Optimisation of the ratio of chitosan, tea polyphenols, and citric acid for avocado preservation and validation of the changes in quality

48 The effect of chitosan, tea polyphenols, and citric acid on avocado storage duration was investigated using a central 49 composite design and response surface methodology by using Design-Expert software. A quadratic polynomial regression 50 mathematical model was constructed with time as the response value to obtain the best concentration combination of 51 chitosan, tea polyphenol, and citric acid for developing the basis of a low-cost, high-efficiency storage preservative for 52 avocados. The results show that the analysis of variance of the regression model of the avocado storage duration has a p = 53 0.0003, indicating that the model reaches an extreme level of statistical significance, and the regression coefficient of R2 = 54 0.9629 indicates a good fit between the model and the equation (96.29%). The optimisation results show that the 55 preservative effect on avocados was the best when chitosan, tea polyphenols, and citric acid were combined at a 56 concentration of 1%, 2% and 2%, respectively, and when the storage duration was 19.2 days. Validation tests confirmed 57 that this composite agent effectively delayed the decrease in hardness, the weight loss rate, the soluble solids, soluble 58 protein and vitamin C （ VC ） content of avocados and decreased their browning index, similar to the predicted results. These 59 results carry practical significance for guiding avocado storage and preservation technology development. 60

Avocado (Persea americana Mill.), also known as butter fruit and alligator fruit, is an evergreen fruit tree from family 68 Lauraceae, genus Persea. It is native to the humid tropical and subtropical regions of Central America and Mexico (Landahl 69 et al., 2009). Avocado pulp is sticky and delicate and has a light fragrance. It is the only fruit with a high oil content and has 70 been nicknamed "forest butter". Avocado has high nutritional value, being rich in fat, protein, vitamins A, C, E, B1, and B2, 71 sodium, potassium, magnesium, calcium, and other vitamins and minerals. Avocado's fat contains up to 80% unsaturated 72 fatty acids, making it an uncommon high-energy but low-sugar fruit. It is used as food in the tropical parts of the Americas. 73 Thus, avocado is a health fruit that has the combined qualities of fruit, grain, and oil (Meyer and Terry, 2008). Avocado has 74 significant health-related functions, including preventing diabetes, lowering the cholesterol and blood lipid contents, 75 protecting the liver, strengthening the stomach, clearing the intestines, and beautifying and moisturising the skin. It is 76 regarded as a treasure among fruits in Europe, America, Japan, and other countries, being known as "forest butter" and "the 77 source of life" (Dreher and Davenport, 2013;Ozdemir, 2004).With the consumer lifestyle changes in recent years, avocado 78 has attracted widespread recognition as a high-energy but low-sugar fruit, and its consumption is steadily increasing. 79 However, avocado is a respiration climacteric fruit with a short after-ripening period disenables its long-term storage and 80 preservation, which limits the development of the avocado industry. Therefore, research on avocado preservation 81 technologies is of particular importance. 82 Tea polyphenols, chitosan, and citric acid are commonly used natural preservatives that can extend the preservation 83 period of foods. The combined use of these three preservatives can enhance the preservative effect, is relatively inexpensive, 84 simple in operation, and non-toxic (Zheng et al., 2020).Tea polyphenols have strong free radical-scavenging capacity, which 85 prevents lipid peroxidation. By scavenging free radicals, the activity of antioxidant enzymes is enhanced, thereby further 86 colour preservation (Wang et al., 2021;Fan et al., 2014). Chitosan forms an extremely thin, uniform, transparent and firm film 89 on fruits, which inhibits evaporation and pathogen invasion, thereby exerting antibacterial effects (Gong et al., 2020;Guo et 90 al., 2021;Liu et al., 2021); it is widely used for preserving fruits and vegetables. Single preservatives are unable to fully meet 91 the food preservation requirements, and multiple preservatives must be combined to achieve a higher food preservation 92 performance Tang and Lu, 2020;Yang al., 2020;Yang al., 2019) .Thus, composite preservatives have become 93 a popular research and development topic. In the present study, tea polyphenols, chitosan, and citric acid were selected for 94 producing a composite with the best preservative efficacy for solving the problem of the short storage period of avocados. 95

