Effect of grafting and harvest stage on the quality of black cherry tomatoes (Solanum lycopersicum cv. OG) cultivated in Vietnam

Black cherry tomato is an important source of nutraceutical compounds; however, the quality of fruits depends on the cultivation technique and maturity stage at harvest. In this study, the physical and chemical properties of non-grafted and grafted tomatoes (Solanum lycopersicum cv. OG) were evaluated at different stages of maturity to observe the effect of grafting on quality of fruits and select the appropriate harvest stage to achieve the highest content of bioactive compounds, especially anthocyanin. The obtained results found that harvesting non-grafted tomatoes on the 30 day after fruit formation would be suitable for storage with the highest anthocyanin level of 4.27 mg CE/100 g and the content of other bioactive compounds (lycopene 24.60 μg/g, vitamin C 42.79 mg/100 g and total phenolic 40.61 mg GAE/100 g). Meanwhile, the optimal harvest stage of grafted tomatoes was shortened, on the 28 day (anthocyanin 4.31 mg CE/100 g, lycopene 38.79 μg/g, vitamin C 55.69 mg/100 g, and total phenolic 38.69 mg GAE/100 g). The grafting technique should be applied for supporting the plant to grow faster as shown by the earlier stage of harvest and the harvested fruit possessed higher content of bioactive compounds.


Introduction
"Black" or "purple" cherry tomatoes are subspecies of Solanum Lycopersicum (Zhang et al., 2018) and exhibit a purplish-brown color on their skin (Mes et al., 2008). Lycopene is the most abundant (about 80-90% of total carotenoid content) and the highest antioxidant natural carotenoid found in tomatoes (Alda et al., 2009). Epidemiological studies have shown that lycopene, a red pigment has the potential to reduce the risk of chronic diseases, most notably prostate cancer (Ford and Erdman, 2012). This red pigment also plays an important role in the prevention of cardiovascular disease (Mordente et al., 2011). Min and Min (2014) observed that consuming a large amount of lycopene-rich foods helped to lower the risk of mortality from Alzheimer's disease in adults. Kaur et al. (2011) also found that lycopene was beneficial in the treatment of Parkinson's disease and other neurological abnormalities by protecting against oxidative stress. In addition to carotenoids, tomatoes are good for other antioxidant compounds such as vitamin C and phenolics which also inhibit reactive oxygen species causing many dangerous diseases (Ilahy et al., 2009). It has been noticed that the content of phenolic compounds and carotenoid pigments, particularly lycopene in black cherry tomatoes are higher than in some red tomato varieties (Zhang et al., 2018). Especially, black cherry tomatoes also can produce a phytochemical of anthocyanin predominantly in the skin (Li et al., 2011). Anthocyanin has been proven to be associated with many health benefits, reduces cancer cell proliferation, protects against cardiovascular disease, prevents obesity and diabetes (Lila, 2004).
Despite containing many bioactive compounds, researches on black cherry tomatoes have been very limited, especially in Vietnam. Moreover, consumers are still sceptical about the anthocyanin component in black cherry tomatoes, because some studies in the world showed that some heirloom varieties result from mutations affecting chlorophyll breakdown and carotenoid content but are not related to anthocyanin production (Gonzali et al., 2009). Therefore, most of the black cherry tomatoes have primarily grown in Vietnamese gardens for visiting and retailing. This wastes a healthy source of black cherry tomatoes and does not create many choices for consumers and also affect the economic benefits for farmers.
Besides that, bacterial wilt, caused by Ralstonia eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources FULL PAPER solanacearum, can be very problematic for all tomato growers as a result of a lack of available resistance genes within cultivated tomato (Rivard and Louws, 2008). The cultivation of grafted vegetable plants has become more popular recently to manage soilborne pathogens (Besri, 2001;Bletsos, 2005). This technique is carried out when the rootstock and scion seedlings are very small, and the two are attached with a small silicon tube or clip (Rivard and Louws, 2006). Grafting with tolerant rootstock is also effective at overcoming abiotic stresses such as salinity (Cuartero et al., 2006), thermal stress (Rivard and Louws, 2008), and excessive soil moisture (Black et al., 2003). Although the use of grafted vegetables is associated with disease reduction and/or abiotic stressors, yield can be increased without the presence of these identified stressors (Yetisir and Sari, 2003). Grafted plants also produced higher fresh and dry matter than control (Yetisir and Sari, 2003).
The physical and chemical properties of tomatoes not only depend greatly on the grafting but also on the maturity stage at harvest. Maturation caused a slight softening in tomato when compared with less mature tomato fruits, which has a significant effect on the quality and influences consumer preferences, storability, shelf life, pathogen resistance, and transportability (Viskelis et al., 2008). Helyes et al. (2006) found that the Brix, carbohydrate, and lycopene content increased during maturation from the green mature to the deep red stage but polyphenol content changed little during tomato ripening. Moneruzzaman et al. (2008) reported that half-ripen tomato showed the highest value of vitamin C and titrable acidity. Therefore, the objective of this study was to evaluate the effect of grafting and harvest stage on the physicochemical properties of a new variety of black cherry tomatoes grown in Vietnam (Solanum lycopersicum cv. OG). These results would help farmers to choose grafting/non-grafting techniques and suitable harvest maturity.

