The mechanism of cholesterol-effect on the quality of green asparagus (Asparagus officinalis L.) spears during low temperature storage

Scientia Horticulturae 231 (2018) 36–42

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The mechanism of cholesterol-effect on the quality of green asparagus (Asparagus officinalis L.) spears during low temperature storage

T

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Xiangyang Wang , Shuang Gu, Beili Chen College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, 310018, PR China

A R T I C L E I N F O

A B S T R A C T

Keywords: Chlorophyll Cholesterol Asparagus Thylakoid membrane

The effectiveness of cholesterol in maintaining the quality of postharvest asparagus was demonstrated in our previous work. In this study, the effect of cholesterol on chlorophyll degradation in green asparagus were determined by analysing chlorophyll content, redox ability of chlorophyll, chlorophyll degradation, chlorophyllprotein binding capacity and fatty acid composition in thylakoid membrane. Results showed that chlorophyll contents were significantly (p ≪ 0.01) reserved by cholesterol treatments. Cholesterol significantly (p ≪ 0.05) reduced chlorophyll degradation in a near neutral aqueous solution and acidic solution, respectively, but failed to inhibit chlorophyll degradation in strong reducing acidic solution (ascorbic acid) and oxidizing solution (hydrogen peroxide). Chlorophyll-protein binding capacity was better maintained under cholesterol treatment compared with the untreated samples, indicating higher content of chlorophyll-protein complexes. In addition, the content of linolenic acid and linoleic acid in thylakoid membrane could be better retained after cholesterol treatment. Hence, the stability of thylakoid membrane and chlorophyll were effectively sustained under cholesterol treatment, providing a new opportunity to preserve the quality of green vegetables.

1. Introduction

suppressed the expression of chlorophyll catabolic enzymes, delaying yellowing of broccoli florets (Fukasawa et al., 2010). Wang et al. (2004) found that pak choy leaves subjected to heat-shock treatment (46 °C–48 °C) for 10 min increased the synthesis of heat-shock proteins, degradation of fructose and glucose less, synthesized sucrose in the outer leaves, and inhibited chlorophyll degradation. In green plants, chlorophylls are embedded within thylakoid membrane (Tovuu et al., 2013; Colbow, 1973), and most of them are attached by non-covalent bonds to proteins existing in the form of chlorophyll-protein complexes (Andersson and Anderson, 1980). As the distribution location of chlorophylls and chlorophyll-protein complexes, the thylakoid membrane is associated with the degradation of chlorophylls, responsible for the light reaction of photosynthesis. Thylakoid membrane showed a stacked structure surrounded by the chloroplast stroma, and the stacked structure presented a positive role for retention of chlorophylls and chlorophyll-protein complexes(Daum and Kühlbrandt, 2011). The disruption of thylakoid membrane structure can induce the release of intercellular acids and enzymes, which come into intimate contact with chlorophylls and chlorophyll-protein complexes, further accelerating the degradation of chlorophylls (Wang et al., 2013). When degraded, pheophytin is formed by the acidic removal of the Mg+ in the chlorophyll molecule and hydrolyzation of the phytol chain by the chlorophyllase enzyme, changing chlorophyll to

Chlorophyll, a green pigment found in plants, algae, and cyanobacteria, is responsible for absorption of energy from light in the process of photosynthesis (Magdalena and Kaczor, 2014). However, because of its light-absorbing properties, chlorophyll is a dangerous molecule and a potential cellular phototoxin (Hoertensteiner and Kraeutler, 2011; Liu and Guo, 2013), this is observed in situations where the photosynthetic apparatus of plants is overexcited, for example in high light conditions. Absorbed energy can then be transferred to oxygen, resulting in the production of reactive oxygen species (ROS) (Liu and Guo, 2013; Hoertensteiner, 2009). Excessive ROS causes peroxidation of membrane lipids, membrane disintegration and eventually cell death (Esfandiari et al., 2010). The most common change in green vegetables is the loss of chlorophyll, which causes a shift in color from brilliant green to yellow or brown in senescent tissues. Thus, the problem of keeping harvested vegetables fresh and green relates directly to the visible symptom of chlorophyll breakdown (Roiser et al., 2015). In recent years, important progress has been made in understanding of physiological mechanism affecting the green color of vegetables. Eum and Lee (2007); Eum et al. (2009) reported decreased lipid peroxidation and yellowing as well as retarded onset of chlorophyll degradation in broccoli florets treated with nitric oxide (NO). Ethanol vapor also

