Effects of ethephon on ethephon residue and quality properties of chili pepper during pre-harvest ripening

Abstract

The application of ethephon was investigated to examine its effects on both ethephon residue and quality properties of chili peppers during pre-harvest ripening with the goal of facilitating maximum commercial harvest along with improving color and flavor. A single ethephon treatment significantly increased L* and a* values and capsanthin concentration, while decreased total chlorophyll contents. Moreover, ethephon treatment induced significant promotion of capsaicin synthesis and reduction of soluble sugar content. While repeated treatments did not increase the total capsaicin content, and the consumption of soluble sugar was accelerated. Additionally, the maximum ethephon residue in chili pepper after ethephon treatment was 21.18 mg kg⁻¹, which is lower than the permissible residue level of 50 mg kg⁻¹ for chili peppers. The ethephon residual decreased with prolonging harvest time of chili peppers. The effects of ethephon treatment on different types of chili peppers were variable. The results of this study indicated that ethephon could hasten the ripening process and increase the quality of chili peppers.

Introduction

Chili pepper (Capsicum annuum L.) is an extremely important seasoning crop in China. China is the largest producer of chilies in terms of acreage and yield in the world. However, the fruits of pepper do not mature at the same time and need to be harvested in batches, so the harvest of mature peppers becomes difficult and the cost is therefore increased. Pre-harvest and post-harvest treatments may augment the maturity proportion of red chili peppers by inducing fruit ripening. Some pre-harvest studies in particular showed that ethylene is involved in the color synthesis of fruit (Ban et al. 2007; Mao and Motsenbocker 2002; Worku et al. 1975; Zhang 2012).

Ethephon (2-chlorethylphosphonic acid) can release ethylene assimilated by fruit and vegetable crops, and eventually facilitate their growth processes (Korsak and Yongseo 2010; Park et al. 2006; Rupinder and Upendran 2009). It has been reported that ethephon plays a role in improving plant quality and facilitating the harvest of plant (Mahajan et al. 2010) and accelerating the color development of fruit (Yang et al. 2009; Zhou et al. 2010). Ethephon was first discovered in 1970s and is one of the most important phytohormones in agriculture due to its excellent biological action. Research showed that ethephon has both acute and sub-chronic toxicities, and can also induce mutations in non-target organisms as well as damage to the environment (Wen-Hui et al. 2006). The main mode for ethephon poisoning is via ingestion (via oral cavity), while rarer cases involve absorption through the skin. Ethephon exposure in strong acidic solutions causes significant irritation and can corrode human tissues. It can enter the gut and decompose into ethylene, resulting in a strong narcotic effect after entering the central nervous system. Ethephon has been reported to exist in maize crops, with the permissible residue level for ethephon in maize set at 0.5 mg kg⁻¹ in China and Japan and 0.05 mg/kg in European Union.

The ethylene concentrations are low in unripe climacteric fruits such as peppers (Mao and Motsenbocker 2002; Worku et al. 1975), tomatoes (Ethephon and Acid 1988), bananas (Vendrell 1985) and peaches (Zhang et al. 2012) but increase during plant ripening. While the ethylene production in red chili pepper is higher than other ripening fruits, the amount is not high enough to induce autocatalysis. The ethylene regulation processes in most plans are induced by 1 mg L⁻¹ or lower concentrations of ethylene (Sawamura et al. 1978). For example, an ethylene concentration of 0.025–0.5 mg L⁻¹ can activate the climacteric function of lemons (Burg and Burg 1962), while Biles et al. reported that a particular concentration of ethylene can induce the ripening of pepper (Biles et al. 1993).

The aim of this study was to investigate the effects of ethephon on the ripening and quality properties (ethephon residue, color, capsanthin content, total chlorophyll and capsaicin contents, and soluble sugar content) of chili peppers during pre-harvest, facilitating the maximum commercial harvest along with improving color and flavor. In addition, a relatively safe application of ethephon for chili peppers was also explored.

