Umami taste and its association with energy status in harvested Pleurotus geesteranus stored at different temperatures

Food Chemistry 279 (2019) 179–186

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Umami taste and its association with energy status in harvested Pleurotus geesteranus stored at different temperatures

T

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Zhiyong Zhang, Xiaoyu Zhang, Guang Xin , Xue Gong, Yudi Wang, Lu Wang, Bingxin Sun College of Food Science, Shenyang Agricultural University, Shenyang 110866, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Methanol (PubChem CID: 887) Potassium dihydrogen phosphate (PubChem CID: 516951) Aspartic acid (PubChem CID: 5960) Glutamic acid (PubChem CID: 33032) Alanine (PubChem CID: 5950) Sulfonyl salicylic acid (PubChem CID: 88597449) Hydrochloric acid (PubChem CID: 313) Guanosine 5′-monophosphate disodium salt (PubChem CID: 21712) Adenosine 5′-monophosphate disodium salt (PubChem CID: 13363962) Adenosine Disodium Triphosphate (PubChem CID: 132399060)

Pleurotus geesteranus has recently been gaining popularity due to its strong umami taste. In the present study, umami taste, energy level, and energy metabolism-related enzymes activity in harvested P. geesteranus, stored at 20, 10, 5, and 0 °C, were investigated to evaluate the relationship between umami taste and energy status. Results showed that the mushroom at 5 °C exhibited significantly higher (p < 0.05) equivalent umami concentration (EUC), higher content of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and higher activity of succinic dehydrogenase (SDH) and cytochrome c oxidase (CCO) in late storage. AMP, associating umami taste with energy, presented a significantly positive correlation with EUC and umami determined by electronic tongue at 5 °C. Furthermore, there were better correlations between umami taste and energy status of mushroom at 5 °C. The results suggest that higher energy status of post-harvest P. geesteranus contributes to better umami taste.

Keywords: Pleurotus geesteranus Mushroom Umami taste Energy status Temperature

1. Introduction Pleurotus genus, commonly known as oyster mushrooms (Ren et al., 2015), are edible basidiomycetes that are consumed and cultivated worldwide. Their yields ranked third, following those of Agaricus and Lentinula (Fernandes, Barros, Martins, Herbert, & Ferreira, 2015). Pleurotus (P.) geesteranus has been gaining attention lately due to its richness of dietary fiber, high quality of proteins and very pleasant umami taste (Zhang et al., 2011). Umami taste has been attributed mainly to the presence of sodium salts of glutamic and aspartic amino acids and 5′-nucleotides (Zhang, Venkitasamy, Pan, & Wang, 2013). Studies of P. geesteranus have predominantly focused on cultivation (Chen et al., 2010) and bioactive components (Song et al., 2018), whereas change in umami taste of fresh fruiting bodies of P. geesteranus, during storage, has rarely been reported.

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Edible mushrooms contain about 90% moisture and have little physical protection against microbial attack and mechanical damage, which make them highly perishable in nature, causing difficulties in their distribution and marketing as fresh products (Li, Qin, Tian, & Wang, 2016; Wang, Hu, Pei, Mugambib, & Yang, 2018). Low temperature is the most common method for their preservation. Furthermore, their energy status is closely related to their biological characteristics, including ripening, browning or other symptoms of senescence in harvested horticultural crops (Jiang et al., 2007; Wang et al., 2018; Yang et al., 2009). ATP, the most important high-energy phosphate compound in organisms, is a major determinant of cell function. In the process of decomposition, ATP is converted into ADP and AMP by the removal of phosphoric acid groups. Evidence shows that AMP has the taste of umami (Yamaguchi, Yoshikawa, Ikeda, & Ninomiya, 1971). The synthetic pathway of purine nucleotides shows

Corresponding author. E-mail address: [email protected] (G. Xin).

https://doi.org/10.1016/j.foodchem.2018.12.010 Received 25 July 2018; Received in revised form 14 November 2018; Accepted 6 December 2018 Available online 10 December 2018 0308-8146/ © 2018 Elsevier Ltd. All rights reserved.

