NL2037655A - Method for Determination of Banana Phospholipase A Activity and Its Application - Google Patents

Abstract

The present invention discloses a method for determining the activity of banana phospholipase A and its application. The method primarily investigates the impact of various factors such as substrate concentration, reaction temperature, buffer solution pH, buffer solution volume, and 5 reaction time on the catalytic activity of banana PLA in hydrolyzing phosphatidylcholine. This optimization aims to enhance the method for determining phospholipase A activity. Furthermore, based on the optimal conditions, the variation trend of banana PLA activity under anthracnose stress is further studied. 10 [Fig 1] 19

Description

Method for Determination of Banana Phospholipase A Activity and Its Application

TECHICAL FIELD

[0001] The present invention relates to the field of enzyme activity detection technology, and more particularly to a method for determining the activity of banana phospholipase A and its application.

BACKGROUND

[0002] Bananas belong to the family Musaceae, genus Musa, monocotyledonous plants with nutritious fruits containing mineral elements, resistant starch, dietary fiber, phenolic compounds, carotenoids, phytosterols, amines, antioxidants, and other nutrients and active ingredients, with great processing potential. With the increasing demand for bananas, as of 2019, the global banana harvested area reached 5.1586 million hectares, with a yield of 117 million tons. At the same time,

China's banana production ranked second globally, with net imports ranking third.

[0003] Banana anthracnose, as one of the main diseases during banana fruit storage and transportation, is characterized by the appearance of black-brown spots on the fruit surface during infection. As the lesions enlarge, fruit hardness decreases, fruit peel cell membranes rupture, membrane permeability increases, membrane lipid peroxidation intensifies, and nutrient loss occurs, resulting in blackening and rotting of the flesh within 2-3 days. Currently, the primary methods for controlling banana diseases still rely on chemical treatments. However, due to increased pathogen resistance and issues such as drug residues, the effectiveness of chemical control has decreased. Therefore, inducing banana's own immune system using biological and chemical means to acquire disease resistance has become one of the research hotspots.

[0004] Phospholipids are the basic molecules constituting cell membranes and serve as the first barrier for cell interaction and response to the external environment. As sensitive environmental sensing organs, cell membranes' molecular receptors can generate cell signal transduction through phospholipases (PL) under biological or abiotic stress, enabling cells to respond differently to external changes. Phospholipases can hydrolyze phospholipids into fatty acids and lipophilic substances such as lysophosphatidylcholine, diacylglycerol, and phosphatidylcholine. Depending 1 on the site of phospholipid hydrolysis, they are classified as phospholipase A (PLA), phospholipase C (PLC), and phospholipase D (PLD). Among them, PLA is an important signal transduction enzyme that selectively cleaves ester bonds at the Sn-2 position of phosphatidylglycerol, regulating the balance of phospholipids in the body and promoting cell differentiation and growth. Studies have shown that when plants face environmental stress, PLA activity changes. By hydrolyzing phospholipids on the cell membrane, its products act on the plasma membrane H+ transport, simultaneously altering the membrane's ion permeability, increasing intracellular Ca?* concentration, thereby adapting to changing environmental and developmental conditions. Under anthracnose stress, changes in PLA activity can affect the ripening process of fruits to some extent.

[0005] Enzyme activity determination is the basis for enzyme research, application, and production. Compared with research on animal PLA, although there is already a considerable amount of research indicating that plant PLA is involved in regulating plant growth, development, and stress response processes, research on enzyme activity determination and mechanism of action of plant PLA is still very scarce. Previous literature has reported several methods for determining

PLA-catalyzed hydrolysis of phosphatidylcholine. Among them, the acid-base titration method is an easy-to-operate method for studying PLA activity. The principle of this method is that due to the hydrolysis of phospholipids by phospholipase, a large amount of free fatty acids is produced, and neutralization titration using NaOH can indirectly calculate parameters such as instantaneous reaction rate based on the consumed volume of NaOH. However, researchers found that further optimization 1s needed when applying the acid-base titration method to determine banana PLA activity.

[0006] Although there are various options for PLA activity determination methods, most studies still use the traditional acid-base titration method. As a classical determination method, the acid- base titration method has advantages such as a wide range of applications, strong reliability, and relative simplicity compared to other complex technical methods. However, traditional acid-base titration methods also have some drawbacks, such as potential interference from other substances during sample processing and low accuracy. Therefore, it is still important to optimize the reaction conditions of existing acid-base titration methods, which will help improve the accuracy of 2 determination results and experimental efficiency, and promote the development of related research and applications.

SUMMARY OF THIS DISCLOSURE

[0007] Addressing the need for optimization in the process of using the acid-base titration method to determine banana phospholipase A activity in existing technologies, the present invention provides a method for determining the activity of banana phospholipase A and its application. By studying the factors affecting the activity of banana phospholipase A, the optimal conditions for determining banana phospholipase A are determined. This method for determining the activity of banana phospholipase A is applied to analyze the relationship between changes in

PLA activity and fruit ripening and decay processes under anthracnose stress at different storage times. It holds significant theoretical and practical significance in scientific research and practical production.

