Proteomic Analysis Unveils the Protective Mechanism of Active Modified Atmosphere Packaging Against Senescence Decay and Respiration in Postharvest Loose-Leaf Lettuce

1. Summary

Dear editor and reviewers:

First, we would like to thank both the editor and the reviewers for their interest in our study and their supportive and helpful comments to improve the manuscript (agriculture-3293397) that entitled “Modified Atmosphere Packaging prolongs postharvest shelf-life of loose-leaf lettuce: Analysis of involved mechanisms using a proteomic approach”. Guided by these comments, we feel that the conceptual innovation and practical applications of our work are now much clearer in our revised text. 

Again, we would like to offer our thanks, and feel this revision has yielded a more technically correct, rigorous, and useful description of our study and its implications.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: I suggest to re-write the title, avoiding the use of “:” I think that the sentence “Modified Atmosphere Packaging prolongs postharvest shelf-life of loose-leaf lettuce” could rest readers´ interest, because the beneficial effect of MAP on lettuce preservation is well known.

Response 1: Let us first take this opportunity to thank the reviewer for his/her helpful comments on our work. Based on your suggestions, we have avoided the use of colons and reworded the title to more accurately reflect the core of our study and its innovations. We have changed the original title “Modified Atmosphere Packaging prolongs postharvest shelf-life of loose-leaf lettuce: Analysis of involved mechanisms using a proteomic approach” to “Proteomic analyses to investigate the effect of Modified Atmosphere Packaging on senescence decay and respiration in loose-leaf lettuce”.

Comments 2: Line 34. Please, replace “loose” with “Loose”

Response 2: Thank you for your kind correction. We are sorry for this mistake and have rewritten it as ‘‘Loose-leaf lettuce is one of the most popular vegetables because of its crisp texture and rich nutritional profile, including vitamin C and polyphenols.”. (Line 37, Page 1)

Comments 3: Line 41. Please, include “and” between “methods [5]” and “synergistic”.

Response 3 Thank you for your careful review of our manuscript. We have added “and” between “methods [5]” and “synergistic” according to your pertinent suggestion to ensure the fluency and accuracy of the sentence. “These approaches encompass chemical treatments [5], physical preservation methods [6], and synergistic combinations of these treatments [7], as well as innovative biological applications, such as the use of green tea extracts [8] and whey protein [9].” (Line 52-55, Page 2)

Comments 4: Line 55. The authors argue that “Nevertheless, the specific conditions for the application of MAP on loose-leaf lettuce remain unclear.”  I think that the MAP conditions for lettuce are well stablished/known.

Response 4: We thank the referee for the reminding. We would like to clarify that, while the optimal Modified Atmosphere Packaging (MAP) conditions of loose-leaf lettuce and its physic-chemical effects have been extensively researched. However, the mechanisms of how MAP specifically affects loose-leaf lettuce at the molecular level of protein metabolism are still not clear. To avoid unnecessary ambiguity, we have revised the relevant statements as “Nevertheless, the mechanisms underlying protein metabolism regulation, including oxidative, wilting, and deterioration processes, during the storage of MAP-treated loose-leaf lettuce remain unclear. This uncertainty limits the broader application of this technology in lettuce storage.” (Line 70-73, Page 2)

Comments 5: Line 79. I think the authors should specify that the gas composition indicated in line 79 was the initial atmosphere. How did you select this atmosphere? This initial atmosphere and the gas composition inside of the package over storage do not match the recommended gas composition for lettuce. The authors should also specify how the film was perforated (section 2.1) and provide the internal gas composition in control packages, as 7 holes of 0.5 mm of diameter are not enough to hypothesize that that atmosphere was regular air.

Response 5: We appreciate the referee’s insightful comments. For determining the optimal Modified Atmosphere Packaging (MAP) conditions during the preservation of loose-leaf lettuce, we conducted a comprehensive experimental optimization phase. This involved varying the concentrations of O2 from 3% to 20%, CO2 from 10% to 30% and N2 from 50% to 87%. Through this meticulous process, we identified the most effective gas mixture as 10% CO2, 3% O2 and 87% N2. The results were reported in our previous research named “Condition Optimization of Modified Atmosphere Packaging Preservation for Lose-Leaf Lettuce” [1]. Therefore, we choose the optimal gas ratios of 10% CO₂, 3% O₂, and 87% N₂ for the loose-leaf lettuce preservation in this study.