Materials and methods

Materials

The chili pepper field used in this study was an agricultural experiment field graciously offered for experimentation by Southwest University (Chongqing, China) and the ethephon was provided by Hu-hui Agricultural Science and Technology Co. Ltd. (Shanghai, China). Methyl alcohol, tetrahydrofuran, acetone, anthranone, vitriol and glucose were procured from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Capsaicin and dihydrocapsaicin were purchased from Sigma (St. Louis, MO). All chemicals and reagents used in this study were of analytical grade.

Field experiment of chili pepper

Eight chili experimental plots were selected from the agricultural experiment field of Southwest University for the study. These eight plots were numbered from one to eight, with six plots designated for ‘LeDeHong’ and the remaining two designated for Pod pepper and Capsicum frutescens. Twenty plants yielding a uniform number of chili peppers were selected in each of 10 experimental plots. Different concentrations of ethephon solutions were prepared from a stock solution of 10 g L⁻¹. All of the experiments began at the first onset of fruit coloration. Randomly, four sets of six ‘LeDeHong’ plots were each sprayed once with 0, 2000, 5000, or 8000 mg L⁻¹ of ethephon. Additionally, two sets of six ‘LeDeHong’ plots were sprayed with 5000 mg L⁻¹ of ethephon, one for twice and the other for three times. ‘Pod pepper’ and ‘Capsicum frutescens’ were each sprayed once with 5000 mg L⁻¹ of ethephon.

Chili pepper sample collection

In this experiment, all the chilies sprayed with various treatments of ethephon were harvested at 7 pm each day, taken to the laboratory and dried. The chili samples were then ground using a high-speed pulverizer and filtered through a 40-mesh sieve to achieve paprika for the following physical and chemical analyses.

Ethephon residues

Ethephon residue was determined by gas-chromatography (Agilent Technologies, Guangzhou, China) as described previously (Dong et al. 2015). Briefly, 5 g of chili pepper samples were added to a 15 mL headspace containing 1 mL acetone and 3 mL of 60% aqueous potassium hydroxide solution and magnetically stirred at medium speed for 2 h at 70 °C. An Agilent 1260 gas-chromatograph was used for GC analysis, with chromatographic separation performed on a 30 m × 0.25 mm, df 0.25 μm DB-1701 capillary column. The inlet temperature was 130 °C with no shunt. The following GC oven temperature program was applied: 40 °C for 4 min, increased to 180 °C at a speed of 100 °C min⁻¹, and 180 °C held for 7 min. The detector used was a flame ionization detector (FID) at 200 °C. Nitrogen was used as the carrier gas at a flow rate of 1 mL min⁻¹ at the following settings: Compensated gas: nitrogen, 15 mL min⁻¹; hydrogen flow rate: 30 mL min⁻¹; Air velocity: 300 mL min⁻¹.

The mass concentration of ethephon was within 0.500–100.0 mg L⁻¹, and its corresponding peak area showed a linear relationship. The linear regression equation was Y = 23621x − 15,330 (Y: peak area, x: mass concentration of ethephon), and the correlation coefficient was 0.9901. According to the 3S/N detection limit of the threefold SNR calculation method, the result is 0.010 0 mg kg⁻¹.

Color analysis

Dried chilies were selected randomly, with three samples analyzed per test group and each sample measured 7 times. The results were expressed as averages.

Total chlorophyll content

The total chlorophyll content was determined as described previously (Patel et al. 2019). Briefly, 1.0 g of fresh chili pepper was homogenized with 20 mL of 80% (v/v) acetone in a centrifuge tube using a homogenizer (IKA Works Guangzhou, Guangzhou, China), followed by centrifugation at 3500g for 20 min. The absorbance of the supernatant was measured at wavelengths of 645, 652, and 663 nm. The total chlorophyll was determined as follows:

$$\text{Total chlorophyll}=\frac{\left(12.7A_{663\mathrm{nm}}-2.69A_{645\mathrm{nm}}\right)\times Volume\times DilutionFactor}{FreshWeight\times 1000}$$

