Physicochemical Quality of Oyster Mushroom for Functional Food

ABSTRACT


INTRODUCTION
The oyster mushroom is a type of mushroom that specifically grows in tropical countries such as Indonesia.Oyster mushrooms are grown on aggregate media one of them is the paddy waste from the paddy cultivation.Therefore, generally at the rice production centre area mostly becoming the centre for mushrooms production.This can be correlated with the availability of biomass as the main growing media for mushroom.
Cultivating high quality of the mushrooms required high-quality seeds, the high-quality media resources as well as the optimal environment due to the mushrooms are sensitive The provision of production house that meets food safety requirements is absolutely necessary to produce safe oyster mushrooms for consumption.Meanwhile the technology development of this mushroom production that meets food safety standards for the isolation of ergothioneine as an antioxidant is a potential matter in exploration of mushroom is still limited.This paper describes the research results of the prototype of FRP house with the air condition variation inside the house and the effect of air condition against mushroom quality.The effect of the IoT implementation in controlling the air condition system as well as minimizing the human contact during oyster mushroom growing during cultivation periods, and to explore the value added of mushroom through the ergothioneine (EGT) analysis as the of antioxidant content in mushroom.The scope of research includes (1) constructing a prototype of FRP house with IoT control system for oyster mushroom cultivation; (2) to evaluate the air condition variation inside the house and the effect of air condition against mushroom growth and quality; (3) Analysis of antioxidant content in mushroom.

MATERIALS AND METHODS
The research was conducted in 2022 at the field laboratory of Post Harvest Research Center of IAARD and analytical laboratory of University of Djuanda.The materials used in this research are analytical grade chemicals.The effectiveness of antioxidant properties on isolated EGT was measured by in vitro method.The main material and controlled apparatus respectively for (1) the construction material for building a prototype of FRP house with IoT control system for oyster mushroom cultivation; (2) The atmospheric air meter apparatus for investigating the air condition variation inside the house and the effect of air condition against mushroom growth and quality; (3) Analysis of antioxidant content in mushroom by HPLC method.The materials used in this research are analytical grade chemicals.The effectiveness of antioxidant properties on isolated EGT was measured by in vitro method.

FRP House
The Fibre Reinforced Plastic (FRP) house constructed (Figure 1) at an area of 650 cm x 350 cm with a light steel alloy frames and the roof is covered by a thin metal sheet roof and all each the wall blanketed by fibre reinforced plastic.(FRP), and white ceramic tile's floor.There are two shelves at inside the FRP house with of 400 cm length and 100 cm width respectively.The FRP House implemented with an Internet of Think (IoT) system (Figure 2) to actuate a set of devices to measure the parameters of air temperature, air humidity, moisture content of the media.The control devices are 30 nozzles of mist sprayer setup along the rack of shelves above the media which are connected with a special hose.Hot air temperatures above 35 o C, caused the exhaust fan is on, whenever a drastic drop of air temperature inside the FRP house below 29 o C, thus the.heater will operate automatically to increase the room temperature.The controller specification showed in Table 1 are set at day and night time with the elapsed time of intervals are enable to monitor the parameters conditions to avoid disturbance during mushrooms growth.
The Arduino Node MCU is an open-source platform based on IoT, which is able to implement various commands with programming languages.The application of the C programming language uses a sketch on the Arduino IDE (Integrated Development Environment), which is capable of programming various kinds of microcontrollers including Node MCU.The Node MCU has a small size compared to Arduino UNO, by using the ESP8266 module, there are GPIO (General-purpose input/output), PWM (Pulse Width Modulation), I2C, UART (Universal Asynchronous Receiver Transmitter), 1-Wire and ADC (Analog to Digital Converter).

Cultivation Process Procedure
Mushroom cultivation process procedure was carried out inside FRP House and utilized the organic components which are mixed and fermentation process for 4 days to be a compost, and then fill into bag log evenly in a plastic sacks assembled inside FRP House.The layers of compost were pasteurized inside FRP house with closed cover condition tightly and then fogging with hot air at temperature 60 to 65 o C for 8 hours.The mushroom seeds were inside over the bag logs of compost and then to be incubated for 3 days at around 29-30 o C of inside air temperature.The treatment during mushroom growth up to harvest periods comprised of the inside air temperature, humidity and soil water content.The treatment during mushroom growth up to harvest periods comprised of the inside air temperature, humidity by thermos-hygrograph and soil water content sensor at every 20 min.The first harvesting conducted two weeks after seedling where the growth mushroom head is indicated by totally white head blooming.