[1] Xu YX, Chen XN, Xu L. Condition Optimization of Modified Atmosphere Packaging Preservation for Loose-Leaf Lettuce [J]. Chinese Agricultural Bulletin, 2016, 32(07): 185-190.

Thank the reviewer very much for pointing out the problem about the oversight regarding the hole diameter. We would like to extend our sincere apologies, after careful checking, we discovered that the unit was incorrect. There were 7 holes of 0.5 cm of diameter on each side of the film, totaling 14 holes. we have rewritten the corrections to the revised manuscript to ensure that the descriptions are accurate.

“The other portion of loose-leaf lettuce served as a control, encapsulated in a high barrier film. A 0.5 cm diameter needle was used to make 7 holes in each side of the film, totaling 14 holes, which was also stored in a refrigerator at 4°C. Samples were taken daily for six days.” (Line 91-94, Page 2)

We apologize again for our carelessness and thank the reviewer very much again for the correction.

Comments 6: Title for section 3.2. In my opinion, the sentence “gas composition of loose-leaf lettuce under MAP” is incorrect, as you probably want to refer to the gas surrounding the lettuce not to the gas composition inside of the lettuce.

Response 6: Thank you for drawing our attention to this important point. We are sorry for our misleading statement. To make it clearly, we have revised the title of section 3.2 to “Weight loss of loose-leaf lettuce and changes in gas composition in MAP group packaging.” (Line 241, page 5)

Comments 7: The results and discussion are so good, however, the conclusions are so basic and expected. It is well known that MAP will alter the TCA metabolism and respiratory chain processes.  I missed if some of the identified proteins were related to browning of lettuce, which is one of the most common factors affecting lettuce quality.

Response 7: We greatly appreciate the reviewer’s positive comments on our results and discussion. We are also grateful for your helpful comments which greatly improved our conclusion. We have supplemented and revised some important results in the conclusion to make it more comprehensive. “In this study, we successfully used physicochemical and proteomic methods to study the quality changes of loose-leaf lettuce in cold storage after MAP treatment. The physicochemical experiments showed that MAP effectively prolonged the storage time of lettuce. With the help of proteomic analysis, it was found that MAP inhibited the apoptotic process by decreasing the expression of protein disulfide bond isomerase and increasing the expression of OB-folding nucleic acid binding site; slowed down the decay process of lettuce by down-regulating the expression of glutamine syntheses; enhanced the defense against cellular oxidative damage and maintained the dynamic balance of the electron transport chain by upregulating the expression of cytochrome c oxidase and the expression of membrane-bound pyrophosphorylase. Controls energy metabolism in the TCA cycle by downregulating dihydrolipoic acid dehydrogenase and malate dehydrogenase; inhibits ATPase synthesis, reduces the rate of electron generation and transfer by downregulating key enzymes in the oxidative phosphorylation pathway. These results provide the theoretical basis for regulating lettuce metabolism and serve as a reference for further quantitative transcriptomic and metabolomics studies.” (Line 477-491, page 13)

Thank the reviewer very much again for the suggestion.

1. Summary

Dear editor and reviewers:

First, we would like to thank both the editor and the reviewers for their interest in our study and their supportive and helpful comments to improve the manuscript (agriculture-3293397) that entitled “Modified Atmosphere Packaging prolongs postharvest shelf-life of loose-leaf lettuce: Analysis of involved mechanisms using a proteomic approach”. Guided by these comments, we feel that the conceptual innovation and practical applications of our work are now much clearer in our revised text. 

Again, we would like to offer our thanks, and feel this revision has yielded a more technically correct, rigorous, and useful description of our study and its implications.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: In section 2.1 Sample collection and treatment, how were the atmospheric conditions of 10% CO2, 3% O2, 87% N2 chosen?

Response 1: We are grateful to the referee for drawing our attention to this important point. For the optimal Modified Atmosphere Packaging (MAP) conditions during loose-leaf lettuce preservation, we conducted a comprehensive experimental optimization phase. This involved varying the concentrations of O2 from 3% to 20%, CO2 from 10% to 30% and N2 from 50% to 87%. Through this meticulous process, we identified the most effective gas mixture as 10% CO2, 3% O2 and 87% N2. The results were reported in our previous research named “Condition Optimization of Modified Atmosphere Packaging Preservation for Loose-Leaf Lettuce” [1]. Therefore, we choose the optimal gas ratios of 10% CO₂, 3% O₂, and 87% N₂ for the loose-leaf lettuce preservation in this study.

