Efficiency of Tragacanth gum coating enriched with two different essential oils for deceleration of enzymatic browning and senescence of button mushroom (Agaricus bisporus)

Abstract The effect of Tragacanth gum (T) coating containing (100, 500, and 1,000 mg/L) Satureja khuzistanica essential oil (S), Zataria multiflora Boiss. essential oil (Z), and (1,000 mg/L) sodium metabisulfite (M) on mushroom (Agaricus bisporus) enzymatic browning and postharvest quality was examined throughout 16 days of cold storage. Mushroom respiration rate, soluble solids content (SSC), percentage of open caps, and sensory quality as well as factors related to browning such polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), and peroxidase (POD) activities were figured out. The significant decrease in respiration rate, cap opening delay, and SSC enhancement was observed after treating mushrooms with TZ and TS. Moreover, TZ‐ and TS‐treated mushrooms prevented enzymatic browning through inhibiting PPO and POD activities and increasing activity of PAL over the storing term. Additionally, the influence of TZ5 and TS5 (containing 500 mg/L essential oil) coatings was validated by sensory evaluation through protecting the overall quality of button mushrooms over the storage. Thus, Tragacanth coating enriched with essential oils might be an encouraging nomination for improving the modality of button mushroom and expanding its shelf life.

Tragacanth gum (T) is one of the three major exudate gums, which possesses a unique range of functionalities (Verbeken, Dierckx, & Dewettinck, 2003). This natural and acidic polysaccharide secretes spontaneously or with a scrape on the different species of Astragalus plant. This gum composed of 30%-40% Tragacanthin (water-soluble and extremely branched) and 60%-70% bassorin (insoluble but water-swellable fraction). Forms and structures of these two constituents are different. The ratio of Tragacanthin and bassorin strongly depends on variety (Azarikia & Abbasi, 2016).
Safety aspects of chemical additives caused increasing interest of application of natural preservative coatings with antimicrobial agents (Jiang, Feng, & Zheng, 2012), such as gum Arabic, aloe vera, chitosan containing thyme essential oil (Bill, Sivakumar, Korsten, & Thompson, 2014), chitosan coating augmented with thyme oil (Jiang et al., 2012), and carboxymethyl cellulose-based coatings containing Zataria multiflora Boiss. essential oil and grape seed extract (Raeisi et al., 2015). These researches have revealed that essential oils (EOs) and extracts of various herbs and spices could be used as natural food preservers due to their antimicrobial acting. Like other biopolymers, Tragacanth gum is known as an appropriate carrier for natural antimicrobial and antioxidant complexes (Taban, Rahimi, Saharkhiz, Hadian, & Zomorodian, 2013).
Zataria multiflora Boiss (Labiatae) is an herbal plant with several thin, hard, and highly ramified stems from family which found naturally in Iran, Pakistan, and Afghanistan. This plant with traditional title of Avishan Shirazi (in Iran) has been used as antiseptic, anesthetic, antispasmodic, and flavor composition in broad range of foods in Iran. The essential oil of Z. multiflora Boiss. contains compounds with important pharmaceutical, antimicrobial, and antioxidant effects (Fathi, Sahari, Zangiabadi, & Barzegar, 2011;Kordsardouei, Barzegar, Sahari, & Ebrahimipour, 2015). Phenolic compounds, such as carvacrol and thymol, are the major components of the Z. multiflora Boiss. essential oil (Z) (Dashipour et al., 2015).
Another plant that is separated through southern part of Iran including Ilam, Lorestan, and Khuzestan provinces is Satureja khuzistanica Jamzad (Lamiaceae). It is a small shrub with branched stem approximately 30 cm high, densely leafy, covered with short white hairs (Moazeni, Saharkhiz, Hoseini, & Mootabi Alavi, 2012). Analgesic and an antiseptic characteristic of plant are considered by the people in southern region of Iran. A main component of wild S. khuzistanica (≤90%) is monoterpene carvacrol (Hashemi, Niakousari, Saharkhiz, & Eskandari, 2012).
The purpose of this study was to investigate the effect of Tragacanth gum coating, individually and/or in combination with Z. multiflora Boiss. essential oil (S) and S. khuzistanica essential oil (Z) and sodium metabisulfite (M) as a chemical preservative (which widely used in the mushroom processing industry), on mushroom tissue browning, some physicochemical, and as well as sensorial changes during 16 days of storage in refrigerator (4 ± 1°C).

| Plant material
Button mushrooms were gathered from a local farm in Shiraz, Iran.
Mushrooms were selected from the similar flower and zone of the shed so as to reduce probable alteration affected by cultivation and environment. During one hour of harvesting, the mushrooms were conveyed to the test center and then kept in dimness at 4 ± 1°C and 90% relative humidity (RH). Mushrooms having good physical shape without any damage were selected to be used in the experiments.