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Corresponding author. E-mail addresses: [email protected], [email protected] (X. Wang).

https://doi.org/10.1016/j.scienta.2017.12.016 Received 16 October 2017; Received in revised form 29 November 2017; Accepted 6 December 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.

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(0.2 g L−1) and cholesterol absorbing (0.5 g L−1) methods were used in this experiment of green asparagus. The dissolution of cholesterol (Bomei Biotechnology Co., ltd., Shanghai, China) used the method of Petersson et al. (2013) with some modifications. Cholesterol (0.2 g, 0.5 g) with purity over 95% was dissolved in 20 mL 99.5% (v/v) ethanol containing 1.0 g Tween-80 and then dried under nitrogen gas. The solid was then re-dissolved in 500 mL distilled water containing 2% (v/v) ethanol, and diluted to 1.0 L with water. Other reagent grade chemicals were obtained from Hangzhou Huipu Chemical Industry Instrument Co., Ltd., Hangzhou, China.

chlorophyllide. Followed by the conversion of chlorophyllide to pheophorbide through the loss of magnesium ion, and pheophytin is converted to pheophorbide through the loss of its phytol chain. The conversion of pheophytin to pheophorbide, results in a yellow-olive colored pigment (Schwart and Von Elbe, 1983; Ozkan and Bilek, 2015). In general, a common feature accompanying senescence of fruits and vegetables is increased membrane permeability after harvest (Marangoni et al., 1996). Therefore, thylakoid membrane is an important factor affecting the degradation of chlorophylls (Sharkey, 2000). Cholesterol as an essential structural component of biological membranes is normally required for maintaining both fluidity and membrane structural integrity (Acimovic and Rozman, 2013; Montagne et al., 2014). Previous research indicated that exogenous cholesterol treatment increased the fluidity of the membrane structure in rice rppt apex under low-temperature stress (Guo et al., 1993). Awad and Graham (2002) found that sperm treated with cholesterol which entrapped cyclodextrin significantly improved sperm activities and survival rate during cold storage. In our previous research, it was noticed that cholesterol exhibited beneficial effect on the reduction of color changes and inhibition of lignin synthesis in green asparagus (Wang et al., 2017). A proposed explanation for this phenomenon is cholesterol inhibited the release of intercellular acids, which alleviated the attack of acid on chlorophyll. On the other hand, cholesterol maintained the integrity and stability of thylakoid membrane, which retarded chlorophyll detaching from the membrane. These might make considerable contributions to the prevention of chlorophyll degradation. With this in mind, the mechanism of cholesterol-effect on better color retention of asparagus need to be further studied. Cholesterol, derived from animal-based food, is a natural and safe preservative. European countries, Asian countries, and Canada do not have an upper limit for dietary cholesterol (Fernandez and Calle, 2010), and the limitation of dietary cholesterol were cancelled by the Scientific Report of the 2015 Dietary Guidelines Advisory Committee in US (Dietary Guidelines Advisory Committee, 2015). Green asparagus (Asparagus officinalis L.), a kind of typical green vegetable, was selected as the experimental material in this study for a better application of cholesterol processing technology. The objective of this study was to evaluate chlorophyll degradation of asparagus spears treated with cholesterol-absorption or cholesterol-coating during a period of 30 days of cold storage, the mechanism of cholesterol-effect on the quality of green asparagus was explored by determining the redox ability and stability of chlorophyll in vitro, and by determining chlorophyll-protein binding capacity, as well as fatty acid composition in asparagus.