Capsanthin content

Capsanthin was extracted by following the method described previously (Barros et al. 2016). Briefly, 5 mL of acetone were added to 5.0 g pericarp tissue sample, shaken and left to stand for 10 min. The mixture was then centrifuged at 3000g for 10 min at room temperature, and the extract was transferred to a clean tube. Samples were then re-extracted twice with 5.0 mL of acetone each time, and the extracts were pooled together and made up to 100 mL with acetone. The absorbance of the sample solution (A) was measured at 460 nm with acetone as a reference along with the absorbance of the standard colorimetric solution (As) at 460 nm. The standard colorimetric solution was made with 0.3005 g potassium dichromate and 34.96 g (NH₄)₂SO₄·CoSO₄·6H₂O dissolved in 1000 mL of sulfuric acid (1.8 mol L⁻¹). ASTA = A × 164 × 0.600/AS/W (mg kg⁻¹), where ASTA is color value and W is sample weight.

Capsaicin content

The amended procedure of Zhang et al. (2013) was applied for determination of capsaicin content in chili pepper. Briefly, 25 mL of methanol-tetrahydrofuran (1:1 v/v) were added to 2 g of ground chili, and the extract was ultrasonically extracted for 30 min twice at 250 W and 45 °C. After filtered through a 0.45 nm organic membrane, the filtrate was analyzed using HPLC. The column used was an Agilent ZORBAX Eclipse XDB-C18 (150 × 4.6 mm). Detection was performed using a variable wavelength UV detector (Agilent Technologies) set at 280 nm. The isocratic mobile phase was methanol/water (75:25 v/v) with a flow rate of 0.8 mL min⁻¹ at 10 μL and 30 °C. Peaks corresponding to capsaicine and dihydrocapsaicin were sorted according to their respective retention times and quantified, respectively.

Soluble sugar content

The soluble sugar content of chili pepper was measured according to the anthranone-sulfuric acid assay described by Liu et al. (2018). Briefly, 1 g of anthrone was prepared immediately prior to use by dissolving anthrone in concentrated sulfuric acid (96% v/v). 2 mL of the prepared anthrone reagent were added to 2 mL aliquots of samples (or dextrose solution), dispensed in a test tube and then incubated at 100 °C for 15 min. Plates were allowed to cool at room temperature for 15 min before absorbance readings were measured at 620 nm.

Statistical analysis

Each sample was measured for a minimum of three replicates and statistical analysis was performed using SPSS 20.0 software. All results are presented as the mean ± standard error, unless otherwise stated. The analysis of variance (ANOVA) was used for variance analyses. Data falling into normal distribution were analyzed using the method of multiple comparisons (Duncan), while data that did not fall into normal distribution were analyzed using the Kruskal–Wallis test. Pearson product-moment correlations were used to determine the representative relationships at < 0.05 and < 0.01 significance levels. Graphical processing was performed using Origin 8.6 software.

Results

Ethephon residue

Ethephon residue analysis was performed after various ethephon spray treatments on chili peppers (Fig. 1). Chili pepper innately had no ethephon present, so there was no ethylene residue detected in untreated chili pepper (Fig. 1a). After ethephon spray treatments, the ethephon residual amount in chili decreased as time elapsed before picking. The permissible residue level of ethephon in ‘LeDeHong’ was determined to be 21.18 mg kg⁻¹ and 18.91 mg kg⁻¹ for ethephon spray concentrations of 5000 mg L⁻¹ and 2000 mg L⁻¹, respectively, on the first day, and the ethephon residual of 5000 mg L⁻¹ group was always higher than the 2000 mg L⁻¹ group (p < 0.05). The ethephon residual in chili pepper significantly increased with increasing amount of spraying treatments (Fig. 1b). When the chili pepper was sprayed with ethephon for three times, the ethephon residual amount in chili reached a maximum of 23.52 mg kg⁻¹. However, as picking time extended, the amount of ethylene residue in each group decreased proportionally. Moreover, the ethephon residue in different types of chili pepper decreased during the whole harvest period, with the ‘Pod pepper’ having significantly higher ethephon residue than the other two types by day 5 (Fig. 1c). An additional ethephon spray test was performed using an ethephon concentration of 8000 mg L⁻¹. We observed that the chili fruit dropped, and its stem withered and died by the third day, indicating that the concentration of ethephon should not be too high when applied to chili peppers.

