1 1 Postharvest Application of Boric Acid on Grape to Improve Shelf 2 life and Maintain the Quality 3 4 Hui Jie Lia,b, Jia Bing Jiaoa,b, Yi Man Fanga,b, Yang Yang Zhanga,b, Da Long Guoa,b 5 a College of Horticulture and Plant Protection, Henan University of Science and Technology, 6 Luoyang 471023, P R China 7 b Henan Engineering Technology Research Center of Quality Regulation and Controlling of 8 Horticultural Plants, Luoyang 471023, P R China 9 Corresponding author Da Long Guo, E mail guod.
Trang 11 Postharvest Application of Boric Acid on Grape to Improve
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4 Hui-Jie Lia,b, Jia-Bing Jiaoa,b, Yi-Man Fanga,b, Yang-Yang Zhanga,b, Da-Long Guoa,b*
5 a College of Horticulture and Plant Protection, Henan University of Science and Technology,
6 Luoyang 471023, P R China
7 b Henan Engineering Technology Research Center of Quality Regulation and Controlling of
8 Horticultural Plants, Luoyang 471023, P R China
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Trang 223 ABSTRACT
24 Boric acid (BA) is commercially acceptable and economically feasible material to enhance shelf-
25 life of pear, orange and other horticultural plants Here, we investigated the effect of BA on the
26 shelf-life and postharvest quality of table grape (cv ‘Kyoho’) Grape berries were immersed in BA
27 solution with different concentrations (0.00 [as control], 0.01, 0.03, 0.05 M) for 10 min and stored
28 at room temperature for 10 days Compared with the control, BA treatment groups maintained
29 higher berry firmness by inhibiting the activity of polygalacturonase (PG) and cellulase, although
30 not all indexes and treatments had advantages after BA treatment At the same time, BA treated
31 grapes maintained higher antioxidant enzyme activities such as catalase (CAT), superoxide
32 dismutase (SOD) and lower metabolic toxic products like superoxide anion ( ) production rate, 𝑂2_
33 malondialdehyde (MDA) and hydrogen peroxide (H2O2) content than control The experiment
34 results showed that postharvest application of BA effectively delay the senescence of grapes
35 compared with the control, and 0.01 M BA treatment had the most obvious effect
36 Keywords: Kyoho, Boric acid, postharvest, antioxidant enzyme
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Trang 345 1.Introduction
46 Grape (Vitis vinifera L.) is one of the most widely consuming fruits all over the world (Li et al.,
47 2018) At present, China has become one of the world's biggest grape producers (Khan et al., 2020)
48 Over 84% of the total land used for grape production in China is cultivated for the table grapes (Sun
49 et al., 2020) However, table grapes, as non-climacteric fruits with thin pericarp and succulent flesh,
50 are easily infected by plant pathogens and exposed to serious water loss, which could result in a
51 high fungal decay rate (Meng et al., 2010) Postharvest senescence and disease have recently
52 seriously restricted the market development of table grapes Therefore, it is necessary to explore
53 some strategies for table grapes storage
54 In this context, different methods such as preharvest and postharvest applications of kombucha
55 (Zhou et al., 2019), short-term high CO2 (Vazquez-Hernandez et al., 2018), Aloe vera gel (Ehtesham
56 Nia et al., 2021), aerosol with calcium-based (Cherviak et al., 2021), edible coatings (1.5% chitosan
57 and 1.0% poly-ε- lysine) (Chen et al., 2019) were used to maintain the firmness and inhibit the decay
58 of fresh table grapes In addition, chemical fungicides are widely used in vineyards to control
59 postharvest diseases of grapes (Ehtesham Nia et al., 2021) However, a large quantity of chemical
60 spraying will lead to adverse effects on consumer health and the environment Hence, more
61 environmentally friendly and economical methods need to be explored urgently to solve these
62 problems preferably