Experimental design
Black cherry tomato (cv. OG) seeds were provided by the F1508 seed store (Ho Chi Minh City, Vietnam) and sown at Nam Long farm, Vinh Long province, Vietnam. After 23 days of sowing, 50 tomato plants were grafted with eggplant root (eggplant "EG 203" was obtained from the Asian Vegetable Development and Research Center). After 29 days of sowing, the remaining 50 tomato plants (without grafting) were transferred to pots for growing. After 13 days of grafting, 50 grafted tomato trees were also transferred to pots for growing. Sowing and growing process were all done at Nam Long farm (Vinh Long province). Marking the time when the flower started to form a fruit. At different stages (from 26-34 days and 24-32 days after fruit formation for non-grafted tomatoes and grafted tomatoes, respectively), fruits were harvested. Black tomatoes were put in perforated PVC and cardboard boxes and transported to the Food Technology laboratory of Can Tho University within 1 hr. Replications of three were made. The photos of fruit were taken and the physicochemical characteristics were determined.

Physical properties 2.2.1 Fruit weight and size parameters
The fruit weight was determined by an analytical balance with the accuracy of 0.0001 g (PR-series, Ohaus, USA). The fruit size was determined by two dimensions, namely height (H) and diameter (D), were measured by using digital calipers with an accuracy of 0.01 mm (MC 01120028, Gaogen, China). The geometric mean diameter (D g ) was calculated by equation 1 (Yıldız et al., 2015).
The surface area (S a ) and aspect ratio (R a ) of the fruit were calculated by using equations 2 and 3, respectively (Yıldız et al., 2015).
The sphericity (S p ) defined as the ratio of the surface area of a sphere having the same volume as that of fruit to the surface area of the fruit was determined using equation 4 (Yıldız et al., 2015).

Respiration rate
Tomatoes (200 g) were put into a 20x30 cm PE zip bag. The oxygen concentration was measured continuously. Respiration rate (R) was determined by equation 5. Where P is the permeability coefficient (mL.cm.cm -2 .h -1 ), A is the surface area of the package (cm 2 ), L is the thickness of PE bag (cm), M is the weight of the sample (kg); y O2 is the oxygen concentration (% v/ v) (o: outside, i: inside).

Firmness
Fruit firmness was determined with a RheoTex (SD 700, Sun Science, Japan). Using a 1 cm -diameter cylindrical probe with a flat end in this case. The force required to press vertically into the middle of fruits for a 4 mm distance was measured and expressed in g/cm 2 .
(1) The whole tomatoes were ground in a blender (MX-GM1011, Panasonic, Japan) for 1 min and the obtained puree was determined TSS content and pH value. The content of TSS was measured by a refractometer (0-32%, Atago, Japan) at 20 o C. The pH value was measured with a pH meter (Edge HI2020-01, Hanna, Vietnam) at 20°C.

Moisture content
The moisture content (W) was determined by drying the sample at 105 o C to a constant weight and calculated using equation 6. Where m o is the weight of the original sample, m is the weight of the sample after drying.

Total sugar content
The total sugar content was determined by the colorimetric method with DNS reagent (Luong, 2003). Tomato puree (3 g) was weighed into a 100 mL flask with 50 mL of distilled water and 5 mL of 36.5% HCl solution. The hydrolysis process was carried out at 60°C for 15 mins. The mixture was neutralized by the 30% NaOH solution to pH 7 then filled up to a volume of 100 mL with distilled water and filtered through a filter paper. The filtrate (2 mL) was added 1 mL DNS reagent and placed in boiling water for 5 mins. The absorbance of the mixture was read at 540 nm using Spectrophotometer UV-VIS (722N, Inesa, China). The total sugar content (S) was determined using equation 7. Where A is the reducing sugar content derived from the standard curve (mg/mL), n is the dilution factor, V is the volumetric flask volume (100 mL), m is the weight of the sample (g).

