Comparison of physico-chemical characteristics of myrtle at different ripening stages
Article Category: Research Article
Published Online: Aug 06, 2020
Page range: 125 - 133
Received: Apr 09, 2020
Accepted: Jul 13, 2020
DOI: https://doi.org/10.2478/fhort-2020-0012
Keywords
© 2020 Xiaohua Kui et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
Myrtle (
The chemical composition of myrtle varies greatly depending on the location, origin and maturity (Xiao et al., 2014; Gu et al., 2018). Exploring the changes in chemical composition and antioxidant activity of fruits at different growth stages is of great significance for further study of the mechanism of action of myrtle. The study found that the contents of phenolic, flavonoids and anthocyanin, as well as antioxidant enzyme activities, varied depending on the developmental stages (Sun et al., 2018). The main reason was that the enzyme activity was different, and the temperature and moisture were also different at different periods. Zhao et al. (2018) studied the nutritional quality and aroma changes of strawberries at different developmental stages and found that contents of anthocyanins, soluble sugars and vitamin C increased with fruit development. Sugar is an important raw material for anthocyanin synthesis. When the sugar content of the fruit reaches a certain concentration, the fruit can have colour. Other studies pointed out that the content of C6-alcohols gradually decreased and the content of esters increased rapidly as the fruit matures, mainly due to the increase in ethyl acetate content (Luo et al., 2012). At present, there is less research on myrtle, focusing on its chemical composition and other nutritional qualities (Ye et al., 2019). Also, it is unclear how the stage of fruit development affects the chemical composition and antioxidant activity of myrtle. In this experiment, the changes in fruit quality and anthocyanin composition at different developmental stages of myrtle fruits were studied to provide a theoretical reference for the selection of high-quality fruit.
Myrtle fruits at different stages of development, namely green (G), colour break (C) and the mature (M), were collected in the orchard located at 150 m above sea level in Yanping Village, Fengshan Town, Huaan County, Zhangzhou City, Fujian Province. The average annual rainfall in the orchard was about 1,700 mm. The climate was characterized by mild and rainy. The plants were grown with 2.0 m spacing within rows and 3.0 m spacing between rows. The colour of all fruits in the green period was green, in the colour break period it was purple-red and in the mature period it was purple-black. Three replicates were set up for each period, with three experimental trees each, for a total of 27 myrtle trees. Each time a sample was collected, fruits with the same maturity, no insect pests and no mechanical damages. Fruits were collected homogeneously from the east, west, south and north of the experimental tree, and 90 fruits were collected at each stage. The collected samples were quickly taken to the laboratory, of which 15 were used for the determination of development indicators and the rest were immediately frozen in liquid nitrogen and stored at −80°C for the determination of the intrinsic quality of the fruit.
Data were collected on the vertical and transverse diameters of 15 fruits in each period, they were measured using a vernier calliper and the average value was calculated. Fruit shape index was obtained by proportioning the fruit length to the fruit width (Eskimez et al., 2019). The average fruit weight of 15 fruits in each period was determined by an electronic scale. Soluble solids content (SSC) was measured using a digital refractometer (mark and model information) and the results were given in percentages (Okatan, 2018).
The determination of the soluble sugar content was carried out in accordance with the method described by Zhu et al. (2019). Of note, 0.2 g of the sample was ground in liquid nitrogen, and the powdered sample was homogenized in 10 mL of water, then activated carbon was added and the sample was placed in a boiling water bath for 30 min (repeated twice). After cooling, the cells were centrifuged, and the supernatant was collected and made up to a volume of 25 mL with distilled water for further analysis. Of note, 0.5 mL of the supernatant was added to a 10 mL tube containing 2 mL of distilled water, and 0.5 mL of enthrone and ethyl acetate were added to the mixture. Five millilitres of concentrated sulphuric acid was slowly added to the mixture. The mixture was boiled in a water bath for 1 min, and after cooling to room temperature the absorbance was measured at 630 nm using a UV–VIS spectrophotometer.