Fig. 1.

Effects of ethephon treatment on ethephon residue of a ethephon concentration, b the number of pesticide applications and c different chili pepper varieties during ripening. Data represent the mean of three independent measurements ± standard error. Bars belonging to the same harvesting time connected by different letters are significantly different

Ethephon is less harmful in the human body since the maximum residue of ethephon in chili in this study (21.18 mg kg⁻¹) was far below the permissible residue level of ethephon in chili pepper from Codex Alimentarius. The standard specifies that the permissible residue level of ethephon is 50 mg kg⁻¹ for dried chili pepper and 5 mg kg⁻¹ for fresh chili pepper. Due to this, a standardized treatment regarding concentration and number of sprays of ethephon was required. The optimal concentration of ethephon was determined to be 2000 mg L⁻¹ with once or twice of spraying according to the ethephon residue results.

Color changes

The trends observed for the brightness value (L*) and the “redness” value (a*) of the chili pepper were used to reflect the chili color after spraying treatment.

As shown in Fig. 2, the values of L* and a* increased with picking time. At later picking time, the L* value for chili sprayed with 5000 mg L⁻¹ of ethephon was higher than those of both the 2000 mg L⁻¹ concentration and the control groups. The L* and a* values in control group increased slightly (p < 0.05) and the color change trend of different chilies was stable (Fig. 2a). The L* value of chili sprayed three times was higher than those sprayed once or twice (Fig. 2b), and there were significant differences observed in the L* value among the three different chili species. As the picking time extended, the differences among the three chili types increased. The L* value of the ‘LeDeHong’ chili was always the highest and the ‘Pod pepper’ was always the lowest (Fig. 2c). The a* value for all the treatment chili groups increased with picking time, and the change trends of a* value observed for different spraying concentrations, times and species were similar to those observed for L* value.

Fig. 2.

Effects of ethephon treatment on color change of a ethephon concentration, b the number of pesticide applications and c different chili pepper varieties during ripening. Data represent the mean of three independent measurements ± standard error

Capsanthin content

As shown in Fig. 3, the capsanthin content in both 2000 mg L⁻¹ and 5000 mg L⁻¹ of ethephon spray groups increased significantly as the harvest time extended (p < 0.05), and the capsanthin content after ethephon treatment was significantly higher than that of the control (p < 0.05, Fig. 3a). As for the effect of spraying times on the capsanthin content, there was no significant difference was observed between the chilies sprayed twice and three times during the later picking time (Fig. 3b, p > 0.05), but the capsanthin content after a single spray treatment was significantly lower than that of the other treatment groups. Furthermore, the capsanthin content for all treatment chili groups increased with picking time, and the capsanthin content of the ‘LeDeHong’ chili was always the highest and that of the ‘Pod pepper’ was always the lowest (Fig. 3c).

Fig. 3.

Effects of ethephon treatment on Capsanthin content of a ethephon concentration, b the number of pesticide applications and c different chili pepper varieties during ripening. Data represent the mean of three independent measurements ± standard error

Total chlorophylls content

The total chlorophyll content (sum of chlorophyll a and chlorophyll b) showed relatively larger changes during ripening of the tested fruit (Fig. 4). The discrepancies in total chlorophyll content of chili were influenced by both different treatment conditions and plant variety. As shown in Fig. 4a, the level of chlorophyll decreased continuously in treated ‘LeDeHong’ during ripening, and significant decrease of chlorophyll content occurred with treatments of 2000 and 5000 mg L⁻¹ of ethephon. The chlorophyll content in the control chili pepper decreased significantly at day 4, and remained lower than that in the treated fruit until the end of the experiment. The chlorophyll contents of the chili pepper treated twice or three times were significantly lower than those of the single treatment peppers, and there was no significant difference in chlorophyll content between the twice and three times of treatments (Fig. 4b). The chlorophyll content of all three varieties of chili pepper decreased after treated, but there was a significant difference in the degradation rate of chlorophyll among different varieties, with the chlorophyll content of the ‘LeDeHong’ type dropping the fastest (Fig. 4c).