63 Boron was accepted as an essential nutrient for all vascular plants, animals and humans Boron
64 regulated the metabolic activities by interacting with magnesium, calcium and vitamin D, which are
65 all necessary for bone metabolism (Devirian and Volpe, 2003) Boron has disinfectant and
66 bactericidal properties which inhibits the fruit decay after harvest and plays a crucial role in
Trang 467 maintaining the rigidity of fruit cytoderm and phenolic concentration (Kaur et al., 2019) It was
68 reported that boron has a good effect on the prevention and control of gray mold of table grapes
69 caused by B cinerea (Qin et al., 2010) Additionally, many studies have revealed that boric acid has
70 chemical properties inhibiting the initial increase of ethylene production (Ahmadnia et al., 2013)
71 and suppressing the activity of ACC synthase and ACC oxidase (Moon et al., 2020) It has also been
72 reported that boric acid enhances the storage life of tomatoes (Wang and Morris, 1992), retains the
73 storability and quality of pear fruits (Kaur et al., 2019), extends shelf life and quality maintenance
74 of Guava (Singh et al., 2017) and improves postharvest quality of Cut Carnation (Ahmadnia et al.,
75 2013), and so on However, little information is available on the effect of boric acid application on
76 the postharvest quality of table grapes during storage
77 Accordingly, the objective of the present study was to evaluate the potential of postharvest
78 treatment of boric acid to extend the shelf life of table grape during ambient temperature storage It
79 was hypothesized that different concentrations of boric acid would enhance the storability and
80 quality of table grape (‘Kyoho’)
81 2 Materials and methods
82 2.1 Plant material and experimental treatment
83 Grape berries of ‘Kyoho’ collecting from a grape vineyard in Luoyang, Henan province, China
84 were employed in this study Grape berries were sampled based on the uniformity in shape and
85 appearance, absence of visible defects All grape samples were harvested after ripening and
86 analyzed at the Henan University of Science and Technology BA solutions at different
87 concentrations (0.00, 0.01, 0.03, 0.05 M) were prepared with distilled water The sampled grape
88 berries were divided into four sets and immersed in four BA solutions for 10 min, then placed at
Trang 589 room temperature for 10 days Samples were taken every 2 days for a total of 6 times, i.e., sampling
90 at 0, 2, 4, 6, 8, 10 days after the treatment, respectively A portion of each sample used for the
91 evaluation of weight loss, berry firmness, and total soluble solid (TSS) content The remain samples
92 were collected and stored at -40 ℃ for subsequent analysis of the physiological indicators
93 2.2 Determination of the weight loss rate, firmness, TSS content
94 The weight of boric acid treated ‘Kyoho’ berries was measured at 0 storage day and 10 storage
95 day using an analytical balance The weight loss rate was calculated by the following formula:
96 Weight loss (%) =Initial weight ‒ final weight
Initial weight × 100
97 The firmness and TSS content of berries treated with BA were measured using the durometer
98 (FT-327, Wuxi, China) and handheld refractometer (WYT-4, Shanghai, China), respectively
99 2.3 Determination of content of ascorbic acid (AsA)
100 AsA was measured according to some modified method (Ge et al., 2015) Frozen tissue was
101 homogenized with 4.0 mL of prechilled 5% metaphosphoric acid and centrifuged at 12,000 rpm for
102 10 min at 4 ℃ The supernatant was used to measure the content of AsA The mixture solution was
103 measured at 525 nm, and expressed as mg AsA/g FW A standard curve with ascorbic acid was used
104 to calculate the content of AsA
105 2.4 Determination of the superoxide anion (𝑂𝟐_) production rate and content of H 2 O 2
106 The modified method (Ge et al., 2015; Yang et al., 2013) was employed to measure the 𝑂2_
107 production rate The absorbance of the extracting solution was recorded at 530 nm A standard curve