Total acid content
The total acid content was determined by the titration method (Yıldız et al., 2015). Tomato puree (10 g) was shaken with neutral water for 1 hr. The mixture was filled to a volume of 50 mL with neutral water and allowed to settle. The supernatant (25 mL) was added 5 drops of phenolphthalein and titrated with the 0.1 N NaOH solution until the mixture had a stable light pink. The total acid content was calculated by equation 8. Where n is the volume of the 0.1 N NaOH solution used to titrate (mL), K is the coefficient of citric acid (0.0064), P is the weight of the sample (g).

Anthocyanin content
The anthocyanin content was determined by the pH differential method (Lee et al., 2005) with some modifications. Tomato puree (5 g) was filled to a volume of 50 mL with ethanol/water (1/1) solvent containing 1% HCl and extracted for 60 mins. The mixture was then separated by a centrifuge at 7000 × g for 10 mins. The supernatant was diluted with two buffers of pH 1.0 and 4.5 and read the absorbance at both 520 and 700 nm versus a blank of distilled water. The anthocyanin content was calculated as cyanidin-3-glucoside equivalent (equation 9). Where A is (A 520nm -A 700nm )pH 1.0 -(A 520nm -A 700nm )pH 4.5, M is 449.2 g/mol for cyanidin-3-glucoside, k is the dilution factor, l is the pathlength (cm), ε is 26900 -molar extinction coefficient for cyanidin-3-glucoside (L × mol -1 × cm -1 ), V is the volume of extract (mL), m is the weight of the sample (g).

Lycopene content
The lycopene content was determined by the low volume hexane extraction method (Fish et al., 2002;Davis et al., 2003). Tomato puree (0.6 g) was mixed with 5 mL of acetone containing 0.05% butylated hydroxytoluene, 5 mL of 95% ethanol, 10 mL of hexane in a vial and extracted for 15 mins on a shaker at a speed of 180 rpm. The mixture was then added 3 mL of deionized water and shaken for 5 mins. The vial was left for 5 mins. The absorbance of the supernatant was read at 503 nm against a blank of hexane. The lycopene content was determined using equation 10. Where A 503 is the absorbance of the extract at 503 nm, m is the weight of the sample (g).

Vitamin C content
The vitamin C content was determined by the titration method (Lam et al., 2004). Tomato puree (10 g) was filled to a volume of 100 mL with 5% HCl solution and filtered through a filter paper. The filtrate (10 mL) was added 5 drops of the 1% starch solution and titrated with the 0.001 N KIO 3 /KI solution until the blue-black color appears. For the control, the sample extract was replaced by the 1% HCl solution. The vitamin C content was calculated using equation 11. Where a and b is the volume of 0.001 N KIO 3 /KI solution used for titration the extract and the control, respectively (mL), 100 is the volume of extract (mL), 0.088 is the weight of ascorbic acid corresponds to 1 mL of 0.001 N KIO 3 /KI solution (mg), m is the weight of the sample (g). The total phenolic content was determined using Folin-Ciocalteu reagent (Teixeira et al., 2013) with some modifications. Tomato puree (5 g) was filled to a volume of 50 mL with 95% ethanol and extracted for 60 mins. The mixture was then separated by a centrifuge at 7000 × g for 10 mins. The supernatant (0.2 mL) was added 1.0 mL of 10% Folin-Ciocalteu reagent, left for 5 mins and then added 1.2 mL of 5% Na 2 CO 3 solution. After 2 hrs, the absorbance was recorded at 750 nm. The total phenolic content was calculated as gallic acid equivalent (equation 12). Where C is the content of gallic acid derived from the standard curve (mg/mL), V is the volume of extract (mL), m is the weight of the sample (g), k is the dilution factor.