[0008] The objective of the present invention is achieved through the following technical solution:

[0009] A method for determining the activity of banana phospholipase A, comprising the following steps:

[0010] 1) Preparation of crude enzyme extract: accurately weighing 3-5g of banana peel tissue into a mortar, add 1-5 times the volume of buffer solution (mL) per sample mass (g) of 5 mmol/L, pH 7.6 Tris-HCI buffer solution, grind in an ice bath for 5 min, filter using a three-layer nylon cloth, centrifuge the filtrate at 4°C, 4000 rpm for 15 min, and collect the supernatant for later use;

[0011] 2) Preparation of substrate solution: preparing a substrate buffer solution containing 2-5 g/L phosphatidylcholine, 2 mmol/L sodium deoxycholate, 5 mmol/L Tris-HCI (pH 7.6), 0.2 mmol/L CaCl:, and 0.2 mol/L NaCl; adjusting the buffer solution to pH 6-9 using 0.4 mol/L NaOH;

[0012] 3) Reaction system: placing the crude enzyme extract and substrate solution at a temperature of 40-60°C for 5 min, take 3 mL of crude extract and 15 mL of substrate solution, mix rapidly, and react at a temperature of 40-60°C for 4-10 h;

[0013] 4) Determination of yield of free fatty acid (FFA): taking 2 mL of the test liquid from the reaction system at different time intervals, diluting with distilled water 25 times, taking 5 mL from the diluted solution into a 50 mL conical flask, titrating in an ice bath with 2 mmol/L NaOH to the 3 initial pH, recording the volume V (mL) of NaOH consumed, and calculating the amount of FFA generated per gram of banana phospholipase A hydrolyzing phosphatidylcholine (umol/g) within a unit time in every gram of banana peel, denoted as pumol/(g-h), representing the activity of banana phospholipase A. The specific calculation formula is as follows:

[0014] FFAproduction = Sade

[0015] PLA activity E = “==,

[0016] Where, C is the concentration of NaOH (mmol/L), V is the volume of NaOH consumed (mL), m is the fresh weight of banana peel (g), t is the reaction time (h), and E is the activity of banana phospholipase A [umol/(g:h)].

[0017] In the present invention:

[0018] In step 1), it is preferred to accurately weigh 5 g of banana peel tissue into a mortar and add 3 times the volume of buffer solution (mL) per sample mass (g) of 5 mmol/L, pH 7.6 Tris-

HCI buffer solution.

[0019] In step 2), it is preferred to prepare a substrate buffer solution containing 4 g/L phosphatidylcholine, 2 mmol/L sodium deoxycholate, S mmol/L Tris-HCI (pH 7.6), 0.2 mmol/L

CaCl:, and 0.2 mol/L NaCl, and adjust the buffer solution to pH=9 using 0.4 mol/L NaOH.

[0020] In step 3), it is preferred to place the crude enzyme extract and substrate solution at a temperature of 55°C for 5 min, take 3 mL of crude extract and 15 mL of substrate solution, mix rapidly, and react at a temperature of 55°C for 7 h.

[0021] The present invention also relates to the application of the above method for determining the activity of banana phospholipase A. Specifically, compared with banana fruits not infected by pathogenic fungi, the activity of banana phospholipase A significantly increases after infection by anthracnose fungi. By measuring the activity of banana phospholipase A in banana fruits, the degree of anthracnose fungal infection can be determined. The greater the activity of banana phospholipase A, the deeper the degree of anthracnose fungal infection in banana fruits.

[0022] Compared with the existing technology, the present invention has the following advantages:

[0023] Plant phospholipase A (phospholipase A, PLA) is a key enzyme in cell membrane phospholipid metabolism. It catalyzes the cleavage of multiple functional groups on phospholipid 4 molecules to generate signaling molecules, participates in regulating plant growth, development, and responses to environmental and biological stress, and possesses significant research value. The method for determining the activity of banana phospholipase A disclosed in the present invention primarily investigates the effects of various factors such as substrate concentration, reaction temperature, buffer solution pH, buffer solution volume, and reaction time on the catalytic activity of banana PLA in hydrolyzing phosphatidylcholine. This optimization aims to enhance the method for determining phospholipase A activity. Furthermore, based on the optimal conditions, the variation trend of banana PLA activity under anthracnose stress is further studied. The results show that the optimized conditions for determining banana PLA activity using the acid-base titration method are as follows: reaction system pH 9, substrate concentration 4 g/L, reaction temperature 55°C, substrate volume 15 mL, crude enzyme extract 3 mL, the ratio of extraction buffer volume to peel weight (mL/g) is 3, and reaction time is 7 h. Under the optimal conditions, the PLA activity of banana peel tissue under anthracnose stress is determined, revealing a positive correlation between the degree of peel disease and PLA activity. As the degree of disease increases, PLA activity increases, significantly exceeding the control group. This indicates that PLA activity may be related to fruit resistance. As the degree of fruit disease deepens, PLA activity increases, phospholipid degradation occurs, cell membrane integrity is compromised, leading to fruit decay.

