Total phenolics, tannin contents, antioxidant properties, protein and sensory analysis of Pleurotus ostreatus , Pleurotus citrinopileatus and Pleurotus djamor cultivated on different sawdusts

: In mushroom cultivation, it is important to be aware of the impact of the growing substrate. This study investigated the cultivation of various oyster mushrooms, including Pleurotus ostreatus , Pleurotus citrinopileatus , and Pleurotus djamor , on different types of wood sawdust. Total phenolic content, condensed tannins, antioxidant activity by ferric reducing antioxidant power assay, protein and sensory evaluations were performed in cultivated oyster mushrooms. Wood sawdust of Fagus orientalis (oriental beech) , Alnus glutinosa (alder)


Introduction
Global food production is currently confronted with a number of challenges, including rising population, the impact of climate change on agricultural productivity, the clear environmental impact of the agricultural-food system, and the uneven economic situation generated by global financing (Ray et al. 2019).As cardiovascular diseases, type 2 diabetes mellitus, and cancer become more widespread, there is a growing need to develop new dietary techniques and foodstuffs that can help prevent these diseases (Masri et al. 2017).So, in this context, Pleurotus spp.mushrooms can provide a valuable contribution as they can support growth and the production of value-added foods by very few uses of biological resources (Sekan et al. 2019, Xiao et al. 2022).Edible mushrooms may be an important alternative to conventional protein sources as they contain high amounts of protein (González et al. 2021).P. ostreatus, in particular, is an edible mushroom with a wealth of functional compounds (Espinosa-Páez et al. 2021), such as, high protein, zinc, chitin, fiber, and vitamins (C, D, and B complex) and amino acids (Sassine et al. 2021).Pleurotus spp.
with around 40 known species is also known as one of the most valuable non-wood forest products (Raman et al. 2021).It has an increasing growth rate in the global marketplace compared to other forest products (Keča et al. 2017).It usually grows spontaneously on the bottom roots of heartwoods.For centuries, those mushroom species have served mankind as a source of health and food (Manzi et al. 1999).However, as in other natural products, the growing period of oyster mushrooms is limited by certain parameters.Therefore, the production of such a valuable non-wood forest product is increased by imitating the growing conditions and ensuring that it is grown in an artificial production environment (Josiane et al. 2018).It has been reported that there are some differences in terms of bioactive compounds content of cultivated mushrooms depending on growing substrate and growing conditions (Aghajani et al. 2018).Studies reported that the antioxidant properties, protein contents, and sensory attributes of oyster mushrooms, which can be easily grown using various lignocellulosic wastes, vary according to the compost they grow on (Yıldız et al.

2017a).
The production parameters of some Pleurotus species (P.ostreatus, P. citrinopileatus, and P. djamor) in different substrates composed different wooden sawdust by beech oriental beech (Fagus orientalis Lipsky), alder (Alnus glutinosa subsp.barbata (C.A.Mey.)Yalt.), chestnut (Castanea sativa Mill.), and walnut (Juglans regia L.) was investigated.The effect of compost material on chemical properties, total phenolic content, condensed tannin content, antioxidant activity, and protein concentration of mushrooms was revealed.Since Pleurotus species are considered to be new and different for Turkey, sensory analysis was also performed to evaluate consumer perception.

Materials and methods
Preparation and cultivation of the oyster mushrooms were carried out in the Laboratories of the Eastern Karadeniz Forestry Research Institute and Forest Biology and Wood Protection Technology Department at Karadeniz Technical University.Extraction, total phenolic content, condensed tannin content, antioxidant activity assays of the cultivated oyster mushrooms were performed in the chemistry laboratories of the same university.Protein content determination was studied in the Central Research Laboratories at Giresun University.Sensory analysis was carried out by panelists from the Food Processing Department of Karadeniz Technical University.

Material
The mycelium of P. ostreatus, P. citrinopileatus and P. djamor were purchased from commercial firms in Turkey (Gürsu Mushroom, Bursa).Waste wood sawdusts were obtained from the workshop of the Furniture Department at Karadeniz Technical University.As a reference to these mushrooms (control), champignon mushroom (Agaricus bisporus) was also included in the study.The champignon mushroom was not produced within the scope of the study and was purchased from a commercial company (Afacan Mushroom, Trabzon).

