Nitrogen fertilizer application rate impacts eating and cooking quality of rice after storage

The effect of nitrogen fertilizer application on the quality of rice post-storage is not well understood. The eating and cooking quality (ECQ) of rice treated with 0 (CK, control), 160 (IN, insufficient nitrogen), 260 (AN, adequate nitrogen), and 420 (EN, excessive nitrogen) kg N/ha was analyzed over 12 months of storage. Results showed that the rate of nitrogen fertilizer application had no significant impact on the changes in taste value during storage. However, EN application significantly increased the hardness (p < 0.05), reduced the gumminess (p < 0.05), and delayed the decline in the viscosity of rice paste by two months after one-year storage, compared with other treatments. In conclusion, although EN application resulted in an inferior texture of rice, it delayed the quality change by two months during storage. It was demonstrated that a rational nitrogen application rate (0–260 kg N/ha) for rice cultivation is particularly important to obtain high ECQ; however, EN may be beneficial for the stability of the ECQ during storage.

sure it is accurate.     Yes -all data are fully available without restriction  Introduction   39 Rice, as a staple food, plays an important role in human diet, and is usually stored for 40 long periods to meet the needs of people owing to its seasonal growth. However, many 41 studies have reported that the storage process may result in the deterioration of the eating 42 quality of rice, including increased hardness and reduced viscosity (Anonymous, 2016a). 43 3 Furthermore, the cooking and gelatinization characteristics may be changed, including 44 increase in water absorption, volume expansion rate, cooking time, and setback, as well 45 as a decrease in trough viscosity, final viscosity, and breakdown (Keawpeng and 46 Venkatachalam, 2015). This deterioration is mostly considered to be related to changes in 47 the interactions between the proteins and starch within the rice grain (Chrastil, 1990 storage of milled rice, which has a high commodity price, but not in rice paddy storage 54 owing to the trade-off between the high cost of the storage method and the relatively low 55 commodity value of rice paddy, which is also the reason why rice paddy is usually stored 56 under natural conditions. To date, no feasible solution to the problem of quality loss during 57 rice paddy storage is available. Except for storage conditions, the initial quality of rice is 58 also a key factor for the end-use quality of rice after storage. Fertilization is important for 59 the establishment of the initial quality of rice, especially the rate of nitrogen application rate. 60 It is well-documented that nitrogen fertilizers have a prominent effect on the eating and 61 cooking quality (ECQ) of rice (Kaur et al., 2016), mainly owing to the increased protein 62 content and decreased amylose content of rice (Cao et al., 2017) resulting from nitrogen 63 application, which causes an increase in the hardness of rice, decreasing its palatability 64 gelatinization characteristics and cooking quality. 66 The effect of nitrogen application rate on the initial quality of fresh rice is well known, 67 but whether this effect persists during a period of storage is still unclear. Previous studies 68 have indicated that fertilization has certain effects on the quality of tomato and potato 69 during storage (Makani et al., 2017;Unlu et al., 2010). However, there are no reports 70 addressing whether different nitrogen fertilization levels affect rice quality during storage. 71 In this study, we explored the dynamic changes in the ECQ during storage of rice produced 72 using different nitrogen application rates, and discussed the likely reasons behind the 73 variations in ECQ after storage, with the objective of providing a theoretical basis for the 74 production and quality-control of high-quality rice from the field to the table. The cooking quality was determined following the method of Gujral and Kumar 122 (Gujral and Kumar, 2003), with minor modifications. Briefly, 2.5 grams of milled rice was 123 boiled at 100 °C for 10 min in a beaker containing 50 mL distilled water. Then, three grains 124 of rice were removed every minute, and pressed between two glass slides until the grains 125 showed no chalky core; the total time was recorded as the cooking time and the 126 measurement was done in three replicates. For volume expansion and water uptake, we 127 measured the volume and weighed 2.5 g of milled rice before and after boiling, according