The determination of the titratable acidity content was carried out in accordance with the method described by Wang and Huang (2015). 0.2 g of the sample was ground in liquid nitrogen, and 10 mL of water was added to the powdered sample, then an appropriate amount of activated carbon was added and the sample was boiled in a water bath for 30 min. After cooling, centrifugation was carried out, the supernatant was collected in another test tube and the process was repeated. The supernatant was aspirated and made up to 25 mL with distilled water for further analysis. The supernatant (10 mL) was placed in a 50 mL Erlenmeyer flask and 3–5 drops of phenolphthalein indicator were added. The mixture was then titrated with a 0.1 N NaOH solution until a pink colour that did not fade within 30 s appeared. The initial and final volumes were recorded to calculate the titratable acidity.
The sample was first ground in liquid nitrogen with the aid of a mortar and pestle, and 0.2 g of the powder was homogenised in 5 mL of 80% chilled ethanol. The extract was centrifuged at 11,000 rpm for 10 min at 4°C, and the supernatant was aspirated, collected and used for calculation of total phenol and flavonoids.
A simple modification of the method of Huang et al. (2009) was performed using a Folin–Ciocalteu colorimetric method to determine the total phenolic content of the sample. Of note, 0.5 mL of sample and different concentrations (20, 40, 60, 80 and 100 μg · mL−1) of standard gallic acid were added to a 5 mL tube containing 2 mL of distilled water and 0.25 mL of Folin–Ciocalteu’s previously added reagent (FCR) (Sigma Chemical, St. Louis, Missouri, USA) and shaken well. After about 5 min, 1 mL of 7% sodium carbonate (Na2CO3) was added to the mixture. The mixture was kept at 25°C for 120 min in the dark. The blank absorbance was then measured at 765 nm using a UV–vis spectrophotometer (TU-1810; Beijing Beifen Ruili Analytical Instruments (Group) Co., Ltd., China). Measurements were made in triplicate. The total phenol content data are expressed in milligrams of gallon acid equivalent (GAE) 100 g−1 berry skin fresh weight (FW). The equation obtained for the calibration curve of gallic acid was
The total flavonoid content was determined by colorimetry according to Huang et al. (2009). Of note, 0.5 mL of sample solution and 0.5 mL of quercetin standard solution (100, 200, 400, 600, 800 and 1,000 mg · mL−1) were added to a 10 mL tube containing 4 mL of water, and 0.3 mL of 5% NaNO2 was added to it. Then, the mixture was allowed to stand at room temperature for 5 min. Of note, 0.3 mL of 10% AlCl3 was added to the mixture. After 5 min, 2 mL of 1 M NaOH was added. The distilled water to a volume of 10 mL was added and mixed well. The absorbance was measured spectrophotometrically at 510 nm against a blank. The equation obtained for the calibration curve of quercetin curve was
Anthocyanin extractions were performed with a protocol similar to that of Fuleki and Francis (1968) with slight modifications. After grinding the sample with liquid nitrogen, 0.5 g of powder was taken, placed in a 50 mL centrifuge tube, added 10 mL of 2% hydrochloric acid methanol solution, ultrasonically extracted for 60 min, centrifuged at 4°C, 12,000 rpm for 10 min and collected the supernatant for extraction.
The method of determining anthocyanin is as follows: 2 mL of anthocyanin extract was drawn, diluted to 4 mL with water and shaken thoroughly. Using 2% methanolic hydrochloric acid as a control, the absorbance was measured at 520nm. To use spectrophotometry, the switch was first turned on and preheated for 20 min in advance. The appropriate wavelength according to the experimental requirements was chosen. The sample to be measured was put into the cuvette and measure.
The chromatographic analysis of individual anthocyanin, delphinidin-3-
The determination of vitamin C content is carried out according to the method by Qiu et al. (2018). Of note, 0.2 g sample was weighed, placed in a 250 mL volumetric flask, added water 100 mL and dilute acetic acid 10 mL to dissolve, added 1 mL of starch indicator solution, immediately titrated with iodine standard titration solution, until the solution is in blue colour that does not fade within 30 s. Each 1 mL of iodine titration solution (0.05 mol · L−1) is equivalent to 8.806 μg of vitamin C.