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Fig. 1. The synthetic pathway of AMP, XMP, GMP from IMP (Zhang, 2006).

2.3. Free amino acid assay

IMP as the precursor of AMP, XMP and GMP (Fig. 1). Therefore, we hypothesized that umami taste might have a direct relationship with energy levels. Studies have demonstrated that adenosine triphosphatase (ATPase), cytochrome C oxidase (CCO), and succinic dehydrogenase (SDH) are key enzymes involved in energy metabolism that regulate ATP synthesis (Jin et al., 2013; Zhou et al., 2014). However, to the best of our knowledge, no report is available on the energy status of postharvest P. geesteranus. Therefore, our study aimed to determine the changes of umami taste and energy status in fruiting bodies of P. geesteranus, stored at different temperature, as well as the relationship between umami and energy. Our report may provide a new direction for studying umami taste of mushroom.

Free amino acid content of the mushrooms was determined as described earlier (Tsai, Weng, Huang, Chen, & Mau, 2006), with some modifications. Fresh samples (3.0 g) were milled finely, extracted with 50 mL of 0.01 M hydrochloric acid, and shaken for 30 min at ambient temperature. The extract was centrifuged at 4500 g for 15 min, the supernatant (5 mL) added to sulfonyl salicylic acid (5 mL), placed for 30 min in the dark, and then filtered through a 0.22-μm hydrophilic membrane. The extracted solutions were analyzed by an automatic amino acid analyzer (L 8900; Hitachi Ltd, Japan). 2.4. Assay of 5′-nucleotides 5′-Nucleotides were extracted and analyzed using the modified method described earlier (Taylor, Hershey, Levine, Coy, & Olivelle, 1981). Different samples were weighed (5.0 g), milled sufficiently, and extracted with 25 mL of distilled water. The suspension was heated to boiling for 1 min, cooled subsequently, and then centrifuged at 4500 g for 15 min. The residues were extracted with 20 mL of distilled water, and the combined filtrates were concentrated by rotary evaporation and re-dissolved in distilled water to a final volume of 50 mL and filtered using a 0.45-μm micro-pore filter membrane before analysis. Waters 1525 HPLC system equipped with UV detector (Waters Corporation, Shanghai, China) was used for the determination of 5′nucleotides. The analysis was completed on a LiChrospher RP-18 column (250 mm × 4.6 mm, 5-μm, Merck) at a flow rate of 1.0 mL/min. The mobile phase consisted of methanol (solvent A) and phosphate buffer (pH 4.2, solvent B). The 5′-nucleotides were monitored at 254 nm, with injection volume of 20 μL and oven temperature of 30 °C. Each 5′-nucleotide was identified using the authentic 5′-nucleotide (Shanghai Yuanye Bio-Technology Co., Ltd, Shanghai, China) and quantified by the calibration curve of the authentic compound.

2. Materials and methods 2.1. Mushroom materials P. geesteranus were obtained from Xinyong Fungi industry Co., Ltd. (Anshan City, China). Mushroom samples were transported to the laboratory at Shenyang Agricultural University, within 2 h of harvest. The mushrooms, used in this study, were selected based on uniformity of maturity, color, shape, and size, and were free of mechanical damage and disease.

2.2. Treatments and sampling P. geesteranus were randomly divided into four groups, each group containing 3 lots (each lot representing a replicate, containing about 500 g), and were respectively stored at 20 ± 1 °C for 3 days, 10 ± 1 °C, 5 ± 1 °C, and 0 ± 1 °C for 12 days. Mushroom samples were placed in sealed polyethylene bags and stored in a refrigerator at constant temperature until the end of the experiment. To assay umamitaste compounds, energy level and activity of energy metabolism-related enzymes, mushroom samples were examined every 3 days during storage, except for those at 20 °C, which were examined every day. The quality of P. geesteranus stored at 20 °C severely deteriorated and lost commercial value after 3 days. Although the samples stored at 10 °C lost commercial value after 12 days, quality of the samples was relatively better when stored at 5 °C and 0 °C. To determine the commercial value of mushroom samples during storage, sensory evaluation was conducted by trained panelists, including that of color, odor, mycelial growth, and spoilage.