108 with sodium nitrite was used to calculate the 𝑂2_ production rate following the reaction equation of
109 𝑂2_ with hydroxylamine The production rate of 𝑂2_ was expressed as nmol/min/g FW
110 H2O2 content of the grape berries was measured spectrophotometrically after reaction with
Trang 6111 potassium iodide (Chakrabarty and Datta, 2007) The reaction mixture was measured at 390 nm, 10%
112 TCA solution was used as control The content of H2O2 was calculated using a standard curve with
113 known concentrations of H2O2
114 2.5 Determination of SOD activity and CAT activity
115 Superoxide dismutase (SOD) and Catalase (CAT) were extracted and assayed according to the
116 modified methods (Sun et al., 2011) Frozen grape berry tissue was extracted for 1 min with 2.0 mL
117 of 0.05 M sodium phosphate buffer (pH 7.8) containing 0.1% (w/v) polyvinyl pyrrolidone The
118 extract solution was centrifuged for 20 min at 12,000 rpm at 4 ℃ The supernatant was collected
119 for the determination of SOD activity and CAT activity
120 SOD activity was determined by measuring its ability to inhibit the photochemical reduction of
121 nitro blue tetrazolium (NBT) A total of 0.5 mL of enzyme solution was added into 3.0 mL of assay
122 reagent consisted of 130 mM methionine, 30 μM EDTA, 750 μM NBT, 20 mM riboflavin in 0.05
123 M sodium phosphate buffer (pH 7.8) The reaction solutions were incubated for 20 min under 4000
124 lux illumination The absorbance of sample was spectrophotometrically measured at 560 nm and
125 0.05 M sodium phosphate buffer (pH 7.8) was used as control The SOD activity was expressed as
126 U/g FW, where 1U is the amount of enzyme that caused 50% inhibition of NBT reduction
127 The assay mixture for determining CAT activity consisted of 0.3 mL of 0.1 M H2O2 prepared by
128 0.05 M sodium phosphate buffer (pH 7.8) and 0.5 mL of enzyme solution The decrease in
129 absorbance at 240 nm was recorded for 2 min at 25 ℃ And the CAT activity was expressed as U/g
130 FW/min, where 1U was defined as the amount of enzyme that caused a change of 0.01 in absorbance
131 per minute
132 2.6 Determination of malondialdehyde (MDA) content
Trang 7133 The modified method (Ehtesham Nia et al., 2021) was employed to measure the MDA content
134 Frozen grape berry tissue was homogenized for 1 min in 5.0 mL of 10% (w/v) trichloroacetic acid
135 The homogenate was centrifuged for 15 min at 12,000 rpm Three milliliter of the supernatant was
136 added to 3.0 mL of 0.67% (w/v) trichloroacetic acid The mixture solution was heated for 20 min at
137 100 ℃, quickly cooled in an ice-bath for 10 min and then centrifuged for 15 min with12,000 rpm
138 at 4 ℃ Absorbances were measured at 532, 450 and 600 nm MDA concentration was calculated
139 as follows: MDA content (mmol /g FW) = [6.45 (OD532 − OD600) − 0.56OD450] × 5 mL/0.5 g
140 2.7 Determination of polygalacturonase and cellulase activity
141 PG and cellulase activity were measured using described methods (Abu-Sarra and Abu-Goukh,
142 2015) The reaction mixture containing 0.5ml crude enzyme, 2.0 ml 0.5% pectin was incubated at
143 37 ℃ for 30 min After the constant temperature reaction, DNS was added, and the mixed solution
144 was boiled for 5 minutes Absorbance was measured at 540nm One pectinase activity unit was 1.0
145 mg galacturonic acid produced by pectin decomposition at 37 ℃ per gram of fresh sample per
146 minute
147 The cellulase activity was determined by the same procedure as PG assay, but the reaction
148 temperature and time was 40 ℃, 60min, and the substrate was 1% carboxymethyl cellulose The
149 cellulase activity unit was 1.0 mg glucose produced by the decomposition of carboxymethyl
150 cellulose at 40 ℃ per gram of fresh sample per minute
151 2.8 Statistical analysis
152 The data presented as mean ± standard deviation (SD) from three replicates were tested using the
153 SPSS 21.0 software Differences at P＜0.05 were considered significant Figures were produced