2.2. Sample treatment Coating and absorption treatment methods were performed on fresh asparagus. Replicates were included in each treatment and three independent experiments were conducted. Coating was carried out at room temperature by dipping the asparagus spears in 0.5 g L−1 cholesterol for 30 min and air-dried at 25 °C, while control samples were treated similarly in a solution without cholesterol. Subsequently, each batch of asparagus was placed into a perforated plastic basket (45 cm × 35 cm × 25 cm) and then packed into 100 cm × 120 cm polyethylene (PE) plastic bag. For the absorption treatment, each batch of asparagus was bundled up and vertically immersed in 0.2 g L−1 cholesterol solution during the storage, the immersion depth was 3 cm, while control samples were treated similarly in a solution without cholesterol. Subsequently, each batch of asparagus was covered with polyethylene (PE) plastic film. After application as described above, all treated asparagus were placed into a refrigerator (MIR-554, Sanyo, Japan) and stored in the dark at 4 ± 0.5 °C and 90% relative humidity. The chlorophyll content was conducted on the first day, then at 6-day interval up to day 30. For the asparagus treated with cholesterol coating, chlorophyll-protein binding capacity and the fatty acid composition in thylakoid membrane were analysised during the storage of day 0 and 3. 2.3. Preparation of chlorophyll extract Preparation of chlorophyll extract was carried out according to the method outlined by An et al. (2006) with some modifications. Briefly, Fresh samples (5.0 g) were homogenized in 10 mL of 80% acetone with a tissue homogenizer (Polytron PT-MR2100, Kinematica AG, Luzern, Switzerland) at a moderate speed for 30 s and centrifuged (12,000g, 15 min) at 4 °C (2–16 K, Sigma, Germany), and then residue was removed. The supernatant was transferred to capped microfuge tubes and stored frozen at −20 °C as chlorophyll extract stock material.

2. Materials and methods 2.4. Chlorophyll content measurement 2.1. Materials and chemicals Absorbance of chlorophyll extract was read at 645 and 663 nm with an UV–vis recording spectrophotometer (UV-2550; Shimadzu, Japan). The total chlorophyll content was calculated by chlorophyll (mg g−1 FW) = [20.2 × Abs 645 + 8.08 × Abs 663]/200 (Inskeep and Bloom, 1985; Ozkan and Bilek, 2015).

Fresh asparagus spears (Asparagus officinalis L. cv. ‘Grande’) were harvested from a local farm in Hangzhou (Zhejiang, PR China), After harvesting, the samples were rapidly placed in crushed ice and transported to the laboratory at Zhejiang Gongshang University within 2 h. Asparagus selected for the study were straight, undamaged, 1.6–2.0 cm in diameter and ∼30 cm in length with closed bracts and no visible signs of injury. Selected samples were randomly separated into samples of approximately 200 g. In preliminary experiments, the overall visual quality of green asparagus treated with cholesterol coating or absorbing by different concentrations was assayed. It was found that the application of cholesterol coating (0.2 g L−1) and cholesterol absorption (0.5 g L−1) were the most effective in terms of shelf life extensions compared with other concentrations. Specifically, cholesterol (0.2 g L−1, 0.5 g L−1) treatment significantly reduced color changes, the losses of fresh weight and retarded chlorophyll degradation. Therefore, cholesterol coating

2.5. Tests in vitro 2.5.1. Determination of chlorophyll degradation in vitro Ascorbic acid (reductant) and H2O2 (oxidant) were selected to study the effect of cholesterol on redox ability of chlorophyll in vitro. According to Table 1, all reagents adding were performed and briefly mixed by vortexing, then placed for 60 min at room temperature. Absorbances of sample solutions were read at 663 nm (Raychaudhuri and Bhattacharyya, 2008), test tube 5 and 1 were regarded as the control groups with/without the addition of cholesterol. Absorbances of test tube 2 and 6 were measured at 0 min, the former was used as A0 of test 37

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was suspended again in quintuple volume of pre-cooled washing solution and centrifuged at 500g for 1 min to get supernatant. Thereafter, it was suspended in the pre-cooled suspension solution (pH 6.9, containing 0.3 M sucrose, 50 mM NaCl, and 50 mM Tricine-HCl). Thylakoid membrane solution were stored at −20 °C for further use.