Antioxidant activity
Antioxidant activity was determined using the DPPH assay (Teixeira et al., 2013) with some modifications. Tomato puree (5 g) was filled to a volume of 50 mL with 95% ethanol and extracted for 60 mins. The mixture was then separated by a centrifuge at 7000 × g for 10 mins. The supernatant (0.1 mL) was added 2 mL of DPPH solution (0.21 mM in 95% ethanol). For the control, the sample extract was replaced with 95% ethanol. The mixture was kept for 1 hr before absorbance reading at 517 nm. The percentage of DPPH free radical scavenging was calculated by equation 13. Where A control is the absorbance of the control, A sample is the absorbance of the sample.

Data analysis
Data analyses were carried out using the Statgraphics Centurion XV (U.S.A.). The significance/nonsignificance of results was determined using the One-Way ANOVA and Duncan test at the 95% confidence level (P = 0.05).

The physical properties of black cherry tomatoes
The color chart of the black cherry tomato "OG" variety at different maturity stages presented in Figure 1. Along with the color change was the change in physical properties and content of nutrients of the fruit. The tomato color gradually converted from green to greenorange to orange-red to deep red during maturation as a consequence of the synthesis of lycopene and depletion of chlorophyll (Arias et al., 2000). However, this change in the fruit skin was not obvious as in the fruit flesh due to the presence of anthocyanin.
The respiration rate of non-grafted tomatoes was still low on the 28 th day after fruit formation (1.54 mLO 2 / kg.h). This value increased gradually and reached its highest on the 32 nd day (1.84 mLO 2 /kg.h) and then decreased slightly on the 34 th day (Figure 2a). Similarly, the respiration rate of grafted fruits decreased from the 24 th day (1.73 mLO 2 /kg.h) to the 26 th day (1.54 mLO 2 / kg.h), then increased to the 30 th day (1.92 mLO 2 /kg.h) and decreased again on the 32 nd day (1.72 mLO 2 /kg.h) (Figure 2b). It was shown that the non-grafted tomatoes reached the maturity after 28 days of fruit formation and the 32 nd day was the full ripening time. For grafted tomatoes, the maturity and full ripening age were on the 26 th and 30 th day, indicating that the maturity of grafted fruits was faster. Tomato is classified as a climacteric ripening pattern, which means that they have a significant rise in respiration and ethylene production rates at the onset of the ripening process (Toivonen, 2007).
The weight and size of fruit are also important quality parameters that necessary for designing the postharvest system including sorting, handling, packaging, storage, and processing. These parameters of black cherry tomatoes were determined at the time of full maturity when fruits reached the maximum size (on the 28 th day for non-grafted tomatoes and on the 26 th day for grafted tomatoes). It was observed that the non-grafted and grafted tomatoes had the same weight and size (Table 1). Furthermore, the shape indexes of them, such as the sphericity (from 96.73% to 97.28%) and the aspect ratio (from 0.95 to 0.96) indicated that the black cherry The firmness decreased rapidly at the early harvest stage and then more slowly at the later harvest stage in both non-grafted and grafted tomatoes but there was no significant difference in firmness between two types of fruits ( Figure 3). The texture and firmness of fruits and vegetables are based on the presence and interactions of different chemical components, like pectins in the middle lamellae and the cellulose/hemicellulose matrix in the primary cell wall, and on physical aspects like arch structure and turgor. The turgor and part of pectin are susceptible to enzymatic degradation during ripening, thereby contributing to the variable part of the firmness (Van Dijk et al., 2006).