MATERIAL AND METHODS 96
Materials and reagents 97 The experimental material was avocado variety Guiyan 10 collected by Guizhou Subtropical Crop Research Institute, 98 Guizhou Academy of Agricultural Sciences, China. Plant experimental research conforms to institutional, national or 99 international standards. We abide by the Convention on biological diversity and the Convention on trade in endangered 100 species of Wild Fauna and flora.A total of 7.5-8 mature avocados with no pests, no damage, and uniform size were 101 randomly selected and transported back to the laboratory on the same day. 102 Food-grade tea polyphenols with 99% active ingredients were obtained from Xi'an Dafengshou Biotechnology Co., 103 Ltd. Food-grade carboxymethyl chitosan with 99.9% active ingredients was obtained from Zhengzhou Jiajie Chemical Co., 104 Ltd. Citric acid with 99% active ingredients was obtained from Wuhan Wanrong Technology Development Co., Ltd. 105

Sample treatment 106
First, avocados were washed with clean water and any surface water was dried. Next, the avocados were soaked in a 107 preservative solution for 3 min; for the control group, no treatment was applied. The processed avocados were stored at 108 room temperature. A total of 10 avocados were processed each time, and the experiments were performed in triplicate. 109

Experimental design 110
The central composite design method in Design-Expert software was used. An experimental design with three factors and 111 three levels was used with the tea polyphenols, chitosan, and citric acid being independent variables and the storage 112 duration being the dependent variable. Table 1

Measurement indicators and methods 119
Measurement of hardness 120 The fruit hardness was measured using a GY-4 digital fruit hardness tester. A thin slice of the pericarp (approximately 1 121 mm in thickness) was pared off at three equidistant locations around the equator of the avocado, and the firmness of the 122 pulp at each location was measured using a fruit hardness tester. The hardness of each fruit was measured at three points, 123 the measurement data was recorded and the average fruit hardness value was calculated. 124 Determination of the weight loss rate 125 The avocados were weighed. 128

134
A sensory grading method was used. Grade 1 indicates no pulp browning, grade 2 indicates a slight pulp browning, 135 grade 3 indicates a significant pulp browning not exceeding 1/2 of the pulp area, grade 4 indicates a significant pulp 136 browning exceeding 1/2 of the pulp area, and grade 5 indicates the browning of all the pulp. 137

Determination of the soluble solid content 138
The soluble solid content was measured using a hand-held digital Brix refractometer. A mass of 5 g of avocado pulp was 139 weighed and wrapped in six layers of gauze. A total of 2-3 drops of avocado juice were squeezed into the test port of the 140 Brix refractometer and the value was read. 141

Determination of the vitamin C content 142
The VC was determined using the 2,6-dichlorophenol indophenol titration method. A mass of 2.5 g of avocado pulp was 143 weighed and placed in a mortar, ground to a slurry in an ice bath, added to a 25-mL volumetric flask and the volume was 144 topped up with a 2% oxalic acid solution. A volume of 10 mL of the filtrate was withdrawn, added to an Erlenmeyer flask 145 and titrated using a standard 2,6-dichlorophenol indophenol solution until a reddish colour that did not fade for 15 s 146 appeared. The amount of dye added was recorded. In addition, 10 mL of a 2% oxalic acid solution were used as a blank and 147 were titrated in the same way (Yu , 2012;Gao , 2006) .The experiment was performed in triplicate. 148 The vitamin C content was determined based on the dye consumption during the titration, was expressed in mg of 149 ascorbic acid per 100 g of fresh weight (FW) of the sample (mg/100 g FW) and calculated as follows: 150

Determination of the soluble protein content 152
The Coomassie Brilliant Blue method was used to determine the protein content. In total, 0.5 g of avocado pulp was 153 weighed and ground to a slurry in an ice bath. Distilled water (5 mL) was added and the mixture was centrifuged at 4°C later use. A total of 1.0 mL of supernatant was collected as a sample, and 5.0 mL of a Coomassie Brilliant Blue G-250 156 solution were added, mixed well and allowed to stand for 2 min. The absorbance of the sample was measured at a 157 wavelength of 595 nm. A standard curve was constructed by determining the absorbance in the same way (Lu and Li, 2012; 158 Li al., 1999).The experiment was performed in triplicate. 159 Based on the absorbance of the solution and the corresponding amount of protein (in mg) based on the standard curve, 160 the soluble protein content in the avocado pulp was calculated as milligrams of soluble protein per gram of fruit tissue 161 (mg/g FW), as follows: 162