Fig. 4.

Effects of ethephon treatment on total chlorophylls content of a ethephon concentration, b the number of pesticide applications and c different chili pepper varieties during ripening. Data represent the mean of three independent measurements ± standard error

Total capsaicine content

Capsaicine and dihydrocapsaicin are two major constituents in chili peppers that promote their spiciness. As shown in Fig. 5a, total capsaicine content increased significantly as the ethephon spray concentration increased (p < 0.05), resulting in not only the chili peppers’ redness, but also an increase in the chili pungency. However, the effect of spraying times did not appear to be significant, as there was no significant difference between the chilies sprayed twice and those sprayed three times during the later picking time (Fig. 5b, p > 0.05). Considering these findings, we concluded that an increased chili pepper spiciness could be assured after once or twice of spray, and there was no need to spray more repeatedly. As indicated in Fig. 5c, the total capsaicine content of the chilies increased gradually as the picking time extended, and significant differences in the total capsaicine content among the three kinds of chilies were observed at the same picking time. While the spiciness of ‘Capsicum frutescens’ was the strongest, the total capsaicin content in its fruit was minimally impacted after ethephon treatment.

Fig. 5.

Effects of ethephon treatment on total capsaicine content of a ethephon concentration, b the number of pesticide applications and c different chili pepper varieties during ripening. Data represent the mean of three independent measurements ± standard error. Bars belonging to the same harvesting time connected by different lowercase are significantly different and different harvesting times on the same cultivars by different majuscules are significantly different

Soluble sugar content

In the control group, the soluble sugar content slightly increased during the first 2 days and then slightly decreased, while the ethephon-treated chili pepper showed a sustained decrease and a significantly lower soluble sugar content than the control fruit. There was no significant difference in soluble sugar content between different treatment groups except at day 4 (Fig. 6a). The peppers treated once showed a higher soluble sugar content than those of the other two groups except on days 0 and 1 (p < 0.05), and there was no significant difference observed between peppers treated twice and three times except on day 3 (Fig. 6b). The effect of ethephon treatment on soluble sugar content in different types of chili pepper was significant (Fig. 6c) with the soluble sugar content of the treated ‘Capsicum frutescens’, while being slightly higher than other chili peppers, decreasing by 56.46 mg kg⁻¹.

Fig. 6.

Effects of ethephon treatment on soluble sugar content of a ethephon concentration, b the number of pesticide applications and c different chili pepper varieties during ripening. Data represent the mean of three independent measurements ± standard error

Discussion

Color additives and flavoring agents from chili peppers are important ingredients for many food products. Chilies provide coloring substances for meat products, condiment mixtures, salad dressings and other processed foods. Chili peppers used for edible pigment are sold by unit weight, with their commercial value determined by the pigment content. By improving and promoting pigmentation of chili peppers, their commercial values would be increased (Worku et al. 1975). Capsanthin is the main carotenoid, accounting for 35% of total carotenoids (Curl 1962; Pepkowitz et al. 1944), and was first isolated and identified by Zechmeister (1927). The red coloring development of capsicum is related to many carotenoids (Curl 1962; Kim et al. 2016), and the ripening of green chilies to the red stage increases the carotene content by more than 30 folds. Several reports have demonstrated that the application of ethephon (2-chloroethylphosphonic acid) can both promote fruit ripening and maturation (Ampa et al. 2016; Ban et al. 2007; Kongsuwan et al. 2017; Mao and Motsenbocker 2002) and increase the color development and respiration of chilies (Krajayklang et al. 2000; Locascio and Smith 1977; Worku et al. 1975).