Extraction of the Active Compound Ergothioneine
Ergothioneine is a complex compound of antioxidant.The isolation of ergothioneine EGT compounds from the harvested fresh mushrooms was carried out by the extraction method refers to the method developed by Ito et al. (2011) and Nguyen et al. (2012) with some modifications.Pre-treatment (blanching and non-blanching), mushroom before drying methods by electric oven drying.
The experimental factors of extraction employed the temperature 40 o C and 60 o C respectively with the variation of the extraction solvent (water and ethanol 30% (v/v), 50% (v/v), 70% ethanol (v/v), and ethanol 100 % (v/v)).The extraction procedure utilized 10 grams of dry mushroom powder and 200 mL of solvent which has been heated in a shaking water bath according to variations in temperature.The solvent along with the solute was continuously agitated at 100 rpm for 1 hour, then cooled at room temperature for 2 hours.Then it was centrifuged at 600 rpm for 15 minutes at 25 o C. The achieved of the process is supernatant which was transferred to a conical tube for further evaporation using a vacuum evaporator at a temperature of 40 o C to evaporate the remaining solvent up to be a paste of EGT extract.Furthermore, the paste of EGT was dried by the freeze-drying method.The dried EGT sample was then weighed to measure as the extraction yield.

EGT Concentration Analysis
Analysis of the concentration of EGT in the oyster mushroom extract used the HPLC method referring to the method used by Kalaras et al. (2017) with some modifications.The mushroom powder (0.5 g) and 7 ml of ethanol was put into a 15 ml centrifuge tube (consisting of 10 mM dithiothreitol, 100 M betaine, 100 M 2-mercapto-1-methyl imidazole) and 3 ml of distilled water was added and homogenized with a vortex for 10 seconds.A total of 2 mL of 1% SDS solution dissolved in ethanol was added and homogenized with a vortex for 10 seconds.Then the suspension was centrifuged for 20 minutes at 4000 rpm and 25 o C. The supernatant formed was transferred to a 15 mL centrifugation tube.Then, 1 mL of the supernatant was put into a micro tube and allowed to stand for 24 hours.The solid formed from this process is then dissolved into 0.5 mL of aqua bides and homogenized using a vortex until it dissolves completely.Samples that have been stored in micro-tubes were centrifuged at 10,000 rpm for 1 minute.The supernatant formed was then filtered using a syringe filter (0.45 100 m) and put into a glass vial for HPLC analysis.
HPLC analysis was performed using column C18.The volume of the solution injected was 10 mL.The mobile phases used mobile phase A (3% acetonitrile, 0.1% TEA, 50 mM dibasic sodium phosphate dissolved in distilled water), mobile phase B (acetonitrile), and mobile phase C (water).The separation was performed (Kalaras et al., 2012) on the elution gradient as follows: 0 minutes 15:85 (B:C), 0-5 minutes set at a slope of 5:15:85 (A:B:C), 5-6 minutes set at a slope of 10:90 ( B:C), 6-8 minutes at 3:97 (B:C), and 8-15 at 3:97 (B:C).EGT was detected at a wavelength of 254 nm with a peak retention time of 5.7 min which was quantified using an external standard curve.
The antioxidant activity of EGT from edible mushroom was carried out by measuring the radical scavenging activity of DPPH referring to the method of Pahila et al. (2019).The analytical procedure comprised of 50 L of straw mushroom extract is added to 50 L of DPPH solution (100 M in 85% ethanol) and stirred using a vortex.After 15 minutes, the solution mixture was put into a 50 L Pyrex NMR capillary tube, then the DPPH radical spin resonance was measured with a spectrometer at a wavelength of 517.The spectrometer settings as microwave power, 1.2 mW; microwave frequency 9149.3MHz; field magnet, 325.5 25 mT; and the sweep time, 30 seconds.Various concentrations of EGT were measured for resonance to construct a standard curve.The radical scavenging activity of DPPH was expressed as mmol equivalent of EGT per mL of sample.

