plants (Veberic, 2016). The establishment of many studies on the constituents of chemicals embedded in plant samples was a result of the antioxidant activity determined by the profile of the phenolic compound (Yoon etal., 2011). In the mushroom species, Pleurotus pulmonarius is among the edible mushrooms that implore great interest in its provision of active compounds that perform a wide range of metabolic activities. Their analysis of the phenolic compound broke it down into a large number of species such as gallic acid, homogentisic acid, procatechuic acid, catechin, chlorogenic acid, vanillin, naringin, myricetin, resveratrol and quercetin (Nguyen et al., 2016).
All the treatments differed significantly in demonstrating phenolic content from 98.96 to 369.44 µg/ml. This finding corresponded with the previous study of Rashidi and Yang (2016) that included Agaricus bisporus (0.63 mg/g), Hypzigus marmoreus (0.67 mg/g), Volvariella volvacea (0.73 mg/g), Flammulina velutipes (0.75 mg/g), P. eryngii (0.44 mg/g), P. ostreatus (0.39 mg/g), Lentinula edodes (0.49 mg/g) and Hericium erinaceus (0.46 mg/g). The current research indicated that the treatment of zeolite from the fourth flush was the largest in total phenolic content (TPC) between treatments. Similarly, zeolite application improved the polyphenols content in wheat as reported from the previous research (Shahbaz et al., 2019). This result also coordinated with the incorporation of zeolite in fertilisation, where it promoted the highest phytochemicals in apricot, especially phenolic compounds (Milošević, Milošević, & Glišić, 2013).
The composition of zeolite affects the chemical content in the fruiting body where zeolite was naturally a good source of K (Milosevic & Milosevic, 2009). The relationship between the amount of K and the phenolic compound was positively correlated (Radi et al., 2003). Thus, this study showed that the nutrient status present in the alkaline materials may induce changes in the phenolic content of mushrooms.
Conclusion: The effect of alkaline materials on the cultivation of Pleurotus pulmonarius manifests the pivotal role in response to the yield and quality of mushroom. The treatment of zeolite considerably increased mushroom yield and biological efficiency and induced a higher level of antioxidant value in radical scavenging activity and phenolics content. In other cases, the treatment of gypsum was beneficial in terms of mycelial growth where it significantly minimised the period of spawn running and initiation of primordia. Our results depicted that the application of zeolite and gypsum as additives in substrates leads to the improvement of mushroom production. However, further research on the market price survey of alkaline materials is required to investigate whether the use of alkaline materials results in significant profit in production. The survey is useful to lead the direction of this sector towards economic manners and can be used to generate high yield prospects in the mushroom industry.
Acknowledgments: The authors express the sincerest gratitude to Universiti Putra Malaysia and the Ministry of Higher Education, Malaysia for providing Geran Putra- Inisiatif Putra Muda (GP-IPM/2016/947200) to conduct the research. We are also thankful to the Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia (UPM) and University Agriculture Park, UPM for the use of laboratory facilities.
Author Contributions: M.P.M.F.R conducted the experiments, analysed the data, and wrote the article. S.A., M.T., A.M. designed the experiment and made critical corrections in this paper. All authors have read and approved the manuscript and have contributed significantly for the paper.
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Appendix 2
SEM View on Mushroom Fruiting Body between Different Treatment of Alkaline Materials in Every Flushes (1-4th Flush)
Scanning electron micrographs on cross-section of stalks of Pleurotus pulmonarius between different treatment of alkaline materials; a) lime (first flush) b) gypsum (first flush) c) zeolite (first flush) d) lime (second flush) e) gypsum (second flush) f) zeolite (second flush) g) lime (third flush) h) gypsum (third flush) i) zeolite (third flush) j) lime (fourth flush) k) gypsum (fourth flush) l) zeolite (fourth flush).
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