Carotenoid content in the sample was determined according to the method by Liang et al. (2019). Of note, 0.2 g of the sample was accurately weighed, ground with liquid nitrogen, placed in 5 m of absolute ethanol containing 0.1% ascorbic acid (m/V) and allowed to stand at 4°C in the dark. Three hundred microlitres of an 80% potassium hydroxide solution (m/V) was added to the extract, shaken and then heated in a constant temperature water bath at 95°C for 45 min. The solution was then added to 5 mL of precooled deionised water and immediately cooled in an ice bath. Five millilitres of petroleum ether was added, and the solution was shaken, extracted and centrifuged at 4°C and 12,000 rpm for 10 min. After 30 min, the supernatant was aspirated. The extraction was repeated three times and the supernatant was combined. The petroleum ether in the supernatant was volatilised by blowing nitrogen gas in a 35°C water bath environment. Then, 3 mL of dichloromethane and methanol in a volume ratio of 1:1 were added and redissolved. The solution was slowly filtered through a 0.22-mm organic filter for HPLC analysis.
Chlorophyll content in the sample was determined according to the method by Liu et al. (2020). Of note, 0.2 g of the sample was ground by an acetone method, extracted with an 80% acetone solution, filtered to a constant volume and then measured by a UV–VIS spectrophotometer.
HPLC mapping was performed using LC-WS100 chromatography workstation software. Results were expressed as mean ± standard deviation (SD). Each value was the average of three replications. The Pearson correlation analysis was subjected to two-tailed analysis of variance using the SPSS 19. A difference between the developmental stages of fruit was considered statistically significant (
Vertical diameter, transverse diameter and single fruit weight are important indicators reflecting the quality of fruit growth. Vertical diameter, transverse diameter, single fruit weight and soluble solids of myrtle fruits from green to colour break and to mature increased significantly (
Physical properties of myrtle fruit at different developmental stages
| Growth stages | Fruit weight (g ± SD) | Vertical diameter (mm ± SD) | Transverse diameter (mm ± SD) | Fruit shape index | Soluble solid content (% ± SD) |
|---|---|---|---|---|---|
| G | 1.20 ± 0.03 c | 14.68 ± 1.49 c | 11.52 ± 0.84 c | 1.27 ± 0.10 ns | 3.40 ± 0.26 c |
| C | 1.87 ± 0.04 b | 16.81 ± 0.81 b | 13.54 ± 0.67 b | 1.24 ± 0.05 ns | 7.23 ± 0.25 b |
| M | 2.74 ± 0.05 a | 18.08 ± 0.66 a | 15.07 ± 0.83 a | 1.20 ± 0.08 ns | 12.17 ± 0.40 a |
Means followed by different letters indicate significant differences at 0.05 level of probability.
SD, standard deviation; G, green; C, colour break; M, mature.
The contents of soluble sugars and titratable acidity showed a different trend during the development of myrtle fruits (Figure 1). Soluble sugar contents of fruits significantly increased (14.30%–17.68%) from green stage to mature stage, while titratable acidity contents decreased significantly at different stages of development from 5.25 to 2.71 mmol/100 g from green stage to mature stage.
Figure 1
Influence of different developmental stages of fruit on soluble sugars and titratable acidity. Bars with different letters differ significantly (
The contents of total flavonoids and total phenolic in myrtle fruit (Figure 2) showed the same patterns of change during development. The content of flavonoids at the green stage was the highest (0.17%), and the colour break and the mature stages were significantly reduced by 0.04% and 0.07% (
Figure 2
Influence of different developmental stages of fruit on total phenolic and total flavonoids. Bars with different letters differ significantly (
With the development of myrtle fruit, the fruit colour gradually changed from green to purple-black (Figure 3). For HPLC analysis, according to the standard samples and references, five anthocyanins were detected in the three developmental stages of myrtle fruits. The type of anthocyanin was determined based on the peak time of the standard sample, followed by Dp, Cy, Pg, Pn and Mv. The contents of Dp, Cy, Pg, Pn and Mv were 2.12, 0.47, 0.45, 0.17, 0.55 μg · g−1, respectively, at the green stage in sequence, 113.79, 120.94, 33.66, 37.26 and 19.50 μg · g−1, respectively, at the colour break stage and 398.76, 1,200.03, 149.00, 371.46, 62.72 μg · g−1, respectively, at the mature stage.