Table 1 The addition amounts of cholesterol and other reagents of chlorophyll stability experiments in vitro (addition amount, mL). Group

1

2

3

4

5

6

7

8

chlorophyll extract 0.5% cholesterol solution 2.5% ascorbic acid or 2.5% HCl or 2.5% H2SO4 or 2.5% H2O2 5.0% ascorbic acid or 5.0% HCl or 5.0% H2SO4 or 5.0% H2O2 ultrapure water (mL)

0 0 0

10 0 0

10 0 5

10 0 0

0 5 0

10 5 0

10 5 5

10 5 0

0

0

0

5

0

0

0

5

20

10

5

5

15

5

0

0

2.7.2. Fatty acid extraction and fatty acid methyl esters (FAMEs) preparation Total lipids were extracted in triplicates by the method of Saini and Keum (2016) with minor modifications. Briefly, Thylakoid membrane solution (2 mL) prepared above was transferred into a 10 mL falcon tube and homogenized with a mixture of chloroform and methanol (1:1 v/v). After homogenization, samples were centrifuged at 5000 g (10 min at 4 °Ctemperature), and the supernatants were pooled in separating funnel and partitioned with 2 mL sodium chloride (0.76% NaCl; w/w). Lower organic (chloroform) phase was collected into the pre-weighted glass tube, dried in a rotary vacuum evaporator, and total lipid content was then determined gravimetrically. From the crude lipids, FAMEs were prepared according to procedures described in spinach (Li et al., 2013a,b) with minor modifications. Briefly, the concentrated asparagus lipids prepared above were re-dissolved in 1.0 mL methanol solution containing 5% concentrated sulphuric acid, vortexed for 1 min, and placed in a 90 °C water bath for 1 h in a tightly capped vial to induce derivatization. Afterwards, samples were removed and allowed to cool down to room temperature. Then, 1.0 mL of deionised water was added to terminate the derivatization reaction. FAMEs were subsequently extracted with 2 mL of cyclohexane, vortexed and centrifuged at 3000 g (10 min at 4 °C temperature), then the supernatant was collected for FAMEs analysis.

The final concentrations of HCl, H2SO4, ascorbic acid, H2O2 and cholesterol in the solutions were only 1/4 of initial adding concentrations.

tube 2–4, and the latter was used as A0 of test tube 6–8. The absorbance A1 and A2 of H2SO4 or HCl treatment were measured at 20 min, the absorbance A3 and A4 of ascorbic acid or H2O2 treatment were measured at 10 min and 60 min, respectively. Degradation rate of chlorophyll was calculated using chlorophyll degradation(%) = 100*(AnA0)/A0. 2.5.2. Stability analysis of chlorophyll in vitro According to Table 1, tube 2 and 5 were placed at room temperature (25 ± 1 °C). Absorbance spectrums of tube 2 and 5 were measured by using a UV–vis spectrophotometer, scaning from 500 to 700 nm wavelength range. The absorbances of sample at different wavelengths was measured at 0, 24, 48, 72 and 96 h during the storage of fresh asparagus. 2.6. Chlorophyll-protein binding capacity measurement

2.7.3. GC–MS analysis of FAMEs FAMEs were analyzed using an Agilent 7890A gas chromatograph (Agilent, Palo Alto, CA) interfaced with a 5975C mass spectrometer, an Agilent DB-17 column was used with Helium (He, flow rate was 20 mL min−1) as carrier gas. Ion source was set up at 230 °C, while the injector temperature was set at 250 °C for split injection at a split ratio of 30:1. The inject volume was 1 μL. The initial column temperature was maintained at 120 °C for 2 min before it was increased by 15 °C min−1 to 250 °C and kept isothermal for 5 min. The ionisation potential of the mass-selective detector was 70 eV and the scan range was 30–450 amu. Analyses were performed in triplicate, identification of compounds were achieved by a mass spectra database search (NIST Library).