2.5. Equivalent umami concentration (EUC) The EUC value reflects the concentration of MSG (monosodium glutamate) equivalent to the umami intensity given by a mixture of MSG and 5′-nucleotides, and is calculated by the following equation (Yamaguchi et al., 1971).

Y=

∑ ai bi +

1218 (∑ ai bi )(∑ aj bj )

where Y is the EUC of the mixture (g MSG/100 g), ai is the concentration (g/100 g) of each umami amino acid [aspartic acid (Asp) or 180

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contents of IMP and XMP were low, the maximum being 0.048 and 0.035 mg/g, respectively. Consequently, their contribution to umami taste was far less than GMP. The contents of flavor 5′-nucleotides decreased during storage, ranging from 0.098 to 0.263 mg/g (fresh weight). Result showed that lower temperature (5 °C and 0 °C) could maintain higher flavor 5′-nucleotides contents (p < 0.05). At 5 °C, the contents of flavor 5′-nucleotides were not less than that at 0 °C, except for at Day 9. At Day 12, there was no significant difference between 5 °C and 0 °C (p > 0.05). Flavor 5′-nucleotides were classified as low (< 1 mg/g, dry weight), medium (1–5 mg/g) and high (> 5 mg/g) (Mau, Lin, Ma, & Song, 2001). Our contents, converted to dry weight contents, were approximately in the range of 0.74–1.99 mg/g. Accordingly, P. geesteranus, in this study, was classified in the low-to-medium range.

glutamic acid (Glu) ], aj is the concentration (g/100 g) of each umami 5′-nucleotide (5′-IMP, 5′-GMP or 5′-AMP), bi is the relative umami concentration (RUC) for each umami amino acid with respect to MSG (Asp, 0.077; Glu, 1), bj is the RUC for umami 5′-nucelotide to 5′-IMP (5′IMP, 1.0; 5′-GMP, 2.3 and 5′-AMP, 0.18), and 1218 is a synergistic constant based on the concentration (g/100 g) used. 2.6. Electronic tongue measurement Analyses were conducted with the Taste-Sensing System SA 402B (Intelligent Sensor Technology, Inc., Kanagawa, Japan), consisting of auto-sampler, reference electrodes, and multichannel lipid/polymer membrane electrodes, as described earlier (Phat, Moon, & Lee, 2016). The mushroom samples were measured (thrice) after the electric potentials of all membranes had been stabilized in standard solutions.

3.3. Equivalent umami concentration (EUC) 2.7. Analysis of enzyme activities in energy metabolism EUC is classified into four levels: I: > 1000 g MSG/100 g (dry weight), II: 100–1000 g MSG/100 g, III: 10–100 g MSG/100 g, and IV: < 10 g MSG/100 g (Mau, 2005). As shown in Fig. 2A, the EUC fluctuated over the range of 6.67 to 18.41 g MSG/100 g (fresh weight), corresponding to 50.5–139.5 g MSG/100 g (dry weight), approximately. Therefore, the EUC of mushroom samples was classified between II and III. Tsai et al. examined Agaricus bisporus harvested at different stages of maturity, EUC ranging from 230 g MSG/100 g to 284 g MSG/100 g (Tsai, Wu, Huang, & Mau, 2007). As shown in Table 2 and Fig. 2A, EUC increased first and decreased later, except at 0 °C. EUC decreased sharply by 51% (20 °C) and 36% (10 °C) during storage. At 5 °C, EUC had the highest value amongst all the conditions in late storage.