The present invention provides a convenient and feasible method for determining banana PLA activity, laying the groundwork for further research into the catalytic mechanism of banana PLA.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 depicts the effect of substrate concentration on the hydrolysis of phosphatidylcholine by banana phospholipase A in the exemplary embodiments of the present invention.

[0025] Figure 2 illustrates the impact of temperature on the hydrolysis of phosphatidylcholine by banana phospholipase A in the exemplary embodiments of the present invention.

[0026] Figure 3 shows the influence of initial pH on the hydrolysis of phosphatidylcholine by banana phospholipase A in the exemplary embodiments of the present invention. 5

[0027] Figure 4 demonstrates the effect of extraction buffer volume on the hydrolysis of phosphatidylcholine by banana phospholipase A in the exemplary embodiments of the present invention.

[0028] Figure 5 displays the process curve of yield of free fatty acid (FFA) during the enzyme- catalyzed reaction in the exemplary embodiments of the present invention.

[0029] Figure 6 presents the changes in the appearance of banana fruits under anthracnose infection conditions in the exemplary embodiments of the present invention.

[0030] Figure 7 depicts the variation of banana phospholipase A activity under anthracnose infection conditions in the exemplary embodiments of the present invention.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0031] The following describes specific embodiments of the present invention in conjunction with examples.

[0032] EXAMPLES

[0033] A method for determining the activity of banana phospholipase A, including the following steps:

[0034] 1 Materials and Methods

[0035] 1.1 Test Materials

[0036] Bananas of the ‘Guijiao 1° variety grown in Guangxi Province were selected as the test samples. These bananas were harvested on October 18, 2022, from Guangxi Meicheng

Agricultural Technology Co., Ltd. at a ripeness level of 7-8. They were transported to Guangxi

Key Laboratory of Fruits and Vegetables Storage-Processing Technology on the same day. Only fruits with consistent ripeness, uniform size, no obvious mechanical damage, and no disease were chosen for the experiment.

[0037] Main Reagents: Soybean phospholipid from Shanghai Macklin Biochemical Technology

Co, Ltd. GR grade; deoxycholic acid sodium salt, calcium chloride from Shanghai Yuanye Bio- technology Co., Ltd., analytical grade; sodium chloride from Sinopharm Chemical Reagent Co.,

Ltd, analytical grade; tris(hydroxymethyl)aminomethane from Beijing Solarbio Science & 6

Technology Co., Ltd., analytical grade; hydrochloric acid from Chengdu Kelong Chemical Co.,

Ltd., analytical grade.

[0038] Main Equipment: Constant temperature water bath, model HH-S4, manufactured by

Jintan Wanhua Experimental Instrument Factory, pH meter, model PHS-3C, manufactured by

Shanghai Yifen Scientific Instrument Co., Ltd.; high-speed freezing centrifuge, model 3-18KS, manufactured by Sigma Corporation.

[0039] 1.2 Experimental Methods

[0040] 1.2.1 Preparation of Anthracnose Spore Suspension

[0041] Anthracnose fungus (Colletotrichum musae) was provided by Guangxi Key Laboratory of Fruits and Vegetables Storage-Processing Technology. The fungus was inoculated on PDA solid medium and cultured at 28°C for 7-14 days. The culture surface was gently rubbed with an inoculating loop to facilitate spore detachment. The obtained spore suspension was transferred to 50 mL conical flasks using a sterile pipette, diluted with sterile deionized water, and adjusted to a concentration of 1x10* cfu/mL for later use.

[0042] 1.2.2 Sample Treatment

[0043] Following the method reported by Li Chunling et al., post-harvest banana fruits were subjected to simulated anthracnose infection treatment. Using a micropipette, 2-20 uL of spore suspension (1x10% cfu/mL) was injected into three wounds, each 4 mm in diameter and 2.5 mm deep, evenly made around the waist of banana fruits to serve as the experimental group. An equal volume of sterile water was injected into wounds as the control group. Fifty fruits were included in each treatment group. After injection, all bananas were placed on open trays and stored for over 15 days at a temperature of 25°C and a relative humidity of 60%. Samples were taken at intervals during banana storage and stored in liquid nitrogen.

[0044] 1.2.3 PLA Activity Assay

[0045] Preparation of crude enzyme extract: Accurately weigh a certain amount of banana peel tissue into a mortar, add Tris-HCI buffer (5 mmol/L, pH 7.6) in a volume proportional to the tissue weight, grind on ice for 5 minutes, filter through three layers of nylon cloth, and centrifuge the filtrate at 4°C and 4000 rpm for 15 minutes. Collect the supernatant for later use.