The binding capacity of chlorophyll-protein was measured according to the methods described by Wang et al. (2004) with some modifications. 8.0 g sodium sulfate (Na2SO4) and 1.0 g magnesium carbonate (MgCO3) were added to 2.0 g fresh weight asparagus. Each sample was ground, added with 10 mL 0.4% ethanol-petroleum ether mixed solvent (1:1), and centrifuged at 8000 g for 10 min, then the liquid was collected. Chlorophyll content was determined by OD645 and OD663. M value was calculated. The residue was added with 10 mL 95% ethanol for 1 h, and chlorophyll content was measured. X value was calculated. Chlorophyll-Protein binding capacity was calculated by the following formula:

Y=

100X M+X

2.8. Statistical analysis

Where Y was chlorophyll-protein binding capacity(not extracted chlorophyll, %); M was total extracted chlorophyll (μg) and X was residual chlorophyll (μg) after being extracted.

All experiments were conducted in triplicate and the average values with standard deviation were used in the analysis. All data were evaluated by one-way analysis of variance (ANOVA) using SPSS 19.0 (SPSS Inc., Chicago, IL, USA), with mean separation at P ≪ 0.05 level of significance by paired samples t-test (P ≪ 0.05).

2.7. GC–MS analysis

3. Results and discussion

2.7.1. Preparation of thylakoid membrane Thylakoid membrane solution extracted from fresh asparagus was selected to investigate the components and functions of thylakoid membrane in asparagus. Thylakoid membrane solution was isolated according to the method of Wang et al. (2016). All procedures were performed at 4 °C, and the sample was kept in an ice bath throughout the whole process. Briefly, 50 g of fresh asparagus was homogenized with 50 mL pre-cooled extraction solution (pH 8.0, containing 0.1 M sucrose, 0.2 mM NaCl, 50 mM Tricine-HCl, and 2.5% PEG) for 1 min to no obvious green debris. The asparagus puree was centrifuged at 4000g for 10 min after filtering through eight-layer gauze, and the chloroplastcontaining pellet was suspended with ten fold volume of pre-cooled washing solution (extraction solution without PEG) and then centrifuged at 3000 × g for 5 min to get precipitation. The precipitation

3.1. The effect of cholesterol on chlorophyll content of asparagus spear The total chlorophyll content of all samples decreased during the storage period (Fig. 1A and B). Nevertheless, cholesterol coating or absorption treatment delayed the decrease of the total chlorophyll content. At the 30th day, the total chlorophyll content of 0.2 g L−1 and 0.5 g L−1 cholesterol-treated samples reduced by 36.1% and 30.2%, while control groups reduced by 58.7% and 56.3%, respectively, and cholesterol-treated samples significantly (P ≪ 0.01) retarded the loss of chlorophyll than that of the control samples. Data presented in our study indicates that cholesterol had a beneficial effect on the reduction of color changes in asparagus. This results were consistent with our 38

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Fig. 1. Effect of cholesterol absorption treatment (A) and coating treatment (B) on chlorophyll contents of green asparagus stored at 4 ± 0.5 °Cfor 30 d. Each data point is the mean of three replicate samples. Vertical bars represent the standard error of the mean.

degradation after 60 min. Similarly, the addition of cholesterol failed to inhibit chlorophyll degradation caused by adding H2O2 (Tables 2B, 2C, ). Meanwhile, H2O2-treated samples exhibited a less loss of chlorophyll than that of ascorbic acid-treated samples, which indicated that the chlorophyll exhibited a better stability to H2O2 than to ascorbic acid. Similar results were also observed by Zhang et al. (2010).