Succinic dehydrogenase (SDH), cytochrome c oxidase (CCO), H+ATPase, and Ca2+-ATPase activities in P. geesteranus were measured with an enzyme-linked immunosorbent assay (ELISA) kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the instructions and information provided by the manufacturer. One unit of CCO activity was defined by an increase in absorbance at 550 nm by 0.01 per minute. One unit of SDH activity was defined by an increase in absorbance at 600 nm by 0.01 per minute. One unit of ATPase activity was defined as the release of 1 μmol of phosphorus per hour. 2.8. Statistical analysis All statistical analyses were performed using IBM SPSS Statistics version 19 (SPSS Inc., Chicago, IL). Data were expressed as the mean ± SD (standard deviation) of three replications. Tukey’s multiple range tests were applied to analyze the mean separations. Differences with p < 0.05 were considered statistically significant.

3.4. Electronic tongue There was a significant correlation between sensory score determined by electronic tongue and human sensory evaluation, which demonstrated that the electronic tongue could reliably characterize the umami taste of mushrooms (Fang et al., 2017). In our study, we measured five taste including umami, salty, sweet, bitter, and sour. Since we mainly focused on umami, results of other tastes were not listed in Table. Sensory scores of saltiness and sourness were much lower than those of the others, ranging from −0.31 to −5.77, and −27.15 to −35.12, respectively. Bitterness of samples increased during storage, ranging from 5.21 to 11.58, while sweetness decreased, ranging from 11.3 to 6.22. Changes in umami taste of mushroom samples were shown in Table 1 and Fig. 2B. In general, values for umami measured by electronic tongue kept rising during storage, from 8.30 to 14.04. The trend was different from that of EUC in our study, there was no significant correlation between them (except for that at 5 °C). The result suggested that some differences do exist between electronic tongue and EUC for describing the changes of umami taste during storage. Clearly, it is necessary to conduct further studies to reveal the causes and discover the most accurate method. The above results of EUC and electronic tongue suggested that, theoretically, we could obtain the maximum umami taste of P. geesteranus during storage.

3. Results and discussion 3.1. Amino acids Generally, 14 to 17 free amino acids have been reported in mushroom (Lee, Jian, & Mau, 2009; Pei et al., 2014; Tseng & Mau, 1999). In our study, we detected 17 free amino acids. Among these, only aspartic acid (Asp) and glutamic acid (Glu) contributed to the characteristic umami taste (Yamaguchi, 1991). Therefore, we mainly focused on these two in P. geesteranus samples. As shown in Tables 1 and 2, Asp and Glu content increased in early storage and decreased later, the peak values being 1.63 and 3.37 mg/g (fresh weight), respectively. The variation rate of MSG-like content slowed down gradually, as temperature decreased from 20 °C to 0 °C. At 5 °C, MSG-like content was significantly higher than that at 0 °C (p < 0.05). α-Ketoglutarate could be converted into glutamic acid (Glu) under the catalysis by transaminase; oxaloacetic acid could be converted into L-aspartic acid (Asp) under the catalysis by glutamic-oxalacetic transaminase (Zhu & Xu, 2016). Therefore, the higher content of Glu and Asp in late storage at 5 °C could be attributed to the higher rate of aerobic respiration which generates more α-ketoglutarate and oxaloacetic acid.

3.5. ATP, ADP and AMP contents and energy charge Studies have shown that ripening, senescence, browning and chilling injury in postharvest fruits are closely associated with deficiencies in cell energy levels (Hu et al., 2015; Lin et al., 2017; Pan, Yuan, Zhang, & Zhang, 2017), which could be revealed from ATP, ADP, AMP contents and energy charge (EC). EC represented the proportion of high energy phosphate compounds in the adenylate pool. EC value was calculated using the formula: EC = ([ATP] + 0.5 [ADP])/ ([ATP] + [ADP] + [AMP]). In our study, ATP, ADP and AMP contents

3.2. 5′-Nucleotides Yamaguchi et al. measured the relative intensity of flavors of 5′nucleotides (AMP, XMP, IMP, GMP), with IMP selected as the standard substance (Yamaguchi et al., 1971). Results showed that the relative umami intensities were 0.13 (AMP), 0.53 (XMP), 1.0 (IMP), and 2.3 (GMP). Table 1 showed that the content of GMP decreased sharply by 33% (0 °C), 44% (5 °C), and 72% (10 °C) between Days 0 and 12. The 181