Compost content, preparation, inoculation and harvesting
Wood sawdust was sterilized in an autoclave at 121 °C for 30 minutes to inactive detrimental organisms.After the cooling period (25 °C), the composts were prepared with 96 % wood sawdust, 3 % mycelium, and 1 % calcitic lime for pH balance in 29 cm x 45 cm bags -4 as 1 kg for each variation as seen Figure 1 (Estrada et al. 2009, Yılmaz et al. 2017b).

Method Extraction procedure
Oyster mushroom and sawdust samples were dried in a laboratory type dryer at 60 °C for 8 hours.After the drying period, the samples were powdered using a blender.5 g of oyster mushroom powder samples were placed in a falcon tube, with 50 mL of methanol (99 %).
The final mixture was stirred using a shaker (Heidolph Promax 2020, Schwabach, Germany) at room temperature for 24 hours.Following extraction, particles were removed using coarse filter paper.

Total phenolic content
Total phenolic content of studied samples was measured using Folin-Ciocalteu method as given by Slinkard and Singleton (1977).400 µL of 0,5 N Folin-Ciocalteu reagent, 20 µL of sample, and 680 µL of distilled water were mixed.After 3 minutes, 400 µL of sodium carbonate solution (10 %) was added.The mixture was then incubated for 2 hours at 20 °C with vigorous shaking.Gallic acid was used as standard to construct the calibration curve.
At the end of the incubation period, absorbance was measured at 760 nm.Total phenolic content of the samples was expressed as mg GAE (gallic acid equivalent)/g sample.

Total condensed tannins
The total condensed tannins were determined according to the Julkunen-Titto method (Julkunen-Tiitto 1985).Varied concentrations of 25 μL oyster mushroom extracts were mixed up with 750 μl of 4 % vanillin and were added 375 μL of concentrated HCl.The absorbance values were read at 500 nm.A graph was drawn with the absorbance values corresponding to the concentration.According to the drawn graph, the condensed tannins of the oyster mushroom samples were calculated.The condensed tannins content of the samples was expressed as mg CAE (catechin equivalent)/g sample.

Antioxidant capacity by ferric reducing antioxidant power (FRAP) assay
Antioxidant activity of the samples was measured using the ferric-reducing antioxidant power (FRAP) method of Benzie and Strain (1996) with minor modifications.The FRAP method is based on the reduction of the Fe(III)-2,4,6-tris(2-pyridly)-S-triazine (TPTZ) complex to form a blue complex Fe(II)-TPTZ in the presence of antioxidants.Varying concentrations of FeSO4·7H2O were prepared for standard curve.50 µL of the sample was mixed with FRAP reagent and incubated for 4 minutes.The results were expressed as μmol FeSO4·7H2O per g of the sample (Benzie and Strain 1996).

Dumas protein analysis
The method is based on determining the amount of protein from the volume of nitrogen gas formed by burning the food at a high temperature.The oyster mushroom powder samples were analyzed using the Costech ECS 4010 Elemental Analyzer after they were completely pulverized.This device determines the element percentages by burning solid or liquid organic compounds at a temperature of 1020 °C -1050 °C.Helium gas is used as carrier gas and oxygen gas is used as combustible gas in the device.For elemental analysis, the powders of the samples were weighed 0,500 to 0,700 mg, poured into 5 mm x 9 mm tin capsules and turned into a prism.Carbon (C), Hydrogen (H), Nitrogen (N) and Sulfur (S) ratios were measured simultaneously by placing the capsules in the device and burning the samples.The peaks of the samples were read in the Costech 4010 program.The amount of protein was calculated by multiplying the amount of nitrogen found by a factor of 4,38 (Crisan and Sands 1978).