previous research that cholesterol significantly reduced the loss of chlorophyll content and maintained post harvest quality of green asparagus (Wang et al., 2017). 3.2. The effect of cholesterol on redox ability of chlorophyll Cholesterol was added combining with H2SO4/HCl, or H2O2 solution in chlorophyll solutions. When H2SO4 (oxidizing acid) or HCl (acid) were added into the chlorophyll solutions, respectively, the colors were converted to brown and precipitates were formed in the solutions. H2SO4 and HCl were not statistically different in the rate of chlorophyll degradation, but they caused color loss significantly faster than the control samples (P ≪ 0.01) with or without the aid of added cholesterol (Table 2A). The chlorophyll exhibited a poor stability to acidity. It is known that the magnesium ion in a porphyrin ring is displaced by hydrogen ions and pheophytin results in acidic conditions (Laborde and Von Elbe, 1994a; Von Elbe and Schwartz, 1996), and replacing the magnesium ion in the porphyrin ring produces a regreening effect. This observation was also in accordance with the research of Gunawan and Barringer (2000), who reported that the color change of broccoli was due to the acid converting chlorophyll to pheophytin, by replacing the magnesium with hydrogen. Senklang and Anprung (2009) reported conflicting results about the pH effect on the green color of pandan leaf extracts; they found that the formation of green color was lower at pH 7 and 8, and highest at pH 5, perhaps the pH effect can have different results with different raw materials and extracts. As was shown in Table 2, the addition of cholesterol significantly retarded chlorophyll degradation in ultrapure water group (P ≪ 0.01). Cholesterol, a component of biological membranes, maintained the integrity of cell membrane, which may prevented the degradation of chlorophyll. However, ascorbic acid significantly (P ≪ 0.01) accelerated the degradation of chlorophylls compared with H2SO4 or HCl treatment with or without the aid of cholesterol, this could be due to its acidity and reductibility. Therefore, cholesterol failed to maintain the green color of the chlorophyll in ascorbic acid. When compared with the control samples, H2O2 (oxidant) inhibited the degradation of chlorophyll with no added cholesterol during 10 min (P ≪ 0.05), but no significant differences were observed in the rate of chlorophyll

3.3. The effect of cholesterol on stability of chlorophyll in vitro Effects of cholesterol on chlorophylls absorption spectra were shown in Fig. 2 and Fig. 3. The chlorophyll absorption spectrum of the combined complexes was very similar to that of the chlorophylls in vitro (Fig. 2), this may be attributed to the suspension formed between cholesterol and chlorophyll. When cholesterol was added to chlorophyll solution, the absorbance at 663 nm had no change basically in 5 min, but a slight decline was observed later due to the dilution of cholesterol. There was no evidence that cholesterol chemically reacted with chlorophyll, and further investigate indicated that cholesterol could inhibit chlorophyll degradation in vitro (Fig. 3). The chlorophyll solution was presented with the red shifting of absorption maxima, when it was placed in the dark for 4 days at 25 °C. Interestingly, the addition of cholesterol dramatically inhibited this change during 4 days, and retarded a reduction in absorbance of chlorophyll in contrast with that of the control group. The maximum absorption value of the control remained 50.8% of initial day, while cholesterol treatment remained 70.5% (Fig. 3A and B). It was clearly indicated that cholesterol effectively delayed chlorophyll degradation in vitro, which was consistent with the conclusion of Section 3.2. 3.4. The effect of cholesterol on chlorophyll-protein binding capacity Effect of cholesterol on chlorophyll-protein binding capacity was presented in Table 3. The binding capacity of chlorophyll-protein were declined during the storage of green asparagus, and the trend showed that the binding capacity of chlorophyll-protein significantly (P ≪ 0.01) increased after cholesterol treatments compared to that of untreated sample at the storage of 3 d. Compared with the initial day, chlorophyllprotein binding capacity of cholesterol treatment just declined by 3.4% while the control samples decreased by 20.0%. which suggested that

Table 2A The effect of cholesterol on chlorophyll degradation in HCl or H2SO4 solution (20 min) (degradation rate, %). Treatments Control Cholesterol 1

water 10.21 ± 1.06 3.31 ± 0.85b

2.5% HCl a1

2.5% H2SO4

5.0% HCl a

a

45.42 ± 2.23 33.58 ± 1.56b

46.01 ± 2.21 33.67 ± 1.88b

5.0% H2SO4 a

43.35 ± 1.95 37.50 ± 1.95b

Results were expressed as the averages of triplicate samples with means ± SD (n = 3) and significant differences (p ≪ 0.05) are indicated with different letters.