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Table 1 The contents of umami compounds of Pleurotus geesteranus stored at 10 °C, 5 °C, and 0 °C. Indicators

Temperature

Content 0d

3d

6d

9d

12 d

10 °C 5 °C 0 °C

0.20 ± 0.02a 0.20 ± 0.02a 0.20 ± 0.02a

0.79 ± 0.03b 0.43 ± 0.02b 0.26 ± 0.01b

1.63 ± 0.09d 0.76 ± 0.05c 0.73 ± 0.02c

1.11 ± 0.07c 1.08 ± 0.08d 0.81 ± 0.02d

0.80 ± 0.02b 1.06 ± 0.06d 0.75 ± 0.02c

Glu

10 °C 5 °C 0 °C

1.64 ± 0.08a 1.64 ± 0.08a 1.64 ± 0.08a

2.65 ± 0.04b 1.74 ± 0.03a 1.81 ± 0.11a

3.11 ± 0.08c 2.23 ± 0.07b 1.80 ± 0.09a

3.37 ± 0.106d 3.13 ± 0.11c 2.29 ± 0.17b

2.42 ± 0.05b 3.26 ± 0.09c 2.38 ± 0.16b

MSG-like

10 °C 5 °C 0 °C

1.84 ± 0.08a 1.84 ± 0.08a 1.84 ± 0.08a

3.44 ± 0.21b 2.17 ± 0.12a 2.07 ± 0.10a

4.74 ± 0.25c 2.99 ± 0.19b 2.53 ± 0.17b

4.48 ± 0.23c 4.21 ± 0.25c 3.10 ± 0.11c

3.22 ± 0.13b 4.32 ± 0.22c 3.13 ± 0.13c

10 °C 5 °C 0 °C

0.032 ± 0.002b 0.032 ± 0.002b 0.032 ± 0.002b

0.046 ± 0.004b 0.031 ± 0.003a 0.029 ± 0.002bc

0.048 ± 0.004b 0.038 ± 0.002b 0.039 ± 0.003d

0.036 ± 0.004a 0.046 ± 0.002c 0.022 ± 0.002a

0.032 ± 0.003a 0.033 ± 0.002ab 0.025 ± 0.001ab

GMP

10 °C 5 °C 0 °C

0.18 ± 0.01c 0.18 ± 0.01c 0.18 ± 0.01c

0.19 ± 0.01c 0.22 ± 0.016d 0.14 ± 0.01b

0.081 ± 0.007b 0.15 ± 0.01b 0.11 ± 0.01a

0.074 ± 0.006ab 0.11 ± 0.00a 0.18 ± 0.011c

0.053 ± 0.004a 0.10 ± 0.01a 0.12 ± 0.01ab

XMP

10 °C 5 °C 0 °C

0.035 ± 0.002c 0.035 ± 0.002c 0.035 ± 0.002c

0.011 ± 0.001b 0.012 ± 0.001c 0.015 ± 0.001c

0.013 ± 0.001b 0.023 ± 0.001c 0.012 ± 0.001b

0.007 ± 0.001a 0.013 ± 0.001b 0.010 ± 0.001b

0.013 ± 0.001b 0.007 ± 0.000a 0.0068 ± 0.001a

Flavor 5′-nucleotides

10 °C 5 °C 0 °C

0.247 ± 0.020c 0.247 ± 0.020c 0.247 ± 0.020c

0.247 ± 0.016c 0.263 ± 0.013c 0.184 ± 0.013ab

0.142 ± 0.011b 0.211 ± 0.014b 0.161 ± 0.008a

0.117 ± 0.008ab 0.169 ± 0.015a 0.212 ± 0.014b

0.098 ± 0.009a 0.140 ± 0.010a 0.155 ± 0.012a

Amino acids (mg/g, FW) Asp

5′-Nucleotides (mg/g, FW) IMP

FW, fresh weight. MSG-like, monosodium glutamate-like, Asp + Glu. Flavor 5′-nucleotides = GMP + IMP + XMP. Means bearing different lower case letters within the same row are significantly different (Tukey, p < 0.05).