Response surface methodology experiments and analysis 165
The central composite design method in Design-Expert software was used to obtain comprehensive single-factor test 166 results (Luo et al., 2021). The three independent variables were designated A (chitosan), B (tea polyphenols), and C (citric 167 acid), and the dependent variable Y (storage duration) was used in an experimental design with three factors and three levels 168 for a total of 17 design points (5 centre points). The mean storage duration (Y) was used as the response variable for 169 regression analysis ( Table 2). The regression equation obtained for the storage duration (Y) from the analysis is as follows: 170 Table 3

Results of tea polyphenol, chitosan and citric acid composite optimisation 224
The optimisation results A = 1.0, B = 2.0, and C = 2.0 were predicted using software analysis, and the value Y = 19.2 was 225 obtained. The analysis showed that when the concentration values of tea polyphenols, chitosan, and citric acid were 1.0%, 226 2.0%, and 2.0%, respectively, the storage duration of avocado was 19.2 days, and the storage and preservation effect was 227 optimal. To validate the reliability of the prediction results, a validation test was performed to analyse the changes in 228 avocado quality during storage. 229

Validation test of composite optimisation 230
The untreated avocados and avocados treated with 1.0% tea polyphenols, 2.0% chitosan, and 2.0% citric acid composite 231 solution were placed at room temperature, and their quality indexes were determined after 10 days in control storage 232 conditions and 19 days after a composite solution treatment. A comprehensive comparison was performed to validate the 233 changes in avocado quality during storage after treatment with the composite preservative. 234 weight loss 235 Figure 4A shows the changes in the weight loss rate of the avocados. The weight loss rate gradually increased with the 236 increasing storage duration. The water loss of the avocados in the control group increased and decreased rapidly in the first 237 10 days. The treatment with the composition preservative solution delayed water loss to some extent and inhibited the 238 increase in the water loss rate. Up to day 19, the weight loss rate in the treatment group was significantly lower than that in 239 the control group.