Sensory analysis
Sensory analysis of the studied samples was performed by a group of 25 semi-trained panelists.The age of the panelists was between 20 and 50 old of both sexes.Mushroom samples were encoded with non-consecutive three-digit numbers.Before microwave cooking, the mushroom samples were divided into four equal parts, and equal amount of salt and oil was added to each sample.The mushrooms were heat-treated in a household microwave oven at 600 W for 4 minutes.All samples were served on the same white plastic plates.Appearance, smell, color, hardness, taste, and overall appreciation for the mushroom samples were evaluated by using the hedonic scale within the range of 1 (bad), 2 (medium), 3 (good), and 4 (very good) points (Figure 3).This method has been preferred due to the fact that it is a technique that gives a fairly fast results, as well as allows the comparison of a large number of samples.Sufficient amount of the mushroom samples to try 3-4 times was served to each panelist.Water was also served to the panelists to rinse their mouths between tasting the consecutive mushroom samples.Because it is the best time for sensory analysis and daylight saving, the panels were performed at 10,00 -13,00 o'clock (Altug-Onogur and Elmaci 2011).

Statistical analysis
All assays were performed in triplicate.The data were recorded as means ± standard deviations and analyzed in a completely randomized design by using Statistical Package for Social Sciences (SPSS) version 23,0 (2016).Statistically significant differences (p < 0,05) among means of experimental results were analyzed by ANOVA and tests of significance were carried out using Duncan's multiple range tests.

Total phenolic content
Total phenolic and condensed tannins contents and FRAP values of mushroom samples were given at Table 1.The highest and the lowest total phenolic contents were found in (in P. ostreatus mushroom cultivated on alder (Alnus glutinosa) sawdust (1,298 ± 0,060 mg GAE/g) and in P. djamor mushroom cultivated on walnut (Juglans regia) sawdust (0,364 ± 0,024 mg GAE/g) (Table 1).In a previous study, it was reported that different growing conditions affect the vitamin and mineral content as well as the polyphenols content of cultivated mushrooms (Muszyńska et al. 2015).P. djamor mushrooms has less total phenolic content in all growing substrates than other mushrooms.Although the total phenolics contents of P. ostreatus and P. citrinopileatus were almost similar, it was found that P. ostreatus had a little higher value than P. citrinopileatus.Yılmaz et al. (2017b) reported that the total polyphenolic content of P. ostreatus samples cultivated on sterilized tea wastes, nonsterilized tea wastes, and espresso wastes were 1,460 ± 0,012, 1,393 ± 0,060 and 1,075± 0,004 mg GAE/g, respectively.It was also reported that P. ostreatus cultivated on tea wastes, alder (Alnus glutinosa), oriental beech (Fagus orientalis), and chestnut (Castanea sativa) had similar total polyphenolic content, as P. ostreatus cultivated on espresso wastes and walnut (Juglans regia L.) (Yılmaz et al. 2017a).Phenolic contents of the samples in our study are higher than some salad greens such as Chicory sp. and Lepidium sativum (1,091 and 1,261 mg GAE/g, respectively) (Uyar et al. 2013).Vo and Ariyo (2013) found that, total phenolic contents of P. ostreatus grown on three different tropical wooden substrates (Cananium sp., african nutmeg (Pycnanthus angolensis (Welw.)Warb.) and kapok (Ceiba pentandra (L.) Gaertn.) were ranged from 0,89 to 2,63 mg GAE/g.This was found between 1,072 ± 0,027 and 1,298 ± 0,060 mg GAE/g for the studied P. ostreatus grown on four different (oriental beech (Fagus orientalis Lipsky), alder (Alnus glutinosa (L.) Gaertn.),chestnut (Castanea sativa Mill.), walnut (Juglans regia L.)) wooden substrates.A study with P. djamor mushroom from Mexico stated that total phenolic content was between 0,95 ± 0,02 and 0,36 ± 0,01 mg GAE/g (Oropeza-Guerrero et al. 2018).It can be stated that the difference in the growing substrate and the species of mushroom influence the total phenolic content of the mushroom.
Table 1: Total phenolic and condensed tannins contents, and FRAP values of mushroom samples.
Means followed by different letters in the same column differ significantly at p < 0,05 (Duncan's multiple range test)