[0046] Preparation of reaction substrate solution: Following the method reported by Lei

Xlangliang et al, prepare a substrate buffer solution containing 2-5 g/L phospholipid, 2 mmol/L 7 deoxycholic acid sodium salt, 5 mmol/L Tris-HCl (pH 7.6), 0.2 mmol/L CaCl:, and 0.2 mol/L

NaCl. Adjust the buffer to the appropriate pH with 0.4 mol/L NaOH.

[0047] Reaction system: Incubate the crude enzyme extract and reaction substrate solution at the appropriate temperature for 5S minutes. Mix 3 mL of crude extract with 15 mL of substrate solution quickly, and incubate the mixture at the appropriate temperature for a certain period.

[0048] Measurement of yield of free fatty acid (FFA): Take 2 mL of the reaction solution at different time points and dilute it 25 times with distilled water. Take 5 mL of the diluted solution in a 50 mL conical flask and titrate with 2 mmol/L. NaOH to the initial pH in an ice bath. Record the volume of NaOH consumed V (mL) and calculate the yield of FFA of banana PLA hydrolyzing phospholipids released per gram (umol/g). PLA activity, expressed as umol/(g:h), represents the amount of free fatty acids produced by PLA hydrolyzing phospholipids per gram of banana peel per unit time. The specific calculation formulas are as follows:

[0049] FFA production = Safes

[0050] PLA activity E = 22

[0051] Where (is the concentration of NaOH (mmol/L), Fis the volume of NaOH consumed (mL), m is the fresh weight of banana peel (g), t is the reaction time (h), and £ is the PLA activity [umol/(g-h)].

[0052] 1.2.4 Effect of Anthracnose Infection on Banana PLA Hydrolysis of Phospholipids

Activity

[0053] A certain amount of banana peel tissue from the experimental group or control group was weighed into a mortar. The tissue was then treated with the optimal volume of 5 mmol/L Tris-HCI buffer (pH 7.6) according to the tissue mass. After grinding on ice for 5 minutes, the mixture was filtered through three layers of nylon mesh. The filtrate was centrifuged at 4°C and 4000 rpm for 15 minutes, and the supernatant was collected for later use. The selected optimal reaction conditions, including substrate concentration, temperature, reaction system pH, and reaction time, were used for subsequent experiments to determine the activity of banana PLA hydrolyzing phospholipids.

[0054] 1.3 Statistical Methods 8

[0055] The experiment was repeated three times, and the results were expressed as mean + standard error. Statistical analysis of the experimental data was performed using SPSS 22 (IBM,

Chicago, USA). The significance of variance between factor levels was analyzed using Duncan's test (P < 0.05). Graphs were generated using Origin 2021 (OriginLab, Northampton,

Massachusetts, USA).

[0056] 2 Results and Analysis

[0057] 2.1 Analysis of Factors Affecting PLA Hydrolysis Activity of Phospholipids

[0058] 2.1.1 Effect of Substrate Concentration on PLA Hydrolysis Activity of Phospholipids

[0059] Referring to the method of Lei Hengliang et al, initial parameters were set as follows: reaction system pH 7.6, temperature 40°C, and buffer volume (mL) / sample mass (g) ratio of 5.

Due to the limited solubility of phospholipids in water, the effect of substrate solution concentration (2 g/L, 3 g/L, 4 g/L, 5 g/L) on the hydrolysis activity of banana PLA on phospholipids was determined, and the dynamic change of yield of FFA with time was recorded.

As shown in Figure 1, under constant conditions, substrate concentration has a significant impact onthe rate of enzymatic reaction. With an increase in substrate concentration, the rate of enzymatic reaction accelerates until it reaches a certain level, after which the reaction rate stabilizes. The FFA content produced by hydrolysis of phospholipids by banana phospholipase A increases gradually with reaction time. Specifically, yield of FFA at substrate phospholipid concentrations of 4 g/L and S g/L is significantly higher than that at 2 g/L and 3 g/L (P < 0.05), with little difference between 4 g/L and 5 g/L. Therefore, subsequent experiments were conducted using a concentration of 4 g/L.

[0060] Figure 1: Effect of substrate concentration on lecithin hydrolysis by banana phospholipase

A.

[0061] Note: Values in the graphs are the mean of three replicate experiments. Error intervals indicate the standard error of the mean. Different letters indicate significant differences among the four lecithin concentrations at the same reaction time (£<0.05).

[0062] 2.1.2 Effect of Temperature on PLA Hydrolysis Activity of Phospholipids

[0063] Figure 2 illustrates the effect of reaction temperature on the hydrolysis activity of banana

PLA on lecithin at a substrate concentration of 4 g/L, reaction system pH 7.6, and buffer volume (mL) / sample mass (g) ratio of 5. The temperatures tested were 40°C, 45°C, 50°C, 55°C, and 9

60°C, and the dynamic change in yield of FFA with time was recorded. As depicted in the graph, yield of FFA significantly decreased at a reaction temperature of 60°C. Below 60°C, an increase in reaction temperature resulted in an increase in yield of FFA, with significant differences observed at each temperature (P < 0.05). Among these temperatures, yield of FFA steadily increased at 55°C and reached the highest level. Temperature has a dual effect on enzymatic reaction: on one hand, within a certain range, increasing temperature accelerates enzymatic reaction, thereby enhancing phospholipase A activity; on the other hand, excessively high temperatures can cause enzyme denaturation and a subsequent decrease in enzyme activity.