Table 2 The contents of umami compounds and energy status of Pleurotus geesteranus stored at 20 °C. Indictors

Content 0d

1d

2d

3d

Amino acids (mg/g, FW) Asp Glu MSG-like

0.20 ± 0.02a 1.64 ± 0.08a 1.84 ± 0.080a

0.31 ± 0.02b 2.19 ± 0.12bc 2.50 ± 0.19b

0.74 ± 0.04d 2.37 ± 0.11c 3.11 ± 0.20c

0.46 ± 0.04c 1.96 ± 0.11b 2.42 ± 0.17b

5′-Nucleotides (mg/g, FW) IMP GMP XMP Flavor 5′-nucleotides

0.032 ± 0.002b 0.18 ± 0.014c 0.035 ± 0.002c 0.247 ± 0.02c

0.043 ± 0.003c 0.16 ± 0.01c 0.033 ± 0.003c 0.236 ± 0.02c

0.060 ± 0.004d 0.12 ± 0.01b 0.014 ± 0.001c 0.194 ± 0.01b

0.023 ± 0.003a 0.06 ± 0.00a 0.024 ± 0.002b 0.107 ± 0.01a

10.43 ± 0.57b

13.69 ± 0.51d

11.83 ± 0.65c

5.11 ± 0.39a

8.50 ± 0.18d

9.67 ± 0.10c

11.63 ± 0.20b

14.04 ± 0.24a

0.098 ± 0.008bc 0.15 ± 0.01c 0.38 ± 0.02d 0.28 ± 0.01a

0.085 ± 0.005b 0.10 ± 0.01b 0.31 ± 0.01c 0.27 ± 0.01a

0.062 ± 0.006a 0.06 ± 0.00a 0.15 ± 0.01a 0.34 ± 0.01b

20.93 ± 1.18c 1.72 ± 0.12b 0.54 ± 0.03a 0.59 ± 0.04c

14.35 ± 1.17b 1.78 ± 0.14b 0.54 ± 0.02a 0.39 ± 0.02b

9.42 1.54 0.68 0.13

EUC (g MSG/100 g, FW) EUC Electronic tongue Umami Energy compounds (mg/g, FW) ATP ADP AMP Energy charge

0.11 0.17 0.25 0.37

± ± ± ±

0.01c 0.01d 0.01b 0.01b

Energy metabolism-related enzymes activity (U/mg protein, FW) SDH 12.15 ± 0.91b CCO 1.22 ± 0.08a H+-ATPase 1.33 ± 0.06c Ca2+-ATPase 0.39 ± 0.02b

FW, fresh weight. MSG-like, monosodium glutamate-like, Asp + Glu. Flavor 5′-nucleotides = GMP + IMP + XMP. Means bearing different lower case letters within the same row are significantly different (Tukey, p < 0.05). 182

± ± ± ±

0.70a 0.06b 0.04b 0.01a

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Fig. 2. EUC and sensory score based on electronic tongue of Pleurotus geesteranus at varying storage temperatures and over time. Means with different lower case letters in the same day are significantly different (Tukey, p < 0.05). 0.26

0.16

A

a

0.14

a a

a

0.12

ab

b

0.10

a

b

b 0.08

c

10°C 5°C 0°C

0.06 0

2

4

b 6

8

10

a

a

0.22

a

b

0.20

b

0.18

b

a

0.16

b

0.14

c c

0.12

10°C 5°C 0°C

0.10 0.08

12

B

a

0.24

ADP content (mg/g FW)

ATP content (mg/g FW)

a

0

2

4

Storage time (d)

b

6

8

10

12

Storage time (d)