[1] Xu YX, Chen XN, Xu L. Condition Optimization of Modified Atmosphere Packaging Preservation for Loose-Leaf Lettuce [J]. Chinese Agricultural Bulletin, 2016, 32(07): 185-190.

Comments 2: In section 3.1. Sensory evaluation of loose-leaf lettuce under MAP, mention is made of the degradation of chlorophyll and browning of the leaves. Why was chlorophyll not quantified?

Response 2: Thank you very much for pointing out this aspect. The chlorophyll content data and its related discussion had been supplemented in the Revised Manuscript (Figure 1C).

“Chlorophyll breakdown is a major cause of leaf yellowing and MAP can inhibit this process [27]. As shown in Figure 1C, the chlorophyll content in lettuce samples under two different storage conditions exhibited a continuous decline over time. However, the rate of chlorophyll degradation under MAP conditions was significantly lower than that in the control group (p ≤ 0.05). By Day 6 of the storage period, the chlorophyll content in the control group had decreased from 0.38 g/kg before storage to 0.12 g/kg, while the MAP-treated lettuce had only decreased to 0.29 g/kg. This suggests that MAP effectively delayed chlorophyll degradation and helped maintain the green coloration of the lettuce.” (Line 229-236, page 5-6)

Comments 3: In the proportion of gases in the atmosphere studied, the effect of each of the gases is not clear (10% CO2, 3% O2, 87% N2). It is suggested to carry out and add a final summary of their contribution to preserving food.

Response 3： We appreciate the reviewer’s valuable suggestion to clarify the specific effects of the atmospheric gas ratios (10% CO2, 3% O2, 87% N2) on food preservation. We have added a final summary of the contribution of each gas for food preservation in MAP.

“By the sixth day of storage, O₂ had continuously decreased from 3% to 1.5%, and CO₂ had continuously increased from 10% to 14.6%. These results are consistent with previous studies, which concluded that in MAP, oxygen levels are controlled at 1-5% to slow food spoilage. High levels of carbon dioxide (usually not exceeding 20%) inhibit micro-organisms and reduce respiration. Nitrogen (N2) serves as an inert gas, preventing oxidation and protecting food from damage [32].” (Line 262-267, page 6)

[32] Powrie, W. D., & Skura, B. J. (1991). Modified Atmosphere Packaging of Fruits and Vegetables. Modified Atmosphere Packaging Of Food. Springer, Boston, MA. ISBN: 978-0-7476-0064-0. https://doi.org/10.1007/978-1-4615-2117-4_7

Comments 4: It is suggested to include information regarding the production and food and commercial importance of lettuce.

Response 4: Thank you for your valuable suggestions, which we have carefully considered and added to the relevant content. In the introductory section, we have added detailed information on the global demand for lettuce, its nutritional value, and its economic significance in the fresh produce market.

“Lettuce, with its many varieties to meet the diverse tastes of consumers, is in high demand on the global market, especially in the United States, Asia, Australia and Europe. China is the world’s largest producer of lettuce, accounting for 56.4% of the global market share [1]. Loose-leaf lettuce is one of the most popular vegetables because of its crisp texture and rich nutritional profile, including vitamin C and polyphenols [2,3]. The high water and fiber content of loose-leaf lettuce helps maintain digestive health, while its low calorie content makes it an excellent choice for weight loss and healthy eating. With increasing awareness of healthy eating, the lettuce industry is becoming more economically viable globally. For example, in Australia where it is worth up to 100 million Australian dollars and exports around 8 million Australian dollars [1].” (Line 34-43, page 1)

[1] Shi, M., Gu, J., Wu, H., Rauf, et al. Phytochemicals, Nutrition, Metabolism, Bioavailability, and Health Benefits in Let-tuce—A Comprehensive Review. Antioxidants 2022, 11(4), 1158. https://doi.org/10.3390/antiox110401158

Comments 5: In the conclusion section, it is stated that the deterioration of lettuce is due to enzymatic activities and the generation of ROS. Microbial activity is not considered. Why was microbiological deterioration not studied under these storage conditions for lettuce?