Therefore, the optimal temperature for banana PLA activity is 55°C.

[0064] Figure 2: Effect of temperature on lecithin hydrolysis by phospholipase A in bananas.

[0065] Note: Values in the graphs are the mean of three replicate experiments. Error intervals indicate the standard error of the mean. Different letters indicate significant differences among the five temperatures at the same reaction time (P <0.05).

[0066] 2.1.3 Effect of Reaction System pH on PLA Hydrolysis Activity of Phospholipids

[0067] Figure 3 illustrates the effect of reaction system pH on the hydrolysis activity of banana

PLA on lecithin at a substrate concentration of 4 g/L, temperature of 55°C, and buffer volume (mL) / sample mass (g) ratio of 5. The pH of the reaction system was varied between 6, 7, 8, and 9, and the dynamic change in yield of FFA with time was recorded. As shown in the graph, the pH of the reaction system has a significant impact on enzymatic catalytic activity. This may be attributed to pH altering the dissociation state of relevant enzyme active site groups, making them suitable or unsuitable for substrate binding, thus affecting the enzyme-catalyzed reaction outcome. The results indicate that as the pH of the reaction system increases, yield of FFA also increases, with significant differences observed in yield of FFA at different pH conditions (P < 0.05). At pH 6 and 7, yield of FFA is minimal and remains relatively constant over time. At pH 8, yield of FFA is higher than at the former two pH levels and increases slowly over time. The maximum yield of

FFA is observed at pH 9.

[0068] Figure 3: Effect of starting pH on lecithin hydrolysis of banana phospholipase A.

[0069] Note: Values in the graphs are the mean of three replicate experiments. Error intervals indicate the standard error of the mean. Different letters indicate significant differences among the pH of the four reaction systems at the same reaction time (P <0.05). 10

[0070] 2.1.4 Effect of Buffer Volume on PLA Hydrolysis Activity of Phospholipids

[0071] Figure 4 depicts the effect of buffer volume on the hydrolysis activity of banana PLA on lecithin at a substrate concentration of 4 g/L, temperature of 55°C, and reaction system pH of 9.

The buffer volume (mL) / sample mass (g) ratio was varied between 2, 3, 4, and 5, and the dynamic change in yield of FFA with time was recorded. The study reveals significant differences in lecithin hydrolysis by banana phospholipase A under different buffer volumes. Initially, in the early stages of the reaction, higher yield of FFA was observed in the reaction system with a ratio of 2, but as the reaction progressed, yield of FFA gradually increased in the reaction system with a ratio of 3.

In the later stages of the reaction, the reaction system with a ratio of 3 exhibited the highest yield of FFA, significantly different from other ratios (P < 0.05). Therefore, in the assay for banana PLA hydrolysis activity on lecithin, the optimal ratio of buffer volume (mL) to sample mass (g) 1s determined to be 3.

[0072] Figure 4: Effect of extraction buffer volume on lecithin hydrolysis of banana phospholipase A.

[0073] Note: The values in the graphs are the mean of three replicate experiments. Error intervals are expressed as standard error of the mean. Different letters indicate significant differences among the four different ratios at the same reaction time (p <0.05).

[0074] 2.1.5 Determination of Reaction Time

[0075] Figure 5 illustrates the effect of reaction time on the hydrolysis activity of banana PLA on lecithin at a substrate concentration of 4 g/L, temperature of 55°C, and reaction system pH of 9, with the buffer volume (mL) / sample mass (g) ratio set at 3. The dynamic change in yield of

FFA with time was recorded. As depicted in the figure, during the initial 7 hours of the reaction, there was an increase in FFA content in the reaction system as the reaction progressed. At the 8th hour, the reaction rate slowed down, with the FFA content recorded as 155.667 + 0.167 umol/g.

A comparison with other time periods reveals that the reaction rate was fastest at 7 hours, indicating that 7 hours is the optimal reaction time.

[0076] Figure 5: Progress curve of yield of FFA in enzymatic reaction.

[0077] Note: The values in the graphs are the mean of three replicate experiments. Error intervals are expressed as standard error of the mean.