Chili fruits appear normal coloring when they are picked at or close to breaker stage, while ethylene treatment could promote the coloring of chilies. The color changes are generally induced by the degradation of chlorophyll, the production of capsorubin and capsanthin, and the esterification of phytoxanthin and carotenoid in chili peppers. In addition, other hormones and pigment precursors are also present, interacting with ethylene to change chili color from green to deep red. While earlier reports indicated that the cuticle of chili acts as a barrier to ethylene (Locascio and Smith 1977), Krajayklang et al. reported that ethylene could penetrate the cuticle since ethylene-treated chili has a high absorption level of internal ethylene (Krajayklang et al. 2000).

Ethephon has been reported to promote color development in chili fruit maturity (Batal and Granberry 1982; Worku et al. 1975). Ethephon treatment before the commercial harvest of chili resulted in increased red area percentage and lightness or chroma (Fig. 2) on the fruit surface. These color enhancements are mainly due to increased concentration of capsanthin (Fig. 3). Drops in chlorophyll content in unripe chili peppers were detected in both ethephon-treated and control chilies, with the chlorophyll content in the control group being lower than that in the ethephon-treated chilies (Fig. 4). The discrepancies of chlorophyll content observed in the chilies may be influenced by both the genotype(s) of plants (Fig. 4b) and their different environmental conditions (Fig. 4a, c). The reduction of chlorophyll content occurred in chloroplasts is due to photosynthesis and the action of enzymes such as chlorophyllase. We found the ethephon treatment substantially increased chlorophyll degradation in chili peppers as compared to those of untreated chilies, similar to the findings of Whale et al. (2008) who reported that chlorophyll degradation accelerates via ethephon treatments in the skin. Ethylene released by ethephon may increase the activities of chlorophyll degradation peroxidase and chlorophyllase, leading to a decrease of chlorophyll a concentration and an accumulation of chlorophyllide a.

The pungent flavor of chili pepper is mainly due to its capsaicin content. Capsaicin is formed by the condensation of short-chain fatty acids with vanillylamine, and is the best nutritional monitoring index for chili pepper quality (Patel et al. 2019). The function of dihydrocapsaicin is similar to that of capsaicin. In our study, the total capsaicine content increased with the maturity of the chili (Fig. 5a), and the ethephon treatments significantly affected the synthesis of capsaicine compared to the control group, except for the 2000 mg L⁻¹ ethephon group at day 1 after treatment (Fig. 5a). Repeated ethephon treatment resulted in no significant increase of total capsaicin in chili peppers compared to a single treatment (Fig. 5b). There are reports indicating that the content of capsaicin in chili peppers is mainly influenced by fruit size, age, growth and storage environment (Estrada et al. 2002). Capsaicin is the condensation product of fatty acids derived from leucine, valine and the intermediate vanillylamine of phenylpropanoid pathway (Aza-González et al. 2011), and its critical control point for synthesis is the capsaicinoid synthase (Han et al. 2013; Lang et al. 2010). Ethephon treatment may increase activities of peroxidase and polyphenol oxidase, which play important roles in the phenylpropanoid biosynthesis pathway, and may produce a significant effect on total capsaicin content of chili pepper.

Conclusion

According to our results, ethephon treatment may accelerate capsanthin production, total capsaicin accumulation and color values (L* and a*) while decreasing the total chlorophyll and soluble sugar contents. Moreover, the treatment produced a lower ethephon residue in chili pepper than the permissible residue level of Codex Alimentarius after 5 days. Additionally, repeated treatments showed no significant impact on color value, total chlorophyll or total capsaicin contents. The effect of ethephon treatment on the characteristics of different chili peppers was variable depending on the chili type. Interestingly, ethephon treatment not only caused chilies to turn red, but also increased their spicy flavor. The results of this study indicate that the recommended ethephon treatment should be a 5000 mg L⁻¹ concentration applied 1–2 times at the pre-harvest ripening stage of chili peppers.

Compliance with ethical standards

Conflict of interest

Authors have no conflict of interest to declare.