Response 5: Thank you for your suggestion regarding the conclusion of our study, particularly concerning the role of microbial activity in lettuce deterioration. The effects of MAP on microbial activity of lettuce has been measured in our previous article “Condition Optimization of Modified Atmosphere Packaging Preservation for Loose-leaf Lettuce” [1]. The results demonstrated that the most significant inhibition of microbial growth was achieved under storage conditions of 10% CO2, 3% O2, and 87% N2. Specifically, after 14 days of storage, the mean bacterial count remained below 106 cfu/g, whereas in the control group, the mean bacterial counts exceeded 106 cfu/g by the 6th day of storage. In this study, we were mainly focused on the enzymatic activities and ROS generation during MAP preservation. And we did not re-measure the microbial activity under the same storage conditions.

[1] Xu YX, Chen XN, Xu L. Condition Optimization of Modified Atmosphere Packaging Preservation for Loose-leaf Lettuce [J]. Chinese Agricultural Bulletin, 2016, 32(07): 185-190.

Thank you very much again for your comment.

1. Summary

Dear editor and reviewers:

First, we would like to thank both the editor and the reviewers for their interest in our study and their supportive and helpful comments to improve the manuscript (agriculture-3293397) that entitled “Modified Atmosphere Packaging prolongs postharvest shelf-life of loose-leaf lettuce: Analysis of involved mechanisms using a proteomic approach”. Guided by these comments, we feel that the conceptual innovation and practical applications of our work are now much clearer in our revised text. All the responses here are in blue. All the changes have been highlighted in red in the revised manuscript.

Again, we would like to offer our thanks, and feel this revision has yielded a more technically correct, rigorous, and useful description of our study and its implications.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: In this study, MAP appeared to extended shelf-life; however, the results are neither fully presented nor discussed thoroughly.

Response 1: We appreciate the constructive comments from the reviewers. To further clarify the relationship between the identified proteins and the shelf-life of lettuce, we have added a discussion on how Modified Atmosphere Packaging (MAP) effects the shelf-life of loose-leaf lettuce at section ‘3.4.2 Molecular function’. We recognize that these enzymes play an important role in scavenging reactive oxygen species (ROS) and maintaining energy homeostasis within the mitochondria, thus protecting the cell membrane to extended the shelf-life of lettuce.

“Peroxidase, cytochrome c oxidase, thioredoxin and glutathione S-transferase are important for scavenging ROS [40], and we found that the expression of all these reductases was up-regulated, suggesting that MAP can eliminate ROS such as hydrogen peroxide and repair oxidized disulfide bonds in lettuce cells by regulating the expression of these enzymes. Malate dehydrogenase is a key enzyme in the TCA cycle, catalyzing the reversible conversion of malate to oxaloacetate, and is also involved in the redox shuttle, which plays an important role in exporting reducing equivalents from the mitochondria and maintaining the dynamic mitochondrial NADH/NAD+ balance [41]. Alkyl hydroperoxide reductase subunit C is capable of directly converting peroxide substrates to water or the corresponding alcohols, thus acting as a detoxifier [42]. The down-regulation of malate dehydrogenase and alkyl peroxiredoxin reductase subunit C expression indicated that NADH/NAD+ was in relative equilibrium in MAP-treated lettuce and ROS production was effectively suppressed. In this way, the damage caused by reactive oxygen species to the cell membrane was reduced and the onset of browning was delayed (Table S2 in revised supplementary materials).” (Line 356-370, page 9)

[40] Hui, K. M., Hao, F. Y., Li, W., Zhang, Z., Zhang, C. Y., Wang, W., et al. Cloning and identification of four Mu-type glutathi-one S-transferases from the giant freshwater prawn Macrobrachium rosenbergii. Fish & Shellfish Immunology 2013, 35(2), 546-552. https://doi.org/10.1016/j.fsi.2013.06.021

[41] Yoshida, K., & Hisabori, T. Adenine nucleotide-dependent and redoxindependent control of mitochondrial malate dehy-drogenase activity in Arabidopsis thaliana. Biochimica et Biophysica Acta (BBA) – Bioenergetics 2016, 1857(6), 810-818. https://doi.org/10.1016/j.bbabio.2016.03.004

[42 Yu, Q., Wu, W., Tian, X., et al. Comparative proteomics to reveal muscle-specific beef color stability of Holstein cattle during post-mortem storage. Food Chemistry 2017, 229, 769-778. https://doi.org/10.1016/j.foodchem.2017.02.015

Thank the reviewer very much again for the suggestion.

Comments 2: Relevant references are missing in several places, for instance, lines 211-213, 284, and 319-321.