FRP House
The prototype of the FRP house is shown in Figure 3. Condition of the air temperature, air humidity and water content of the media inside the FRP house during mushroom cultivation are monitored automatically.The variation of air temperature around 29-39 o C is relatively stable, while condition at day, night and rainy, resulted in high fluctuated of air humidity 40-98% with the water content of the media around 39-99%.
The devices inside the mushroom's FRP house consists of circuits, input blocks and output blocks which are joint by the Nodemcu microcomputer to control the air condition inside mushroom's FRP house.The input block is a soil moisture sensor and BME280 as a temperature, humidity and barometric detector.The sensor will generate an input value for the Nodemcu microcomputer, this value will be processed by Nodemcu which produces an output value to the connected devices.While the output block is a receiver a value generates by sensor through Nodemcu, such as a heater will operate if the temperature inside the mushroom's FRP house is low and the mist-maker will turn off if the humidity inside the mushroom's FRP house is sufficient.Nodemcu has a Wi-Fi module that is able to save the values and data in the database, this can make it easier to use a smartphone to access mushroom's FRP house condition even the values or data remotely.The IoT control system is setup for operating the hot air exhaust with the mist sprayer process taking place in parallel if the air temperature at above 39 o C with humidity below 60%.And when the opposite condition occurred (below 29 o C), thus the water heater will actively operate spraying hot air vapor into the FRP House.This methods of IoT control mechanism will reduce the human intervention so that the growth process of mycelium and pinhead of fungal embryos can achieve optimally.

Effect of FRP House on Mushroom Quality
Air conditions and lighting inside mushroom's FRP house day and night during the mushroom growth (Figure 4) contibute for the effect to the quality of harvested fresh mushroom.The results of quality analysis indicated that the range of phenotype length of 2.5-10 cm, diameter of 3-10 cm and weight 10-120 gram (Figure 5), in general the mushrooms growth from organic media are longer and heavier than non-organic growth media with more uniform dimensions except for the diameter of phenotype.Figure 4 is to show the effect of air condition mentioned by sensors to the growth of the mushroom.The hardness test and the moisture content of the mushrooms were carried out by comparing the mushrooms at the farm level as shown in Table 4, indicating that the mushrooms in the FRP house were harder and less water content than that ones at the farmer mushroom's housing at the farm level.

Micro Laboratory Analysis
The results of micro-laboratory analysis (Table 5) showed that the fresh harvested mushroom are negative from total fungus and salmonella.

Active Compound Ergothioneine
The results of the analysis on functional compounds of EGT in oyster mushrooms using the HPLC method, a graphic recording of the results of HPLC analysis of dried mushrooms with standard ethanol solvent.The average recording results from the EGT chromatography results were 0.674 mg/g as shown in Table 6.The extraction process breaks down the bonds among the molecules of the material so that EGT and phenol compounds are produced with varying concentrations depending on the type of solvent used (Table 7).The results of the standard solution analysis showed that the EGT compound had a retention time of 3.33 ± 0.06 minutes, while the mushroom sample showed a retention time of 3.28 to 3.30 minutes.