39

44.69 ± 2.04a 39.44 ± 1.98b

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Table 2B The effect of cholesterol on chlorophyll degradation in ascorbic acid or H2O2 solutions (10 min) (degradation rate, %).

1

Treatments

Water

2.5% ascorbic acid

5.0% ascorbic acid

2.5% H2O2

5.0% H2O2

Control Cholesterol

8.32 ± 1.24a1 0.78 ± 0.22b

44.12 ± 2.22b 50.19 ± 2.50a

54.36 ± 2.43a 53.79 ± 1.55a

3.03 ± 1.01a 3.31 ± 0.50a

5.08 ± 1.31a 4.24 ± 0.56a

Results were expressed as the averages of triplicate samples with means ± SD (n = 3) and significant differences (p ≪ 0.05) are indicated with different letters.

Table 2C The effect of cholesterol on chlorophyll degradation in ascorbic acid or H2O2 solutions (60 min) (degradation rate, %). Treatments Control Cholesterol 1

water

2.5% ascorbic acid a1

16.84 ± 1.28 5.06 ± 0.35b

5.0% ascorbic acid

b

a

52.07 ± 2.25 60.00 ± 2.03a

55.56 ± 2.33 59.25 ± 2.45a

2.5% H2O2

5.0% H2O2 b

14.27 ± 1.36 20.29 ± 1.54a

16.32 ± 1.42b 22.36 ± 1.63a

Results were expressed as the averages of triplicate samples with means ± SD (n = 3) and significant differences (p ≪ 0.05) are indicated with different letters. Table 3 The effect of cholesterol on Chlorophyll-Protein binding capacity.

1

Group

Chl-Pro binding capacity (%)

0d Control 3d Cholesterol 3d

72.87 ± 3.45a1 58.30 ± 3.24b 70.37 ± 2.68a

see the legend in Table 2.

chlorophyll-protein bond, which may be caused by the disruption of thylakoid membrane. To our knowledge, excessive ROS causes peroxidation of membrane lipids is the main factor of chlorophyll degradation (Esfandiari et al., 2010). Cholesterol, a component of biological membranes, maintained the integrity of cell membrane, which may prevented the peroxidation of membrane lipids, and inhibited the degradation of chlorophyll. In order to prove this result, further investigations about the changes in thylakoid membrane during the storage of cholesterol-treated asparagus were necessary.

Fig. 2. Effect of cholesterol on the absorbance of chlorophyll solution at 5 min.

cholesterol played a protective role in maintaining chlorophyll-protein binding capacity. In general, Chlorophyll-protein complexes can improve the stability of chlorophyll and inhibit the degradation of chlorophyll (Mayfield and Huff, 1986; Peng et al., 2013). In green plants, chlorophylls are embedded within thylakoid membrane, and 80% of them are attached by non-covalent bonds to proteins existing in the form of chlorophyll-protein complexes (Wang et al., 2016). As the important components of thylakoid membrane, chlorophylls and chlorophyll-protein complexes play a key role for retention of thylakoid membrane function, especially for chlorophyll-protein complexes, since they are involved in light absorption and excitation transmission of thylakoid membrane (Kirchhoff et al., 2008). The decline of chlorophyll-protein binding capacity represented the cleavage of

3.5. The effect of cholesterol on fatty acid composition of asparagus thylakoid membrane The content of unsaturated fatty acids represents the fluidity of thylakoid membrane and further affects chlorophylls and chlorophyllprotein complexes (Sharkey, 2000). The fatty acid composition expressed as percentage of the total fatty acid content of green asparagus was shown in Table 4. It mainly consisted of tridecylic acid (13:0), palmitic acid (16:0), linoleic acid (18:2) and linolenic acid (18:3) (Figures not shown). For all the samples, the contents of 18:2 and 18:3 were higher than those of other fatty acid components at the initial day,

Fig. 3. The absorption spectra of chlorophyll solution of the control (A) and cholesterol treatment (B) stored at 25 ± 1 °C for 4 d.