| Tragacanth gum and coating treatments
The best kind of gum was obtained from a local shop, washed, dried, milled, and determined the optimum concentration of gum solution (0.6%), and ten different treatments were prepared based on Nasiri, They were placed in polyethylene (HDPE) containers and were kept for 16 days at 4 ± 1°C and 95% RH pending further analysis, and every 4 days, three replicates from each treatment group were analyzed.

| Respiration rate
Respiration rate of samples was measured using method described by Li, Zhang, and Yu (2006).

| Soluble solids content
Mushrooms were crushed in a pounder. The juice was pressed and analyzed for the soluble solids content (SSC). The measurement was carried out in a CARL Zeiss refractometer model 78969 (Germany) at 25°C.

| Percent open caps and overall acceptability
The principles of evaluating the percentage of open caps were in accordance with development of the umbrella-like form of the cap followed by detachment of veil. The percent open caps were measured using known number of mushrooms based on Equation (1): where N oc = number of opened cap mushrooms; N t = total number of mushrooms.
The consumer acceptance of the product was studied using 10 panelists with age ranging from 25 to 40 years. Panelists were trained based on the method described by Ares, Parentelli, Gámbaro, Lareo, and Lema (2006). Mushrooms were served in closed, odorless plastic containers at room temperature. After opening polyethylene bags, evaluations were performed within 2 hr in order to avoid loss of offodors. The overall acceptability based on color, texture, and percent opened caps were done by round-table basis using four-point scale where 1 = poor, 2 = fair, 3 = good, and 4 = excellent (Jiang et al., 2011).

| Determination of protein content and PPO, PAL, and POD activities
Enzymatic activity determined as explained by Gao et al. (2014).
The protein content determination was carried out as the method of Bradford (1976), with application of bovine serum albumin as a standard.
Polyphenol oxidase (EC 1.10.3.2) activity was investigated using incubating 0.5 ml of enzyme extract in 2.5 ml of buffered substrate (100 mM sodium phosphate, pH = 6.4, and 50 mM catechol), then detecting the change of absorbance at 398 nm. The quantity of enzyme caused 0.01 increases in absorbance per minute under the specified conditions defines one unit of PPO activity. The specific PPO activity was represented as U/mg protein.
Three ml of the reaction mixture, containing 0.8 ml supernatant and 50 mM l-phenylalanine in sodium borate buffer (200 mM, pH = 8.8), was incubated at 37°C; after 90 min, the reaction was interrupted by ice water. The amount of enzyme caused 0.01 absorbance rise in 1 hr under the assay conditions defined as one unit of PAL activity. The specific PAL activity was expressed as U/mg protein.
Peroxidase (EC 1.11.1.7) activity was analyzed by determining the absorbance of guaiacol. 50 mM sodium phosphate buffer (pH = 6.0), 5 mM guaiacol, 5 mM H 2 O 2 , and 50 μl of tissue extract constructed the reaction mixture. One unit of POD activity was distinguished as the quantity of enzyme that leads to change of 0.01 absorbance per minute at 470 nm under the specified conditions. The specific POD activity was defined as U/mg protein.

| Statistical analysis
Experiments were conducted in triplicates. Data were analyzed using SPSS (version 16; IBM, USA). Analysis of variance (ANOVA) followed by Duncan's multiple range test was used to distinguish the treatments at p < 0.05.
Consequently, interaction of gum and EOs could decrease respiration rate without increment of off-flavor.

| Effect of gum coating containing essential oils on percentage of open caps and overall acceptability
After 16-day storage, the proportion of opened caps mushrooms increased in all samples (Table 1). Control (C) and M-treated samples had higher opened caps mushrooms, 82.2% and 80.0%, respectively.
Over the same period, the percentage of opened caps mushrooms coated with TZs, TSs, and T were in the range 66.7%-75.6%.
According to Jiang (2013), mushroom cap opening leads to increased water loss, which in turn leads to mushroom dryness. Cohesive forces of water and other hydrophilic molecules, such as proteins responsible for the well position of the caps and veil in mushrooms, diminished over the storage, as a result of water loss increment. TG coating alone and in combination with each of the essential oils attenuated water loss, the cap opening of mushrooms was lower, and the least changes (66.7%) were related to mushrooms coated with TZ10.
Overall acceptability (based on color, texture, and percentage samples opened caps) reduced during storage (Table 2)

| Effect of gum coating containing EOs on PPO activity
The activity of PPO increased during the storage period. PPO activity of control mushrooms increased swiftly without peaks and was considerably greater than samples coated with TG and TG containing essential oils (Figure 3). TG coatings might be slightly permeable especially to oxygen, thus preventing PPO to access to oxygen. Browning has been studied in other fruits, and discoloration correlated well with PPO activity. The inhibition of the PPO activity by coating fruits and vegetables was reported by numerous investigators: chitosan coating on litchi fruit (Jiang, Li, & Jiang, 2005), carrageenan coating on fresh-cut banana ( Therefore, in the presence of oxygen, polymerization and creation of the unwanted brown pigments took place (Amin, 2016). In general, treatments with essential oils prompt a decrease in the activity of PPO. The studies conducted by Amin (2016)  TG containing essential oils preserved more ascorbic acid content (Data not shown). This may be attributed to the pH lowering action of ascorbic acid particularly at high concentration, which decreases the pH below the optimum value in turn inhibiting the catalytic action of PPO (Sapers, 1993).