Response 2: Thank you for the kind and pertinent suggestions. The related discussion and reference have been supplemented in the Revised Manuscript.

“Chlorophyll breakdown is a major cause of leaf yellowing and MAP can inhibit this process [27].” (line 228-229, page 5)

[27] Torales, A. C., Gutiérrez, D. R., Rodríguez, S. D. C. Influence of passive and active modified atmosphere packaging on yel-lowing and chlorophyll degrading enzymes activity in fresh-cut rocket leaves. Food Packaging and Shelf Life 2020, 26, 1-10. https://doi.org/10.1016/j.fpsl.2020.01.001

“In the low-oxygen environment of the package, oxidation is reduced and browning enzymes are inhibited [28].” (line 236-237, page 6)

[28] Ren, Z., Liu, Y., Huang, J., An, L., Zhang, Y., Yang, W., & Lei, T. Low oxygen concentration alleviates banana peel browning by inhibiting membrane lipid oxidation and polyphenol oxidase activity. International Journal of Food Science and Technology 2024, 59(5), 3350-3359. https://doi.org/10.1111/ijfs.15213

“The data were retrieved from the Uni_Asteraceae library with the assistance of Mascot software [37].” (line 312-313, page 8)

[37] Noor, Z., Ahn, S. B., Baker, M. S., Ranganathan, S., & Mohamedali, A. Mass spectrometry–based protein identification in proteomics—a review. Briefings in Bioinformatics 2021, 22(3), 1620–1638. https://doi.org/10.1093/bib/bbab016

“In the transporter activity category, only 7% of the differentially expressed proteins were enriched. However, key categories such as signal transduction and transport are crucial. The differential proteins within these categories are important factors reflecting the mechanisms of damage, including apoptosis, membrane rupture, and organelle cleavage, in the large, fast-growing lettuce plants of the control group [39].” (line 349-353, page 9)

[39] Monterisi, S., Zhang, L., Garcia-Perez, P., et al. Integrated multi-omic approach reveals the effect of a Graminaceae-derived biostimulant and its lighter fraction on salt-stressed lettuce plants. Scientific Reports 2024, 14(1), 1-10.

https://doi.org/10.1038/s41598-024-07667-8

We sincerely appreciate you again for your constructive comments.

Comments 3: The table presenting proteins identified in this study is not cited in manuscript.

Response 3 Thank you for your kind and pertinent suggestions. We are very sorry for omitting this part of the graph. The protein data identified from the LC-MS/MS analysis of the loose-leaf lettuce have been supplemented in Figure S1, Table S1, and Figure S2 of the revised supplementary materials.

Figure S1. Schematic diagram of Q-Exactive mass spectrometry

Table S1. Basic information chart of proteome identification

“A total of 298,152 secondary spectra were identified, with 45,611 matching spectra, 6,092 peptides matched, and 1,794 proteins identified (Figure S1 and Table S1 in revised supplementary materials).” (Line 311-312, Page 8)

Figure S2. Protein ratio distribution (MAP vs Control) (With regard to protein abundance levels, a difference level of greater than 1.2-fold at p < 0.05 was selected as the threshold for statistical significance. Red dots represent proteins with an up-regulation, while green dots indicate proteins with a down-regulation.)

“A total of 374 distinct proteins were identified, of which 185 were found to be up-regulated and 189 down-regulated (Figure S2 in revised supplementary materials).” (Line 315-317, Page 7)

We apologize again for our carelessness and thank the reviewer very much again for the correction.

Comments 4: Label Figure 1A for clarity

Response 4: Thank you very much for your correction. The group label has been supplemented in Figure 1A. (Line 225, Page 5)

Figure 1. Effects of MAP on the sensory state (A), overall visual quality score (B) and chlorophyll content (C) in postharvest loose-leaf lettuce stored at 4°C for 6 d.

Comments 5: The figure legends are incomplete Table 1 contains two columns labeled 'number'; please specify the distinction between them..

Response 5: The figure legends are incomplete Table 1 contains two columns labeled ‘number’; please specify the distinction between them.

Response 5: Thank you for your careful review and valuable comments. Due to the table width limitation, the display in the table was misleading. The first column is “Serial number”, the third column indicates the “Target protein number”, and the fourth column represents the “Background protein number”. We have adjusted “Table 1” to ensure that all information is displayed on a single line. Thank you very much. (Line 429, Page 11)

Comments 6: Lines 45-46: The authors highlighted the necessity of preventing oxidative browning and decay by safe and efficient methods. However, they did not mention in the introduction that these two disorders are critical concern for extending the shelf-life of lettuce.