169
It has been reported that the activity of α-amylase in rice is higher under higher nitrogen 170 application rates (Zhu et al., 2017a). Therefore, for our study, it is possible that nitrogen 171 9 application increased the activity of α-amylase in rice, and accelerated the degradation of 172 amylose during storage. However, this effect seems to be dependent on nitrogen 173 application rate only in the case of EN, which is significant (p < 0.05). Moreover, since 174 amylose content is closely related to the ECQ of rice ( For CK group, the taste value of rice stored for 12 months treatments decreased from 63 195 to 60 than without storage (Fig 4). Notably, nitrogen application had a greater impact on 196 the taste value of rice than storage under the conditions of this study. Accordingly,197 controlling the rate of nitrogen application for cultivating rice is particularly important for 198 maintaining the high eating quality of rice during storage. In all the experimental groups, the effect of protein/amylose ratio on the ECQ of rice was 300 greater than that of the amylose and protein contents alone (Table 1) implying that greater emphasis must be placed on this index when considering the changes 305 in rice quality during future storage operations. Besides, the protein/amylose ratio had a 306 significantly (p<0.05) negative correlation with breakdown, and the peak and trough 307 viscosity (Table 1). Apparently, the change in peak and trough viscosity of rice, delayed by 308 excessive nitrogen application (Fig 4), may be related to the change in protein/amylose 309 ratio. In this study, this ratio was at a high level in the EN treatment (data not shown), which 310 means that high protein/amylose ratio may be beneficial in the stability of rice. 311 Nonetheless, the observation is worth further investigation and validation in other rice 312 varieties. 313 To our knowledge, this is the first report on the effect of nitrogen fertilizer levels on rice 319 quality during storage. Results showed that excessive nitrogen application significantly 320 increased the hardness and reduced the gumminess during late storage. AN and EN 321 applications delayed the change in the viscosity of rice paste. In addition, during storage, 322 the protein/amylose ratio showed a better correlation to the ECQ of rice than the protein or 323 amylose contents alone. The results demonstrated that fertilizer application rate affected 324 the quality of the agricultural product during storage by influencing the initial quality of the 325 raw material. Hence, more attention should be paid to the effects of pre-harvest field 326 fertilization on the quality changes of agricultural products during storage, so as to better 327 ensure the high-quality of agricultural products from the field to the plate.         We wish to express our gratitude to the three reviewers for their professional opinions and suggestions. However, after carefully going through the reviewers' comments, we found that a few questions mentioned by the reviewers were based on the field of agronomy and crop production. Although the present study involves the application of nitrogen in the field, we mainly focused on the differences of eating quality changes during storage due to the different quality of raw materials. Nitrogen fertilizer was applied at different rates only to obtain the raw materials with different qualitative characteristics. The research topic of this paper is pertinent to the field of food science, which may be one of the reasons why the reviewers did not approve of the study. Further, we believe that the research topic of this article is suitable for your journal. Hence, we wish to resubmit the article to your journal, hoping that you will give us another chance to get the article reviewed and published. We also hope that experts from the field of food science will be invited to review the article. Finally, based on the opinions and suggestions of the three reviewers, we have made various modifications to the manuscript. The queries of the three reviewers have been answered as follows: Reviewer #1: Title: It seems to me that title should changes like as, 1. "Nitrogen fertilizer and storage time impacts on cooking and eating quality of rice". We thank the reviewer for their suggestion. However, this paper mainly focuses on the effect of nitrogen application rate on the change of eating quality of rice during storage. Nitrogen application is the treatment in the field before rice harvest, and storage time is the treatment condition during storage after rice harvest. If the two phrases are placed parallelly, as in the suggested title, it may not convey our intended meaning. Thus, we think that it will be more appropriate to use the original title. We apologize for our negligence on this issue. We have now changed the sentence as follows："In the present study, the eating and cooking quality of rice treated with 0 (CK), 160 (IN), 260 (AN), and 420 (EN) kg N/ha over 12 months of storage was investigated." has been changed to "The eating and cooking quality (ECQ) of rice treated with 0 (CK, control, 160 (IN, insufficient nitrogen), 260 (AN, adequate nitrogen), and 420 (EN, excessive nitrogen) kg N/ha were analyzed over 12 months of storage." at the Line 25 & 27.

Line 43 & 45: Express the same thing. It is better to compile both of the two sentences.
We have made the correction according to the reviewer's comment, and have deleted some content from the sentences, "During storage, the ratio of protein/amylose showed a significant negative correlation with taste value, springiness, cooking time, and water uptake. In rice with the nitrogen treatments, the ratio of protein/amylose showed a significant negative correlation with peak viscosity, trough viscosity, and setback."