246
Hardness 247 The hardness of the avocados decreased with an increasing storage duration ( Figure 4B). The hardness of the avocados was 248 initially measured at 10 kg/cm2, but the treatment with the composite preservative solution significantly inhibited the 249 decrease in hardness. At 10 days of storage, the hardness of the control group avocados was 2.5 kg/cm2. At 19 days of 250 storage, whereas the hardness of the treatment group avocados was 3.5 kg/cm2 ,the hardness remained at a high level. 251 Soluble Solids 252 Figure 4C shows that the soluble solid content of the avocados in the control group increased continuously. At 10 days of 253 storage, the maximum content of soluble solids was 7.22%. After treatment with the composite preservative solution, the 254 soluble solid content increased slowly over the first 10 days, rapidly increased to a peak at 7.24% on day 15, and then 255 remained unchanged between days 15 and 19. 256 Soluble Protein 257 Figure 4D shows that the soluble protein content of the control group increased rapidly during storage, reaching 5.72 mg/g 258 on day 10. The treatment with the composite preservative solution delayed the change in the soluble protein content. At day 259 group. On day 19, the soluble protein content increased to 6.21 mg/g, indicating that the composite preservative solution 261 controlled the rapid soluble protein content increase. 262 Vitamin C 263 Figure 4E shows that the VC content of the avocados during the storage period gradually increased as the avocados matured. 264 After the control treatment, the VC content increased rapidly to a peak of 37.26 mg/100 g on day 8. After treatment with the 265 composite preservative solution, the VC content increased significantly more slowly, indicating that the tea polyphenol, 266 chitosan and citric acid composite solution could delay the time at which the VC content reaches a maximum, thereby 267 maintaining the quality of the avocados. 268 Browning Index 269 Figure 4F shows the changes in the browning index of the avocados. During the storage period, there was no significant 270 browning in the control or the treatment group avocados in the first 5 days. Subsequently, the browning index of the 271 avocados in the control group increased continuously, reaching 21.28%. After the treatment with the composite preservative 272 solution, the browning of the avocados was significantly controlled. At day 19 of storage, the browning index was only 273 2.85%. Figure 5A shows that the skin of the avocados was bright green and smooth in the early stages of storage. By day 10, 274 the colour changed to yellowish-green, and folds appeared on the avocado skin, accompanied by brown spots( Figure 5B). Avocado is a respiration climacteric fruit with strong post-harvest metabolism, a short after-ripening period, and a short 284 preservation period. It matures and softens within 5-7 days at room temperature, and is susceptible to microbial infections 285 that cause anthracnose, brown rot, stalk rot, and other diseases. The skin colour of avocados rapidly changes to brown, the 286 pulp decays, the quality and flavour deteriorate rapidly and they lose commercial value, which cause severe economic 287 losses (Wang et al., 2010;Cao et al., 2013). Currently, the avocado preservation technologies used in China are not advanced, 288 which severely limits the development of the avocado industry (Bao, 2017) .Avocado preservation methods are broadly 289 divided into two categories: chemical and physical preservation. Chemical preservation primarily uses fungicides to prevent 290 fruit rot and delay ripening. Physical methods primarily include heat treatment, low temperature storage, low-pressure efficacy; for chemical preservation, the selection of a suitable preservative is essential. Currently, many studies on produce 293 storage and preservation techniques have been conducted, but relatively few have focused on avocado. Wei et al. studied the 294 inhibitory effects of various fungicides on ulcers and anthracnose in avocado, and the results showed that 45% prochloraz 295 and 10% difenoconazole are effective in inhibiting anthracnose and ulcers, respectively (Wei, 2015;Wei, 2014). A study by 296 Huang et al. found that SporGon at a low concentration could delay the post-harvest decay of Guiken III and Hass 297 avocados (Huang et al., 2010). Treating avocado fruit with a wax containing 0.4% thiabendazole and 0.1% benomyl reduced 298 the incidence of post-harvest diseases and extended the shelf life by 2-3 days (Sisler et al., 1996) .A study by Zhong et  polyphenols and citric acid were combined at concentration values of 1%, 2%, and 2%, respectively. The comprehensive 317 comparison results showed that the level of influence of each factor on the avocado storage duration in decreasing order is: 318 citric acid > tea polyphenols > chitosan. The composite solution of 1% chitosan, 2% tea polyphenols and 2% citric acid 319 effectively prolonged the avocado storage duration. The validation test results showed that a composite solution of 1.0% tea 320 polyphenols, 2.0% chitosan, and 2.0% citric acid significantly extended the storage duration of the avocados and controlled 321 the deterioration of their quality. Compared with the control group at 10 days of storage, the treatment group at 19 days of 322 storage, which was treated using the composite solution, showed a slower decrease in the weight loss rate, and the hardness 323 and the soluble solid, soluble protein, and VC contents did not decrease, indicating that the original quality was maintained. 324 Moreover, the browning index was lower than 5.0%, thus the reliability of response surface optimization results is verified. 325 326 CONCLUSION 327 The central composite design method of design expert software was used, with a (tea polyphenols), B (chitosan), C (citric 328 acid) as independent variables and Y (storage time) as dependent variables. The results showed that the order of the factors concentration combination for the preservation of avocado was the mixture of 1% chitosan, 2% tea polyphenols and 2% 331 citric acid, and the storage time of avocado was 19.2 days. 332 In order to verify the reliability of the prediction results, the quality changes of avocado during storage were tested. 333 The hardness, soluble solids, soluble protein and Vc content of avocado were still higher after 19 days treatment with 1.0% 334 tea polyphenols, 2.0% Chitosan and 2.0% citric acid. The weight loss rate decreased slightly and the browning index 335 increased slowly. These results were similar to the predicted ones, further confirming the reliability of the response surface 336 methodology optimisation results. Thus, the composite solution of 1% chitosan, 2% tea polyphenols, and 2% citric acid 337 effectively prolonged the post-harvest storage duration of avocados.