Figure 3
High-performance liquid chromatographic results of anthocyanins at different developmental stages of myrtle fruit. G, Green; C, colour break; M, mature.
The anthocyanin contents of myrtle at different stages were shown in Table 2. It was observed that during the development of myrtle fruit, the anthocyanin content increased significantly at different periods (
Content of anthocyanins (μg · g−1) in myrtle fruit at different developmental stages
| Growth stages | TAC (μg · g−1 ± SD) | Dp (μg · g−1 ± SD) | Cy (μg · g−1 ± SD) | Pg (μg · g−1 ± SD) | Pn (μg · g−1 ± SD) | Mv (μg · g−1 ± SD) |
|---|---|---|---|---|---|---|
| G | 3.76 ± 0.26 c | 2.12 ± 0.18 c | 0.47 ± 0.02 c | 0.45 ± 0.07 c | 0.17 ± 0.04 c | 0.55 ± 0.04 c |
| C | 325.15 ± 20.02 b | 113.79 ± 7.56 b | 120.94 ± 8.99 b | 33.66 ± 1.67 b | 37.26 ± 2.22 b | 19.50 ± 1.60 b |
| M | 2,181.97 ± 70.50 a | 398.76 ± 17.81 a | 1,200.03 ± 34.85 a | 149 ± 4.34 a | 371.46 ± 10.26 a | 62.72 ± 5.33 a |
Means followed by different letters indicate significant differences at 0.05 level of probability.
SD, standard deviation; G, green; C, colour break; M, mature; TAC, total anthocyanin content.
Correlation analysis of vertical diameter (VD), transverse diameter (TD), soluble sugar content (SSC), titratable acidity content (TaC), total phenol content (TPC), total flavonoids content (TFC), chlorophyll a content (CaC), chlorophyll
| VD | TD | SSC | TaC | TPC | TFC | CaC | CbC | TCC | Vit.C | TAC | CC | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| VD | 1 | |||||||||||
| TD | 0.953** | 1 | ||||||||||
| SSC | 0.884** | 0.830** | 1 | |||||||||
| TaC | −0.875** | −0.811** | −0.968** | 1 | ||||||||
| TPC | −0.884** | −0.827** | −0.966** | 0.896* | 1 | |||||||
| TFC | −0.873** | −0.822** | −0.989** | 0.967** | 0.966** | 1 | ||||||
| CaC | −0.749* | −0.711* | −0.905** | 0.950** | 0.769* | 0.880** | 1 | |||||
| CbC | −0.726* | −0.706* | −0.879** | 0.919** | 0.732* | 0.840** | 0.992** | 1 | ||||
| TCC | −0.740* | −0.710* | −0.895** | 0.939** | 0.755** | 0.865** | 0.999** | 0.997** | 1 | |||
| Vit.C | −0.562 | −0.605 | −0.726* | 0.798** | 0.625 | 0.770* | 0.832** | 0.800** | 0.820** | 1 | ||
| TAC | 0.873** | 0.811** | 0.893** | −0.788* | −0.964** | −0.887** | −0.621 | −0.588 | −0.609 | −0.420 | 1 | |
| CC | 0.694* | 0.767* | 0.812** | −0.842** | −0.718* | −0.823** | −0.880** | −0.872** | −0.878** | −0.938** | 0.554 | 1 |
Correlation is significant at the 0.05 level.
Correlation is significant at the 0.01 level (two-tailed).