Line 47 & 48:
Need to re-write. Need to improve the write-up.
We have re-written this part according to the reviewer's suggestion, and have changed the statement "EN had both positive and negative effects on the rice quality during storage. Further investigation is needed to elucidate the mechanism of the changes in rice eating quality during storage." to "It was demonstrated that a rational nitrogen application rate (0-260 kg N/ha) for rice cultivation is particularly important to obtain high ECQ, but EN might be beneficial for the stability of the ECQ during storage." at the Line 32 & 34.

Keywords: It is better to use only "Nitrogen".
We have changed the phrase from "Nitrogen fertiliser application" to "Nitrogen". Introduction: 7. If use the spelling fertiliser, then everywhere use the same. But I think it is better to use the spelling "Fertilizer" in the whole text. We thank the reviewer for their suggestion. We have now changed all instances of the word "fertiliser" to "fertilizer" following American English conventions.

Line 59: check the grammar of this sentence.
We are very sorry for the issues associated with language. Since we have now rewritten the whole introduction according to the suggestions by other reviewers, we have deleted the sentence, "the main reasons for this negative effect are the increased protein content and decreased amylose content of rice [2] produced by application of nitrogen." Materials and methods: 9. Line 82, 83, 86 & 87: Need to re-write these sentences. We have rewritten this part according to the other reviewer's suggestions, and have changed it to "The following treatment groups were set up for our studies: a control (CK) group without nitrogen treatment (0 kg N/ha), and three treatment groups with insufficient (IN; 160 kg N/ha), adequate (AN; 260 kg N/ha), and excessive (EN; 420 kg N/ha) nitrogen, based on the typical nitrogen fertilization of 260 kg N/ha used by local farmers. Except for the nitrogen application rates, standard rice cultivation procedures were followed as done by the local farmers. The rice variety was Yanfeng 47, which is one of the main local varieties. The seeds were transplanted on May 25, and harvested on October 8 in 2018.". at the Line 77 & 87.
Results and discussion: 10. Please check the terms "gumminess", "trough viscosity", "setback" is appropriate for the rice scientists!! We thank the reviewer for pointing this out. We have explained the meanings of these terms, and have inserted relevant references. For example, the following statements have been added: "The texture of the rice cakes (hardness, gumminess, and springiness, defined for instrumental texture analyses in the study by Champagne et al. [1]) was determined with the Texture Analyzer (Brookfield Engineering Laboratories, MA, US) according to Zhang et al. [2]." at the Line 109 & 111.
"The pasting parameters of milled rice flour, such as peak viscosity, trough viscosity, breakdown, final viscosity, setback, peak time, and pasting temperature (Fig 2), were determined with a rapid viscosity analyzer (RVA-4, Newport Scientific, Australia)." at the Line 133 & 134 and the Line 138 & 139. Considering the reviewer's suggestion, we have deleted Table 1, and have added the following sentences to express this: "Among the ECQ indices evaluated, nitrogen application rates and storage time only showed significant interaction effects on gumminess, trough viscosity, and setback (p < 0.05; data not shown), indicating that nitrogen application rate has a significant impact the ECQ of rice during postharvest storage. Thus, controlling the nitrogen application rate in the fields is important for maintaining the ECQ of rice during storage." at the Line 150 & 154.

Table 2: Need to add the footnote for clarify the terms.
Considering the reviewer's suggestion, we have changed the Table 2 to the Figure 4. And we have now added the footnote to clarify the terms as follows: "CK, control group (0 kg N/hm 2 ); IN, insufficient nitrogen (160 kg N/hm 2 ); AN, adequate nitrogen (260 kg N/hm 2 ); EN, excessive nitrogen (420 kg N/hm 2 ). For each parameter, the mean values of three replicates ± standard deviation followed by different lowercase letters in the same column differ significantly as per Duncan's test (P < 0.05) as a function of storage time. Different uppercase letters in the rows denote significant differences as per Duncan's test (P < 0.05) as a function of different nitrogen application rates." the Line 227 & 233.
Conclusion: 13. It should be necessary for the expression at a glance for the nitrogen application rate and storage time effect on rice quality. It is also necessary to focus on the output of the whole experiment.
We have now rewritten this part according to the reviewer's suggestion, reorganized the structure, and highlighted the important conclusion for this part as follows: "To our knowledge, this is the first report on the effect of nitrogen fertilizer levels on rice quality during storage. Results showed that excessive nitrogen application significantly increased the hardness, and reduced the gumminess during late storage. AN and EN applications delayed the change in the viscosity of rice paste. In addition, during storage, the protein/amylose ratio showed a better correlation to the ECQ of rice than the protein or amylose contents alone. The results demonstrated that fertilizer application rate affected the quality of the agricultural product during storage by influencing the initial quality of the raw material. Hence, more attention should be paid to the effects of pre-harvest field fertilization on the quality changes of agricultural products during storage, so as to better ensure the high-quality of agricultural products from the field to the plate." at the Line 310 & 320.