0.55 0.50

C

a

0.40

a

b

0.30

c 0.20

0

2

4

0.35

a

b

0.25 0.20

10°C 5°C 0°C

0.15

6

b

b

b

0.30

c

10°C 5°C 0°C

0.15 0.10

c

c

a

a

a

b

0.25

D

a

0.40

b

b

0.35

a

0.45

a

a

0.45

Energy charge

AMP content (mg/g FW)

0.50

8

10

0.10

12

Storage time (d)

0

2

4

c c

6

8

10

12

Storage time (d)

Fig. 3. ATP (A), ADP (B), AMP(C) content and energy charge (D) of Pleurotus geesteranus at varying storage temperatures and over time. Means with different lower case letters in the same day are significantly different (Tukey, p < 0.05).

183

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2.0

26

a

a

A

SDH activity (U/mg protein)

22 20 18

a

16

a

a

b

14

ab

12 10

b b

b

8

4 0

2

4

b

c

10°C 5°C 0°C

6

2

b

8

10

a

1.6

1.4

b b

b

b 10°C 5°C 0°C 0

2

4

6

8

10

12

Storage time (d)

Storage time (d) 2.1

1.0 0.9

a

a

1.5

C

a

a

Ca2 -ATPase (U/mg protein)

1.8

H -ATPase (U/mg protein)

ab

b

ab

1.2

0.8

12

a

a

1.0

6

B

a

1.8

CCO activity (U/mg protein)

24

a b

a 1.2

ab

b

0.9

b c

c

0.6 10°C 5°C 0°C

0.3 0

2

4

D

a

0.8

0.6

a

8

10

0.5

Storage time (d)

10°C 5°C 0°C 0

2

4

a

b

b

b

0.4

0.2

12

a

ab

0.3

6

a

a

0.7

b

b

6

8

10

12

Storage time (d)

Fig. 4. Activities of energy metabolism-related enzymes in Pleurotus geesteranus at varying temperatures and over time. Means with different lower case letters in the same day are significantly different (Tukey, p < 0.05).

pathway, couples the reduction of ubiquinone (Q)1 to the oxidation of succinate (Li, Jarukitt, Li, Du, & Luo, 2016). As shown in Fig. 4A, SDH activity of mushroom samples changed slightly during storage at 5 °C and 0 °C, whereas SDH activity significantly increased at Day 1 (20 °C, Table 2) and Day 6 (10 °C, Fig. 4A). CCO activity (Table 2, Fig. 4B) was higher than its initial value during storage. The fluctuating trends of SDH and CCO activity at 5 °C and 0 °C were similar to those of blueberries treated with cold temperature (Zhou et al., 2014). In late storage, SDH and CCO activity at 5 °C was significantly higher than that at 0 °C (p < 0.05). Results indicated that P. geesteranus stored at 5 °C had higher rate of TCA cycle and oxidative phosphorylation, resulting in production of more energy in the mitochondrion. This could be demonstrated by higher ATP, ADP and AMP contents (Fig. 3).

of mushroom samples stored at 20 °C decreased rapidly by 71%, 65% and 40%, respectively (Table 2), whereas EC (Table 2) of samples stored at 20 °C increased by 26% at Day 3. This phenomenon suggested that spores would be liberated soon. Similar trends were seen in ATP and ADP contents of cold-stored litchi fruit held at 25 °C, while EC value rose in late storage time (Liu et al., 2011). As shown in Fig. 3, ATP (A), ADP (B), and AMP (C) contents at 5 °C were significantly higher than that at 10 °C in late storage (p < 0.05), whereas, EC (Fig. 3D) of mushroom samples stored at 0 °C had higher value than at 5 °C in late storage (p < 0.05). High EC value suppressed the production of ATP while promoting its utilization (Zhu & Xu, 2016). This could probably explain why ATP content at 5 °C was higher than at 0 °C in late storage. The decreasing tendency in ATP content and EC value at 10 °C was similar to that in litchi fruit treated with tea seed oil (Zhang et al., 2017).