Table 7. Recapitulation of EGT characterization data from edible mushrooms
The extraction process using 70% ethanol produced higher concentrations of EGT and phenol than the extraction process using 96% ethanol.This shows that 70% of ethanol solvent is more effective used to bind EGT and phenol compounds in oyster mushrooms than that in pure solvents.This result is in line with the previous study conducted by Nguyen et al. (2012).The process of extracting the Flammulina velutipes mushroom using 70% ethanol was able to produce EGT and phenol compounds with higher concentrations than that the extraction using 50% and 100% of ethanol, with values of 2.05 ± 0.18 mg/g and 3.65 ± 0, 08 DW, consecutively.
The extraction process that has been carried out can break down the bonds between the molecules of the material so that EGT and phenol compounds are produced with varying concentrations depending on the type of solvent used (Table 8).The results of the standard solution analysis showed that the EGT compound had a retention time of 3.33 ± 0.06 minutes, while the straw mushroom sample showed a retention time of 3.28 to 3.30 minutes.The extraction process for active compounds (Table 9), such as EGT and phenol, is influenced by several factors, such as solvent polarity and process conditions.A mixture of solutions has the ability to bind the active compound better than the pure solvent.This is due to the mixing of nonpolar solvents with water which can increase the polarity index of the solvent so that the extraction efficiency increases.
In addition, the increase in extraction temperature was positively correlated to the increase in antioxidant activity and polyphenols (flavonoids and total phenol), while the increase in extraction time was not positively correlated to the increase in antioxidant activity and polyphenols.In addition, the presence of agitation during the extraction process also contributes to increasing the extraction yield.This happens because the agitation process can increase the rate of diffusion and minimize the barriers to mass transfer from the solid surface into the solution (Muhamad et al., 2014).Table 10 showed that the role in synthesizing EGT is the methyltransferase enzyme which is encoded by the EGT gene and specifically remodels histidine into EGT compounds (Stampfli et al., 2020).The results of another study stated that the biosynthesis of EGT is a complex synthesis process involving various types of amino acids and carbon sources.However, in principle there are three types of amino acids that play an important role in EGT biosynthesis, namely methionine, histidine, and cysteine.Histidine acts as a starting material that plays a role in the whole series of EGT biosynthesis.Cysteine serves to provide a source of sulfur atoms, while methionine which contains the protein S-adenosyl-l-methionine (SAM) acts as a precursor in reactions involving methyl groups.In supporting its growth, cells can use dextrose, fructose, glycerol, maltose, and sucrose as carbon sources.A suitable carbon source for EGT biosynthesis is glycerol because it is able to maintain cells in a longer stationary growth phase.In the glycolysis cycle, glycerol is broken down into glycerol-3 phosphate by the enzyme glycerol kinase.Furthermore, glycerol-3 phosphate is broken down by the enzyme glyceraldehyde-3 phosphate dehydrogenase to glyceron-P which is then overhauled by triosephosphate isomerase to glyceraldehyde 3 phosphate (GAP).GAP acts as a precursor in the glycolysis process to produce pyruvic acid which will also be a substrate in the Krebs cycle.Consumption of large amounts of pyruvic acid can stimulate enzymatic reactions of amino acids to produce EGT (Yu et al., 2020).

CONCLUSSION
The research results showed that the cultivation of oyster mushroom in an FRP house with ceramic floors and plastic walls and an IoT management.The temperature of the air inside was between 29 and 35 degrees Celsius, and the relative humidity was between 60% and 90%.The average texture of fresh oyster mushroom was soft.The average length, width, and height of the fresh mushroom were 41.5 mm, 60.0 mm, and 29.5 mm, respectively.The microbiological test confirmed that there was no salmonella infection in the collected mushrooms.The stage of mushrooms have less total fungus than the bloom-stage mushrooms.The extraction standard method employs an EGT content of 0.674 mg/g as determined by chromatography data analysis.The oyster mushrooms can be consumed as a healthy meal, and the study of EGT showed also very prospective as one of immunotherapeutic food.

Figure 1 .
Figure 1.Prototype of FRP house model of straw mushroom (dimentions 3.0 m x 5.0 m x 3.0 m)

Figure 2 .
Figure 2. Architecture of IoT of production house in oyster mushroom

Figure 3 .
Figure 3. (a) The prototype of the FRP house, (b) with IoT circuit, and (c) panel control box

Figure 4 .Figure 5 .
Figure 4. Fluctuation of air temperature, RH, and moisture content inside FRP House

Table 2 .
Temperature and humidity sensor testingTable3is a comparison graph of sensor values with measuring instruments.Digital measuring tools used to compare sensor values such as mini-Hygrometer, lux meter and carbon detector.The test was carried out by taking samples 30 times by getting an average temperature difference of 0.91 °C, humidity 0.89%.Furthermore, the light sensor gets an average difference of 19.76% and gas content of 10.13 ppm.The second test evaluates the performance of the tool and obtains an accuracy rate of success of 93.3%.

Table 3 .
Light sensor and CO 2 content

Table 6 .
Results of chromatogram graphic data processing from mushroom extract

Table 8 .
Methods and varying concentrations type of solvent for EGT

Table 9 .
Results of chromatograph analysis of mushroom samples