Response 6: We thank the referee for focusing our attention on this important point. The protection effect of oxidative browning and decay on extending the shelf-life of lettuce was supplemented in the introduction.

“Nevertheless, the high leaf-to-stalk ratio and delicate tissue of lettuce, particularly loose-leaf varieties, make it highly susceptible to mechanical damage during storage and transport. Such damage contributes directly to the deterioration of the product and triggers a series of chemical reactions. Exposure to oxygen after damage triggers a reaction in which the polyphenolic compounds in the lettuce undergo enzymatic oxidation. This enzymatic browning results in the formation of quinone derivatives, which further polymerise to form brown pigments, exacerbating the deterioration of the visual and nutritional quality of the lettuce [4].” (line 43-50, Page 1-2)

[4] Zhan, L., Li, Y., Hu, J., et al. Browning inhibition and quality preservation of fresh-cut romaine lettuce exposed to high-intensity light. Innovative Food Science & Emerging Technologies 2012, 14, 70-76. https://doi.org/10.1016/j.ifset.2012.01.006

Thank you again for your constructive comment.

Comments 7: Lines 55-56: Include any previous studies on the effect of MAP on loose leaf lettuce.

Response 7: We greatly appreciate the reviewer’s comment. We have improved the introduction with a comprehensive overview of the MAP effect on preservation of loose leaf lettuce.

“Thus, MAP is widely used to extend the shelf-life of postharvest vegetables and fruits. It has been shown that MAP treatment significantly affects the mechanical properties of lettuce leaves, maintains the crispness of lettuce during storage [17], improves the anti-oxidant capacity of the product, increases the quality of the product and prolongs the shelf life of the product [18].” (line 66-70, page 2)

[17] Soltani Firouz, M., Alimardani, R., Mobli, H., et al. Effect of modified atmosphere packaging on the mechanical properties of lettuce during shelf life in cold storage. Information Processing in Agriculture 2021, 8(3), 485-493.https://doi.org/10.1016/j.inpa.2021.03.003

[18] Islam, M. Z., Lee, Y. T., Mele, M. A., et al. Effect of modified atmosphere packaging on quality and shelf life of baby leaf lettuce. Quality Assurance and Safety of Crops & Foods 2021, 11(3), 749-756. https://doi.org/10.3920/QAS2020.0173

Comments 8: Lines 69-72: Delete this portion.

Response 8: Thank you very much for your comments. Revised accordingly.

Comments 9: Line 192: Revise as ‘Results and discussion’.

Response 9: Thank you very much for your correction. “Results” has been revised as “Results and discussion”. (line 209, page 5)

Comments 10: Line 195: Is ‘enzymatic oxidative browning’ a component of loose-leaf lettuce quality? Please specify.

Response 10: We thank the referee for focusing our attention on this important point. Yes, it is. Enzymatic oxidative browning plays a significant role in the postharvest quality of loose-leaf lettuce. Primarily, this phenomenon primarily occurs after cutting or following mechanical damage, leading to notable alterations in the lettuce’s stem, leaf ribs, and leaf color [1]. The color shift is intricately linked to the activity of enzymes like polyphenol oxidase (PPO) and peroxidase (POD). Upon damage, the exposed polyphenolic compounds react with oxygen through the catalytic action of these enzymes, yielding quinines that polymerize to form pigments and initiate browning [2]. This process impacts the visual appeal of the lettuce and potentially its taste and nutritional value. Therefore, inhibiting enzymatic oxidative browning is crucial for preserving the freshness and market value of loose-leaf lettuce.

[1] Peng, H., Luo, Y., Teng, Z., et al. Phenotypic characterization and inheritance of enzymatic browning on cut surfaces of stems and leaf ribs of romaine lettuce. Postharvest Biol. Technol. 2021, 181,1-9. https://doi.org/10.1016/j.postharvbio.2021.111653.

[2] Peng, X., Li, R., Zou, R., et al. Allicin Inhibits Microbial Growth and Oxidative Browning of Fresh-Cut Lettuce (Lactuca sativa) During Refrigerated Storage. Food Bioprocess Technol. 2014, 7, 1597–1605. https://doi.org/10.1007/s11947-013-1154-0.