[0078] 2.2 Impact of Anthrax Infection on Banana PLA Activity 11

[0079] As depicted in Figure 6, with the increase in storage time, the degree of browning on the banana peel intensifies, and the fruit gradually decays. Under the infection of anthrax, the diameter of lesions on the experimental group's banana fruits gradually increases, accelerating the rate of fruit decay, while the diameter of lesions on the control group's fruits remains relatively stable, with a slower decay rate. Under the optimal conditions for banana PLA activity determination, i.e., substrate concentration of 4 g/L, temperature of 55°C, reaction system pH of 9, buffer volume (mL) / sample mass (g) ratio of 3, and a reaction time of 7 hours, the impact of anthrax infection on banana PLA activity was assessed. The results, as shown in Figure 7, indicate that with the increase in storage time, both the experimental and control groups exhibited a significant increase in PLA activity. Moreover, the PLA activity in banana fruits inoculated with anthrax was much higher than that in non-inoculated fruits. This suggests that changes in PLA activity are related to the ripening process of bananas, with PLA activity increasing with storage time. Additionally, anthrax infection has a significant impact on PLA activity in banana fruits, leading to a substantial increase in PLA activity.

[0080] Figure 6: Changes in the appearance of banana fruits under anthrax infection.

[0081] Figure 7: Changes in PLA activity of bananas under anthrax infection.

[0082] Note: The values in the graphs are the mean of three replicate experiments. Error intervals are expressed as standard error of the mean.

[0083] 3. Conclusion and Discussion

[0084] PLA is an important hydrolytic enzyme widely present in the cells of animals and plants, participating in various biological processes such as cell membrane metabolism and signal transduction. Several methods have been developed to measure PLA activity in biological samples.

Li Feng et al. discovered that fluorescently labeled phospholipase substances can be catalyzed by

PLA: to produce a fluorescent signal. Thus, real-time fluorescence detection methods can monitor changes in PLA: activity in real-time and determine its activity at the cellular level. Li Hongli et al. used model bile and synthetic fluorescent-labeled substrates to measure the activity of porcine pancreatic PLA;, providing important information for the clinical treatment of gallstones. Mirsky et al. indirectly measured PLA activity by monitoring changes in membrane potential based on the hydrolysis reaction of ester bonds between phosphatidylethanolamine and phosphatidylcholine.

Jiménez M et al. achieved real-time monitoring of PLA; activity using continuous 12 spectrophotometry by directly monitoring the absorbance or emission light signals produced during enzyme-catalyzed reactions. Le Zhen et al. used the agar plate method to study PLA activity, measuring the enzyme activity standard curve of snake venom sPLA: based on the diameter of transparent circles and holes.

[0085] Despite the availability of multiple methods for measuring PLA activity, most studies still rely on traditional titration methods. As a classic method, acid-base titration has advantages such as wide applicability, strong reliability, and relative simplicity compared to other complex techniques. However, traditional acid-base titration also has some limitations, such as susceptibility to interference from other substances during sample processing and low accuracy.

Therefore, it is still crucial to optimize the reaction conditions of existing acid-base titration methods, which will help improve the accuracy of measurement results and experimental efficiency, and promote the development of related fields of research and application. In order to better optimize the reaction conditions of acid-base titration and make it more applicable in detecting plant PLA activity, this study aims to explore the optimal conditions for measuring banana PLA activity through acid-base titration from five aspects: the acid-base balance of the reaction system (pH value), reaction temperature, reaction concentration, ratio of extraction buffer volume to peel mass, and reaction time. These optimization results can provide important references and foundations for further research on plant PLA activity and related biological processes under certain conditions.

[0086] In this experiment, the optimal conditions for the determination of banana PLA activity by acid-base titration were obtained as follows: reaction system pH 9, reaction temperature 55°C, substrate concentration 4 g/L, substrate solution volume 15 mL, crude enzyme extract volume 3 mL, and the ratio of extraction buffer volume to peel weight (mL/g) as 3, with a reaction time of 7 hours. Under these conditions, the highest yield of FFA reached 154.78 £ 0.111 umol/g, and the phospholipase activity was 22.111 + 0.016 pmol/(g-h). Similarly, under the same reaction time, Li

Hongli et al. used acid-base titration to measure the PLA activity in rice as 11.428-14.286 umol/(g-h), while Deng Zhi et al. used spectrophotometry to measure the PLA activity in rubber latex as 39.857-43 umol/(mL-h). These optimal conditions were then used to detect PLA activity in banana fruits under anthrax disease stress. The results showed that after anthrax infection of banana fruits, the PLA activity increased significantly compared to fruits not infected by the 13 disease. Furthermore, as the severity of the disease increased, the PLA activity of the fruits also increased continuously. This may be because under the attack of the disease, the cell membranes of the fruits are damaged or altered. In order to repair the damaged cell membranes and release signaling molecules and metabolites beneficial for resisting pathogen invasion, banana fruits increase the activity of PLA enzymes, causing hydrolysis of phosphatidylcholine and other phospholipids to release free fatty acids, solute products, and secondary signaling molecules to participate in plant defense responses. Moreover, the increase in PLA activity may also be due to the perception and response of banana fruits to signaling molecules produced by pathogenic microorganisms or stress. These signaling molecules may regulate the expression and activity of

PLA enzymes to modulate plant defense responses. Therefore, we believe that the changes in PLA activity in fruits are closely related to the degree of disease infection. These findings provide important experimental evidence for further research on the relationship between PLA activity and plant defense responses.