154 using GraphPad Prism 9.0
Trang 8155 3 Results
156 3.1 Effects of BA treatment on the weight loss and firmness of ‘Kyoho’ berries
157 In the 0.01 M BA treatment group, the weight loss rate was significantly lower than the control,
158 while there was no significant difference among the other groups (Fig 1A) These data indicate that
159 the three treatments of boric acid are not all beneficial to the index of water loss
160 The firmness of grape berries gradually decreased during storage (Fig 1B), and it was
161 significantly higher in the 0.01 M BA treatment group than that of berries in the other BA treatment
162 and the control groups This suggests that 0.01 M BA treatment could effectively prevent the
163 reduction in grape quality and firmness during storage (Fig 1B)
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165 Fig 1 Effects of boric acid (BA) treatment on the weight loss rate(A)and firmness(B) of ‘Kyoho’
166 grape berries The concentrations of boric acid treatment were 0.00, 0.01, 0.03 and 0.05M,
167 respectively Vertical bars indicate mean ± standard deviation (SD) n = 3 replicates The bars
168 followed by the same letter are not significantly different at P＜0.05
169 3.2 Effects of BA treatment on TSS content and AsA content
170 The variation trend of TSS after BA treatment with different concentrations was consistent with
171 that of the control (Fig 2A) Among the BA treatment groups, the 0.03 and 0.05 M BA treatment
Trang 9172 group showed the highest TSS contents (Fig 2A)
173 The AsA content of 0.01 M BA treatment grape berries were significantly higher than the control
174 during all storage days (Fig 2B) In addition, the content of AsA in 0.03 M BA treatment was also
175 significantly higher than the control except at the 8th storage day (Fig 2B)
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177 Fig 2 Effects of boric acid (BA) treatment on the TSS (A)and AsA content (B) of ‘Kyoho’ grape
178 berries The concentrations of boric acid treatment were 0.00, 0.01, 0.03 and 0.05M, respectively
179 Vertical bars indicate mean ± standard deviation (SD) n = 3 replicates The bars followed by the
180 same letter are not significantly different at P＜0.05
181 3.3 Effects of BA treatment on MDA content, superoxide anion (𝑂𝟐_) production rate and H 2 O 2
182 content
183 Changes in the MDA content of grapes were shown in Fig 3A The MDA content of berries
184 increased during the first 4 days of storage, reaching the peak on day 4, and then declined from day
185 4 to day10 At the peak levels, the MDA content was the highest in the 0.03 M BA treatment group
186 and the lowest in the 0.01 M BA treatment group (Fig 3A) During all storage times other than at
187 the 10th storage day, the MDA content in 0.01 M BA treatment was significantly lower than the
188 control These data showed that BA inhibited the production of MDA, and it slowed the senescence
Trang 10189 rate of grape berries effectively at a concentration of 0.01 M (Fig 3A)
190 The superoxide anion ( ) production rate of grape berries showed the same increase or decrease 𝑂2_
191 trend in all the treatment groups, and the BA treatment groups was lower than the control group on
192 the whole especially in 0.01 M BA treatment group (Fig 3B) Additionally, the 0.01 BA treatment
193 had the lowest hydrogen peroxide content in grape berries among all the treatments (Fig 3C)
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195 Fig 3 Effects of boric acid (BA) treatment on MDA content (A), superoxide anion ( ) production 𝑂2_
196 rate(B) and H2O2 content(C) of ‘Kyoho’ grape berries The concentrations of boric acid treatment
197 were 0.00, 0.01, 0.03 and 0.05M, respectively Vertical bars indicate mean ± standard deviation
198 (SD) n = 3 replicates The bars followed by the same letter are not significantly different at P＜
199 0.05
200 3.4 Effects of BA treatment on SOD and CAT activities