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Table 4 The effect of cholesterol on thylakoid membrane fatty acid composition of green asparagus (at ambient temperature).

1

Storage days

palmitic acid* (%) 16:0

tridecylic acid* (%) 13:0

linoleic acid* (%) 18:2

linolenic acid* (%) 18:3

0d Control 3d Cholesterol 3d

21.19 ± 2.13c1 50.35 ± 3.52a 36.97 ± 2.45b

1.84 ± 0.12b 5.17 ± 0.54a 4.63 ± 0.34a

41.43 ± 1.86a 26.40 ± 1.45c 35.99 ± 2.02b

35.54 ± 2.00a 12.92 ± 1.22c 22.41 ± 1.45b

see the legend in Table 2.

which represented the unsaturation degree of fatty acids. Unsaturation degree of fatty acids can affect the stability of thylakoid membrane by changing the fluidity of membrane (Wang et al., 2016). Hence, variations in the structure, components, and functions of thylakoid membrane can be reflected by changes in the contents of 18:2 and 18:3. At the 3 d of storage, the contents of 18:2 and 18:3 in cholesterol-treated asparagus were decreased to 35.99% and 22.41%, respectively, which were significantly (P ≪ 0.01) higher than that of the control samples (26.40% and 12.92%, respectively). However, the contents of 13:0 and 16:0 in untreated samples were higher compared with those of cholesterol-treated samples at 3 d. This phenomenon indicated that the fluidity and stability of thylakoid membrane could be better retained after cholesterol treatment compared with those of the control samples, which was similar to the finding of Dong et al. (2016), who reported that stigmasterol, as one of sterols, probably maintained the integrity of cell membrane, which prevented a quick superficial dehydration and delayed asparagus shriveling and thus quality deterioration. Cholesterol played a key role in maintaining the function of anterior fatty zone and its integrity (Qian et al., 2014), which may explain the mechanism of cholesterol inhibiting the chlorophyll degradation in postharvest green asparagus.

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4. Conclusions The phenomenon of cholesterol inhibiting chlorophyll degradation of postharvest green asparagus during low temperature storage was verified in the study. Asparagus treated with cholesterol significantly exhibited high chlorophyll contents over a chilled storage period of 30 days. Cholesterol was effective for reducing chlorophyll degradation in a neutral or acidic solution, it also maintained the stability of chlorophyll-protein complexes and inhibited the decline of unsaturated fatty acid in thylakoid membrane of asparagus. These results indicated that cholesterol alleviated the attack of acid on chlorophyll, decreased the acidic removal of Mg+ in the chlorophyll molecule, which contributed to repress chlorophyll degradation and stay green. Meanwhile, cholesterol inhibited the decline of unsaturated fatty acid in thylakoid membrane, which maintained the integrity and stability of thylakoid membrane, then retarded chlorophyll detaching from binding protein in thylakoid membrane. This might be the explanations of cholesteroleffect on better color retention of green asparagus. Cholesterol was of great benefit to keep asparagus green, which could be used as a new type of additive applied in postharvest fruit or vegetable. Moreover, some foods such as egg yolk rich in cholesterol may also be used as a safe preservative in future. Acknowledgments This work were supported by Non-profit Applied Research Project of Zhejiang Province, P.R. China (GN18C200026), and First-class Discipline of Food Science and Engineering of Zhejiang Gongshang University (2017SIAR204). References Acimovic, J., Rozman, D., 2013. Steroidal triterpenes of cholesterol synthesis. Molecules 18, 4002–4017. An, J.S., Zhang, M., Lu, Q.R., Zhang, Z.G., 2006. Effect of a prestorage treatment with 6-

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