It was observed from Figure 4 that the contents of total chlorophyll, chlorophyll
Figure 4
Influence of different developmental stages of fruit on chlorophyll
The contents of carotenoids and vitamin C at the different developmental stages of myrtle fruits were determined (Figure 5). In the green fruits development period, no carotenoid component was detected and it reached the highest at the mature stage (16.90 μg · L−1) of the fruit development. The content of vitamin C reached the highest (7.5 μg · 100 g−1) at the green stage and the lowest (3 μg 100 g−1) at the colour break stage. The difference from the green stage to the colour break stage was significant (
Figure 5
Influence of different developmental stages of fruit on carotenoids and vitamin C. Bars with different letters differ significantly (
Fruit size is an important indicator of fruit appearance quality and is closely related to the development of fruit vertical and transverse diameters. After fruit setting, the fruit showed a certain growth dynamic after continuous cell division and cell expansion. So, the growth progress of vertical and transverse diameters is parallel (
Fruit colour is an important indicator of fruit quality. Anthocyanin is the main pigment that determines fruit colour and is also the main symbol of fruit ripeness (Qi et al., 2018). The changes in fruit colour are closely related to the type and content of anthocyanins. The colour of Pg usually is brick red, Dp, Pt and Mv exhibit blue-violet and Cy and Pn exhibit a purplish red colour (Zhang et al., 2011). In this experiment, five anthocyanins were detected, which were Dp, Cy, Pg, Pn and Mv. In this study, it was observed that the main anthocyanin component in myrtle was cyanidin after the colour change. Cui et al. (2013) found six anthocyanins in the fruits of myrtle, among which the main component was cyanidin. This was basically consistent with the results of this study. The only difference was that no petunia was detected in this study. This may be attributed to differences in the growth conditions and cultivar. Yang et al. (2017) also found five anthocyanins in their work and the content of anthocyanin increased significantly with the development of ripening, which was consistent with the results of this experiment.
Organic acids are an important part of fruits quality and together with sugar they form fruit flavour. Most fruits have gradually increasing contents of soluble sugar during fruit development, and when matured the organic acid content decreases (Wen et al., 2001; Shaw and Wilson 1981; Zhou et al., 2015). Su et al. (2019) reported that as grapefruit mature, the soluble sugar content gradually increased, and the titratable acidity content decreases gradually which is an agreement with our findings.
Flavonoids and polyphenols were widely found in plants. They not only have important physiological functions on plants but also have strong anti-oxidation, anti-mutation, anti-arteriosclerosis, antitumour and antiviral effects (Guo et al., 2008; Song et al., 2000). Xia et al. (2016) found that the contents of total phenols and flavonoids in the development of apricot were the lowest in the early developmental stage which gradually increased to maximum and decreased with the increase of fruit ripening. Su et al. (2018) showed that the flavonoids and total phenols showed a downward trend with fruit development through the changes of physiological indices during pear fruit development. This is consistent with the results of this study. On the one hand, the contents of flavonoids and total phenols were closely related to the rapid growth period of fruit development. As the fruit gradually approaches maturity, the fruit surface began to appear red, reaching a lower maturity. Due to the rapid growth of the fruit, the supply absorption capacity was weak. On the other hand, fruit trees provided energy during fruit ripening, and the content of soluble sugar and soluble solids increased sharply. Phenolic substances were required to induce the differentiation of related cells, so the content of flavonoids and total phenol gradually decreased to maintain the nutritional balance of the tree.
Vitamin C is a water-soluble substance with antioxidant and anti-free radical effects. It was found in many fruits and vegetables but cannot be synthesised by the human body (Mo et al., 2018). Zhu and Du (2019) measured the content of vitamin C in the development of persimmon and found that the vitamin C content in persimmon gradually increased as the fruit develops and reached the highest level at maturity. This is in contrast with the results obtained in this experiment. In this study, vitamin C content reached the highest in young fruits. The reason may be due to differences in the varieties. Also, in our study carotenoids were not detected in the green fruit stage and slowly formed after the turning period. This is basically consistent with the findings of Li et al. (2006). The only difference is that carotenoids were detected at an early stage of wolfberry development in Li’s research.
Our research showed that the physicochemical properties of myrtle varied greatly at different developmental stages. Five anthocyanin components were detected, and each component gradually increased during the three stages of myrtle development. The contents of soluble sugar, soluble solids and carotenoids decreased as the fruit matures. The content of titratable acidity, phenols and chlorophyll decreased with the developmental stage. This study will help us to better understand the effect of myrtle development stage on physicochemical characteristics.