Protein content
Protein content of P. ostreatus, P. citrinopileatus, P. djamor and A. bisporus were given in

Sensory analysis
Since oyster mushrooms are usually cultivated on oriental beech (Fagus orientalis) wood substrate, sensory analysis was performed only using mushrooms produced in oriental beech (Fagus orientalis) sawdust and also commercial A. bisporus (Table 3) (Akçay andDoğan 2019, Başyiğit andSakaldaş 2021).Oyster mushrooms can absorb the flavor of the compost.
Therefore, the mushrooms grown in an unusual compost such as walnut (Juglans regia L.), chestnut (Castanea sativa) and alder (Alnus glutinosa) wood substrates were not included (Omarini et al. 2010, Yokota et al. 2016).Mushrooms grown on oriental beech (Fagus orientalis) compost were used in the sensory analysis, since alder (Alnus glutinosa), walnut (Juglans regia) and chestnut (Castanea sativa) shavings could add a different flavor to the mushroom.P. citrinopileatus and P. djamor samples were scored lowest on all the evaluated sensory characteristics except color.The smell characteristics of P. citrinopileatus and A. bisporus mushrooms were found to be quite different by the panelists.The hardness, taste, and general appreciation scores of A. bisporus, P. ostreatus and P. djamor mushrooms were similar and statistically different from P. citrinopileatus mushrooms (p < 0,05).Although P.
ostreatus had the highest score in general appreciation, no statistically significant difference was detected between the samples (p > 0,05).General appreciation scores showed that A.
bisporus and P. ostreatus mushrooms gained more appreciation than P. citrinopileatus (Table 3).Previous findings demonstrated that Pleurotus species produced using plant wastes had the highest mushroom flavor and sour intensity ratings.(Omarini et al. 2010).Nayak et al. (2015) evaluated sensory attributes of 15 % (w/w) A. bisporus added fish meatball samples.They reported that the sensory evaluation scores of the mushroom-added samples were higher than the control (non-added) sample.El-Refai et al. (2014) were also found that sensory analysis of 8 % (w/w) P. ostreatus added beef meatballs had good sensory characteristics.Wu et al. (2022) reported that Sausage containing P. ostreatus had better sensory scores than sausage samples without P. ostreatus (Wu et al. 2022).
Means followed by different letter(s) in the same column differ significantly at p < 0,05 (Duncan's multiple range test)

Table 2 .
Bengü et al. (2019)11)ghest protein concentration(13,84 %), in this study.Similar protein concentration (13,75 %) was measured in P. ostreatus cultivated on alder (Alnus glutinosa) sawdust (Table2).This value was 13,67 % in P. djamor cultivated on walnut (Juglans regia) sawdust.The lowest protein concentration (9,86 %) was determined in P. citrinopileatus cultivated on oriental beech (Fagus orientalis) sawdust.Protein contents of Pleurotus species grown in selected wood sawdusts were determined between 9,86 -13,75 %.It was 13,84 for the of commercially obtained A. bisporus.Therefore, it can be concluded that the protein content of both mushroom groups is close to each other.In another previous study,Khan and Tania (2012)determined the protein ratio in P. eryngii and P. ostreatus to be between 11 -17 %.Jaworska et al. (2011)found that as a proportion of total amino acids in fresh form of P. ostreatus is 10 -12 %, in frozen form 10 -11 %, and in canned form 11 -12 %.Bengü et al. (2019)performed Dumas protein method of cultivated P. ostreatus (22,04 It was also determined that the protein ratio of the mushroom is influenced by the substrate in which it is cultivated, even if the mushroom is of the same species. %) and A. bisporus(22,22 %)samples, which values they were multiplied by the conversion factor of 6,25.In the current study, the protein percentage of P. ostreatus was, and of A. bisporus 13,84 %.In another study, protein percentage of P. ostreatus samples cultivated on tea and espresso wastes were 13,16 % and 13,05 % respectively(Yılmaz et al. 2017a).It has been determined that the content of protein found in mushrooms varies depending on what specie of mushroom.