3.7. H+-ATPase and Ca2+-ATPase activities 3.6. CCO and SDH activities H+-ATPase, a P-type proton pump, couples ATP hydrolysis and transports H+ ions across the membrane, generating electrochemical gradients used for trans-membrane transport of substances (Sondergaard, Schulz, & Palmgren, 2004). Ca2+-ATPase is a primary regulator of Ca2+ efflux from cytosol to extracellular matrix (Guan,

CCO, the terminal enzyme in the mitochondrial respiratory electron transport chain, plays a crucial role in aerobic metabolism and energy production during oxidative phosphorylation (Kan, Wang, & Jin, 2011). SDH, a respiratory chain enzyme in the tricarboxylic acid (TCA) cycle 184

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References

Fan, Dou, Zhanh, & Li, 2006). Generally, Ca2+-ATPase activity was seen to increase first and decrease later (Table 2, Fig. 4D). The peak value of Ca2+-ATPase activity at 20 °C was lower than that at 10 °C, 5 °C, and 0 °C. It might be interpreted as chilling-injury stress response of mushroom samples in cold storage. In contrast, Ca2+-ATPase activity of samples stored at 0 °C was significantly lower (p < 0.05) than that at 10 °C and 5 °C, probably because of the inhibition of enzyme activity at lower temperatures. Recently, a study showed that H+-ATPase activity substantially increased at early storage time and decreased thereafter in pitaya fruit pretreated with methyl jasmonate (Li et al., 2018). However, in our study, H+-ATPase activity at 5 °C and 0 °C (Fig. 4C) increased noticeably upon late storage. This difference might arise from the different signaling pathways of different treatment processes.

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3.8. Correlation between umami taste and energy status Pearson correlation coefficient is generally used to reflect the statistical dependence between two variables; correlation is considered significant at p value < 0.05. The relationship between EUC, umami intensity (measured by electric tongue) and energy status were analyzed in this study. Statistical analysis demonstrated that EUC was significantly (p < 0.05) related to ATP (at 10 °C) and AMP content (at 20 °C and 5 °C). Umami was significantly related to ATP (at 20 °C) and AMP content (at 5 °C), besides correlating well with the content of GMP and Glu. In addition, the correlation between umami taste and energy metabolism-related enzymes was also analyzed. Results showed that SDH activity of P. geesteranus stored at 5 °C and 0 °C markedly correlated with EUC (p < 0.05). CCO activity exhibited strong correlation with Glu content (p < 0.05), except for that at 0 °C. Taken together, we proposed that umami taste might be closely related to energy level, and SDH and CCO were probably important enzymes that affected umami taste in harvested P. geesteranus; and further experiments would be required to validate these hypotheses. 4. Conclusion In summary, the above data indicated that umami compounds and energy level of mushroom stored at 20 °C decreased rapidly, resulting in severe deterioration of mushroom quality. Umami taste and energy level at 10 °C declined quickly after 6-days storage while P. geesteranus stored at 5 °C exhibited remarkably (p < 0.05) higher EUC value, higher energy level, and higher activity of energy metabolism-related enzymes (except for Ca2+-ATPase) than that at 0 °C in late storage. Meanwhile, umami taste of samples markedly (p < 0.05) correlated with energy compounds (ATP, AMP) and energy metabolism-related enzymes (SDH, CCO). In our experimental condition, 5 °C was deduced as the optimum temperature to maintain umami taste at a relatively higher level and postpone quality deterioration in postharvest P. geesteranus. Furthermore, higher energy status could contribute to higher umami taste of mushroom during storage. However, further evidence would be required to show how energy status directly related to the umami taste at the molecular level. Acknowledgments This work was financially supported by Edible mushroom resources exploitation and the key technology development in efficient processing (No. 2018YFD0400200) and Liaoning Province, Shenyang Agricultural University, high-end talent introduction fund project (NO. SYAU20160003). Conflict of interest The authors declare that there are no conflicts of interest. 185

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