| Effect of gum coating containing essential oils on POD activity
In all treatments, pattern of POD activity was similar during 16 days of storage, reaching a peak and then declined. The POD activities in TZ-and TS-coated mushrooms were significantly lower than that in C ( Figure 4). Unpleasant quality alterations, such as discoloration, offflavor, and nutritional destruction through handling, transportation, F I G U R E 2 Effect of essential oilincorporated TG coating on soluble solids content of button mushrooms stored at 4°C for 16 days. Vertical bars represent standard errors of means. For abbreviations see Figure 1 A  11.9 ± 4.1 Ad 37.1 ± 2.5 ABCc 58.3 ± 5.6 ABCb 77.0 ± 1.6 BCa C 17.8 ± 3.6 Ad 42.2 ± 3.9 Ac 64.5 ± 3.9 Ab 82.2 ± 3.9 Aa a Mean of three replications ± standard deviation. Means in the same row with different small letters are significantly different (p < 0.05). Means in the same column with different capital letters are significantly different (p < 0.05). C: control (without coating); M: 1,000 mg/L sodium metabisulfite; T: coating with Tragacanth gum; TM: TG coating containing 1,000 mg/L metabisulfite; TS1: TG coating containing 100 mg/L of Satureja khuzistanica essential oil (S); TS5: 500 mg/L S; TS10: 1,000 mg/L S; TZ1: TG coating containing 100 mg/L Zataria multiflora Boiss. essential oil (Z); TZ5: 500 mg/L Z; TZ10: 1,000 mg/L Z.
TA B L E 1 Effect of Tragacanth gum coating containing two essential oils on percentage of open caps of samples packaging, processing, and storage of fruit and vegetables, are consequences of enzymatic browning. These reactions resulted mostly from enzymes such as peroxidase (Mousavizadeh, Sedaghathoor, & Khorami, 2011). The POD association with enzymatic browning and antioxidant defense has been revealed by former investigation (Lin et al., 2017). The increase in peroxidase is a sign of fruit quality deterioration and is relevant to the enzymatic browning process because o-phenols could encourage fruit and vegetable products to be darkened over the processing and preservation and could display reducing function in the enzyme reaction (Amin, 2016). The quality features and mushroom decay may be affected by different enzymatic changes in chemical constituents. Also, POD could affect odor and taste and deteriorate eating and nutritional qualities of mushrooms (Baardseth, 1979;Fasidi & Kadiri, 1991). For that reason, it is required to inactivate the peroxidase enzyme to minimize deterioration.
It is acceptable that edible coatings practice as protective barrier on the surface of fresh product, diminish oxygen supply, slow down ripening and senescence, and, subsequently, postpone browning of fruits and vegetables. Several preservation methods, based on chitosan coating (Eissa, 2007) and alginate coating (Jiang, 2013), restricted activities of POD. In addition, application of essential oils resulted in the decrease in the peroxidase enzyme activity, which may account for the inhibition of the mushroom browning. Alikhani, Sharifani, Azizi, Hemati, and Mousavizadeh. (2009)

| Effect of gum coating containing EOs on PAL
Despite PPO, PAL activities of all samples decreased continuously during storage ( Figure 5). PAL is the major enzyme in the biosynthesis of varieties of phenylpropanoid compounds, such as simple phenols, anthocyanin, flavonoid, and lignin (Hiratsuka et al., 2001;Ju, Yuan, Lieu, & Xin, 1995). It is stated that the accumulation of phenols and anthocyanins equated the PAL activity enhancement in some fruits (Wang et al., 2009 (Gao et al., 2014). TZ10, TZ5, TS10, and TS5 were more effective than other treatments in enhancing PAL activity.

| CON CLUS IONS
Inhibition of enzymatic browning is the most important concern in the mushroom's industry. As the food industry tends to decrease the use of chemical preservatives in food products, TG coating contained each essential oils could be considered as a potential source for shelf-life extension of cold-stored mushroom. TZ and TS treatments slowed down respiration rate, postponed cap opening, and SSC addition compared with gum-coated, M-treated, and control mushrooms. Furthermore, TS5 showed greater influence to inhibit discoloration of button mushrooms. Consequently, these natural products have promising effect to protect the quality and safety of button mushroom. TZ and TS coatings could prevent enzymatic browning through inhibition the activities of PPO and POD, increasing PAL activity during the storage time.

ACK N OWLED G M ENT
The authors acknowledge the financial support provided by the