The chemical properties of black cherry tomatoes
The moisture content varied from 92.73 to 94.86% and 92.45 to 94.23% for non-grafted and grafted fruits, respectively with higher values in the first harvest stage, followed by reductions in the content throughout the ripening cycle (Figure 4). During the ripening of the fruit, chemical compounds are synthesized reaching maximum content at the end of ripening, therefore the content of moisture decreases gradually (Schulz et al., 2015).
As could be seen in Table 2, the content of total sugar and TSS of non-grafted tomatoes increased by harvest stage and peaked on the 32 nd day, after that, it tended to decrease on the day of 34 th . When the harvest stage was extended from 26 to 32 days, the increase in total sugar content was due to the conversion of starch during fruit ripening (Prasad et al., 2018), however, after the fruit was fully ripening, the hydrolysis process no longer took place and the sugar participated in the respiration leading to a decrease in total sugar (Bashir and Abu-Goukh, 2003). Similarly, the total sugar and TSS content of grafted fruits increased from the 24 th harvest day and reached the highest value on the 30 th day, then decreased on the 32 nd day (Table 3). These results were also consistent with the obtained results of Mini (2017) for the "PKM-1" tomato variety.    The total acid content and pH changed in two opposite directions (Tables 4 and 5). The total acid content increased with the harvest stage until the fruit was fully ripening and reached its highest values, 0.578% and 0.506%, for non-grafted and grafted tomatoes on the 32 nd and the 30 th day, respectively. This component tended to decrease later. The predominant acid in tomatoes is malic acid and is utilized as a substrate for respiration and esterification, creating a special aroma for fruits, which is the reason for the decreasing trend in acidity in the later stage (Mini, 2017). Conversely, the pH value reached the lowest on the 32 nd day (4.21) and the 30 th day (4.35) for nongrafted and grafted tomatoes corresponding to the time of the highest total acid content.
The bioactive compounds such as anthocyanin, lycopene, vitamin C, and other phenolics were all valuable components in black cherry tomatoes, which determine the antioxidant activity of fruits. During ripening, these components changed in different directions (Tables 6 and 7).
It was worth noting that the non-grafted black tomatoes reached the highest anthocyanin content on the 34 th day of harvest stage (4.32 mg CE/100 g) but there was no statistically significant difference between the two days of 30 th and 32 nd . The anthocyanin accumulation process takes place in three stages: the first phase with a rapid increase in the concentration, a transitional phase characterized by a decreasing accumulation rate until a maximum concentration, and a final phase of decreasing concentration (Schulz et al., 2015). At the first stage of harvest, the content of lycopene was very low (8.74 μg/ g) which increased almost 5 times during ripening and reached 42.28 μg/g on the 34 th day. Increased level of lycopene in tomatoes with the harvest stage is due to the ripening advancement of fruits and conversion of    (Mini, 2017). The vitamin C content also increased from 30.18 mg/100 g on the 26 th day to 55.69 mg/100 g on the 32 nd day, and then decreased to 49.47 mg/100 g on the 34 th day. In tomato fruits, ascorbic acid content increases with maturity and stage of ripening, however, once fruit reaches the fully ripe stage, ascorbic acid content starts to decline (Mini, 2017). A decrease in total phenolic content with ripening was observed. This content decreased sharply from the 26 th day (48.74 mg GAE/100 g) to the 32 nd day (38.69 mg GAE/100 g). The phenolic content on two harvest days of 32 th and 34 th was not different significantly. Phenolic acts as an antibody of a plant, in the early stages of development, many antibodies are needed to resist nature. After adapting to the environment, plants do not synthesize phenolic compounds. Meanwhile, during ripening, phenolic can be oxidized by polyphenol oxidase (Ayaz et al., 2008). The antioxidant activity of non-grafted tomatoes expressed by the DPPH free radical scavenging activity reached the highest level on the 32 nd harvesting day (76.59%), after which there was a decreasing trend on the harvest day of 34 th but they were not different significantly. The phenolic compounds were a major contributor to the antioxidant activity of plants (Song et al., 2010), so a decrease in phenolic content would affect the antioxidant activity of black cherry tomatoes. However, the antioxidant activity of tomatoes is derived from a mixture of antioxidant compounds, including carotenoids, ascorbic acid, and total phenolic (Bhandari et al., 2016). It was observed that the changing trend of bioactive compounds in grafted tomatoes was also similar. However, the anthocyanin content was found highest on the 30 th day (4.45 mg CE/100 g) but not significantly different compared to the 28 th and 32 nd days.

Conclusion
The appropriate harvest stage of black cherry tomatoes (cv. OG) was on the 30 th day for non-grafted fruits and on the 28 th day for grafted fruits, at this time, the anthocyanin content was maximum while the other bioactive compounds remained low. If collected at the fully ripening stage, the fruits would be vulnerable in transport and storage. Tomato is a climacteric fruit, so that, it will ripen and the remaining compounds may continue changing under storage conditions. Anthocyanin is synthesized on plants during growing under the sunlight, this component does not increase after harvesting. The grafting helped the plant to grow faster as shown by an earlier harvesting stage and the harvested fruits had a higher level of bioactive compounds such as anthocyanin, lycopene, and vitamin C than the non-grafted fruits. It is necessary to further investigate the impact of grafting on the resistance to bacterial wilt of the black cherry tomato plants to better understand the importance of this technique.   Arias, R., Lee, T.C., Logendra, L. and Janes, H. (2000). Correlation of lycopene measured by HPLC with the L*, a*, b* color readings of a hydroponic tomato and the relationship of maturity with color and lycopene content.