[0087] Here's the reference list formatted:

[01] JIE M M, KE Y P. Discussion on the Deep Processing of Bananas in China. Tropical Agricultural

Science, 2013, 33(03): 75-78.

[02] YANG E LIU C C, DENG GM, HE W D, LI CY, YI Q J, SHENG O. Research progress on nutritional quality and functional characteristics of bananas. Agricultural Science, 2022, 49(10): 146- 154.

[03] HONG JM, HEY S, ZHENG FY, ZHENG Y Y, ZHANG S. Research progress in banana product processing technology. Chinese Journal of Agriculture, 2016, 32(34): 180-186.

[04] HU C J. Analysis of the Changes and Trends in the World Banana Market. China Tropical

Agriculture, 2020, (06): 39-41.

[05] HUANG Y Y, XU X J. The Current Situation and Development Trends of the Global Banana

Industry. Tropical Agricultural Engineering, 2021, 45(05): 34-38.

[06] TAO X Z. QIU H B. LIL, JIANG J P. Study on the correlation between Phospholipase activity, membrane permeability changes and ethylene release during banana ripening. Journal of Horticulture, 1985, (03): 145-150.

[07] WU X J. CHEN Q, LIANG P G, ZHAO C G, LUAN HY, XIE Z Q. FENG X Q. XIANG Y,

YANG ZX. HE X M. Membrane lipid metabolism of banana under Anthrax stress. Journal of Southern 14

Agriculture, 2021, 52(05): 1325-1333.

[08] ZHOU Y K, CHEN X Y, ZHENG F C. Research progress on Biological pest control of banana

Anthrax. Chinese Journal of Agriculture, 2008, (04): 328-331.

[09] CHEN H P, CHEN X M. CAI SY. Changes in reactive oxygen species metabolism during post ripening of banana fruits. Joumal of Horticulture, 2000, (05). 369-370.

[10] TAN W P, PANG X Q, ZHANG Z Q, HUANG X M. The relationship between BABA induced disease resistance and accumulation of reactive oxygen species during storage in banana fruits. China

Agricultural Science, 2014, 47(16): 3290-3299.

[11] GONZALEZ-MENDOZA VICTOR M, SANCHEZ-SANDOVAL M E, CASTRO-CONCHA

LIZBETH A, HERNANDEZ-SOTOMAYOR S M TERESA. Phospholipases C and D and Their Role m Biotic and Abiotic Stresses. Plants (Basel), 2021. 10(5).

[12] SHI J F, ZHANG Q, SONG S Q, LIU J. Research progress on Phospholipase and its regulation of seed vigor. Journal of Southern Agriculture, 2022, 53(09): 2612-2623.

[13] ZHANG L, ZHANG H. Overview of Phospholipase A2 and its functions. Journal of Qiqihar

Medical University, 2004, (02): 178-180.

[14] LEI X L, ZENG X C. Study on the determination method of rice Phospholipase A activity. Journal of Jiangxi Agricultural University, 2011, 33(05): 851-854.

[15] LIL. HEX M. LIC B, SUN J, LING D N, SHENG JF, RAO CY, XIAO ZS, LIJ M, ZHENG

FJ, LIU GM, TANG YY. Effects of anthracnose infection on postharvest quality changes and disease resistance related enzyme activities of bananas. Modern Food Technology, 2017, 33(09): 83-90.

[16] LI CL, WANG Q G, XU LN, CHEN Q M. Study and identification of antagonistic antibacterial activity of a mango Anthrax. Food Industry Technology, 2012, 33(11): 147-150.

[17] ZHANG J L, ZHU G H. Comprehensive Utilization of Barley Roots (1) - Study on Extraction

Conditions and Partial Properties of Phospholipase. Western Grain and Oil Technology, 1999, (01): 44-47.

[18] LI M, YANG J G, YANG B. Phospholipase A_ A Study on the Method for Determining Enzyme

Activity. Modern Food Technology, 2007, (08): 80-82.

[19] FENG LI, MANABE KELLY, SHOPE JOSEPH C. WIDMER STANTON, DEWALD DARYLL

B, PRESTWICH GLENN D. A real-time fluorogenic phospholipase A(2) assay for biochemical and cellular activity measurements. Chem Biol, 2002, 9(7): 795-803. 15

[20] LI H L, WANG Z Q, YANG Q, SUN LM, ZHU Y Y, AN X Q. Phospholipase A in model bile

A New Method for Activity Determination. Journal of Nanjing Normal University (Natural Science

Edition}, 2009, 32(03): 138-140.

[21] MIRSKY V M, CHERNY V V, SOKOLOV V 5, MARKIN V S. Electrostatic assay of phospholipase A activity: an application of the second harmonic method of monitoring membrane boundary potentials. J Biochem Biophys Methods, 1990, 21(4): 277-284.

[22] JIMENEZ M, CABANES J, GANDIA-HERRERO F, ESCRIBANO J , GARCIA-CARMONA

F , PEREZ-GILABERT M. A continuous spectrophotometric assay for phospholipase A(2) activity.

Anal Biochem, 2003, 319(1): 131-137.

[23] LE Z, CHEN X M, YAO S H, FENG J Q, ZHU X P, CHEN M. Anti snake venom Phospholipase

A of water extract of sappan_ 2. Activity Study. Modern Agricultural Research, 2018. (12): 23-26.

[24] ZHANG J. The effect of Resveratrol on airway inflammation and airway remodeling in chronic asthma model mice and its mechanism. Nanjing Medical University, 2012.

[25] TONG X X, ZHAO J S, LIU J F. Comparison of two measurement methods for Snake Venom

Phospholipase A_2. Journal of Hebei Medical University, 1999, 20(06): 372.

[26] ZHAO M M, XUE Z L, HUANG Z Y, SUY N, MA Q Y. Comparison of three methods for determination of Phospholipase A1 activity. Food and Fermentation Technology, 2012, 48(03): 74-77.

[27] DENG Z, LIU S Z, XIAO X Z. Purification and enzymatic characterization of phospholipase A2 from latex of Hevea brasiliensis. Guihaia, 2010, 30 (06): 876-880.

[0088] The foregoing description sets forth only the preferred embodiments of the present invention, and it should be understood that various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention as set forth in the claims. 16

Claims (5)

Conclusions 1. A method for determining the activity of banana phospholipase A, including the following steps: step 1) preparation of crude enzyme extract: accurately weigh 3-5g of banana peel tissue in a mortar, add 1-5 times the volume of buffer solution (mL) per sample mass (g) of 5 mmol/L, pH 7.6 Tris-HCl buffer solution, grind in an ice bath for 5 minutes, filter with a three-layer nylon cloth, centrifuge the filtrate at 4°C, 4000 rpm for 15 minutes, and collect the supernatant for later use; step 2) preparation of substrate solution: prepare a substrate buffer solution containing 2-5 g/L phosphatidylcholine, 2 mmol/L sodium deoxycholate, 5 mmol/L Tris-HCl (pH 7.6), 0.2 mmol/L CaCl, and 0.2 mol/L contains NaCl; adjust the buffer solution to pH 6-9 using 0.4 mol/L NaOH; step 3) reaction system: place the crude enzyme extract and substrate solution at a temperature of 40-60°C for 5 minutes, take 3 mL of crude extract and 15 mL of substrate solution, mix quickly, and react at a temperature of 55°C for 7 o'clock; and step 4) determination of the yield of free fatty acids (FFA): take 2 mL of the test liquid from the reaction system at different time intervals, dilute with distilled water 25 times, take 5 mL of the diluted solution into a 50 mL conical flask, titrate in an ice bath with 2 mmol/L NaOH to initial pH, record the volume V (mL) of NaOH consumed, and calculate the amount of FFA produced per gram of banana phospholipase A hydrolyzing phosphatidylcholine (umol/g) within unit time in each gram of banana peel, denoted as umol/(g:h), and represents the activity of banana phospholipase A; the specific calculation formula is as follows: Efficiency of FFA = Dn. PLA activity E = === 17 where, ( is the concentration of NaOH (mmol/L), I is the volume of NaOH consumed (mL), m is the fresh weight of banana peel (g), t is the reaction time (h) , and £ the activity of banana phospholipase A [umol/(g-h)]. The method for determining the activity of banana phospholipase A according to claim 1, characterized in that, in step 1), 5 g of banana peel tissue is accurately weighed in a mortar and 3 times the volume of buffer solution (mL) per sample mass (g) of 5 mmol/L, pH 7.6 Tris-HCl buffer solution is added. The method for determining the activity of banana phospholipase A according to claim 1, characterized in that, in step 2), a substrate buffer solution is prepared containing 4 g/L phosphatidylcholine, 2 mmol/L sodium deoxycholate, 5 mmol/L Tris-HCl ( pH 7.6), 0.2 mmol/L CaCl2, and 0.2 mol/L NaCl; the buffer solution is adjusted to pH=9 using 0.4 mol/L NaOH. The method for determining the activity of banana phospholipase A according to claim 1, characterized in that, in step 3), the crude enzyme extract and the substrate solution are placed at a temperature of 55°C for 5 minutes, 3 mL of crude extract and 15 mL of substrate solution are taken, mixed quickly, and reacted at a temperature of 55°C for 7 hours. An application of the method for determining the activity of banana phospholipase A according to any one of claims 1 to 4, characterized in that, compared to bananas not infected by pathogenic fungi, the activity of banana phospholipase A increases significantly after infection by anthracnose -fungi; by measuring the activity of banana phospholipase A in bananas, the extent of infection with anthracnose fungi can be determined; the greater the activity of banana phospholipase A, the deeper the degree of infection with anthracnose fungi in bananas. 18