Overall comments: This manuscript English language is poor. It is better to improve the presentation of total English language.
Considering the reviewer's suggestion, the article has been proofread and edited by "Editage" after revision, to address any issues associated with language.
We express our thanks for your helpful comments.

Reviewer #2:
The manuscript presents a research study on the impact of nitrogen application at different doses on rice cooking time and nutritional quality which is/could be an interesting topic to focus on in East and south Asia and sub-Saharan Africa but:

The introduction is very poor and does not expose really the problematic, no questions to answer, no assessment of previous studies performed targeting the same subject, no bibliography, etc.
We have now rewritten the introduction according to the reviewer's suggestion, and added statements focusing on the importance, relevant research progress, and existing problems for controlling the deterioration of rice quality during storage. We have also explained how nitrogen application can change the initial quality of rice, which may affect the rice quality during storage, and have added relevant references. The revised statements are as follows at the Line 39 & 73 : "Rice, as a staple food, plays an important role in human diet, and is usually stored for years to meet the needs of people owing to its seasonal growth. However, many studies have reported that the storage process may result in the deterioration of the palatability of rice, including increased hardness and reduced viscosity [3]. Furthermore, the cooking and gelatinization characteristics may be changed, including increase in water absorption, volume expansion rate, cooking time, and setback, as well as a certain decrease in trough viscosity, final viscosity, and breakdown [4]. This deterioration is mostly considered to be related to changes in the interactions between the proteins and starch within the rice grain [5][6][7][8][9].
Numerous efforts have been made in recent years to reduce the deterioration of rice quality due to storage. Low temperature and humidity [10,11], as well as vacuum or nano packaging [12] have proven to be beneficial in maintaining rice quality during storage. The methods have been applied in the storage of milled rice, which has a high commodity price, but not in rice paddy storage owing to the trade-off between the high cost of the storage method and the relatively low commodity value of rice paddy, which is also the reason why rice paddy is usually stored under natural conditions. To date, no feasible solution to the problem of quality loss during rice paddy storage is available. Except for storage conditions, the initial quality of rice is also a key factor for the end-use quality of rice after storage. Fertilization is important for the establishment of the initial quality of rice, especially the rate of nitrogen application. It is well-documented that nitrogen fertilizers have a prominent effect on the eating and cooking quality (ECQ) of rice [13], mainly due to the increased protein content and decreased amylose content of rice [14] resulting from nitrogen application, which causes an increase in the hardness of rice, decreasing its palatability [15][16][17], and also alters the rice gelatinization characteristics and cooking quality.
The effect of nitrogen application rate on the quality of fresh rice is well known, but whether this effect persists after a period of storage is still unclear. Previous studies have indicated that fertilization has certain effects on the quality of tomato and potato during storage [18,19]. However, there are no reports addressing whether different nitrogen fertilization levels affect rice quality during storage. In this study, we explored the dynamic changes in the ECQ after storing rice produced using different nitrogen application rates, and discussed the likely reasons behind the variations in ECQ after storage, with the objective of providing a theoretical basis for the production and quality-control of high-quality rice from the field to the table."

The materials and methods section are not well organized; Nothing was mentioned on the materials, what kind of germplasm (released varieties, cultivars, advanced lines, …)
We apologize for our negligence on this issue. We have now added the relevant statement in the "Materials and methods" section to indicate the rice variety, as follows: "The rice variety was Yanfeng 47, which is one of the main local varieties. The seeds were transplanted on May 25, and harvested on October 8 in 2018." at the Line 85 & 87.
3. The different nitrogen fertilizer treatments were adjusted based on the farmer's fertilization rate (which farmer, small or big farmers) and this should not be the case. The treatments should be defined based on the optimal recommended rate (i.e. 0, 50%, 100%, 150%, 200%). As reported in previous studies the economically optimum and ecologically optimum N rates in Asia were estimated to 180-285 kg/ha and 90-150 kg/ha, respectively. Which reference did the authors used and why? We thank the reviewer for their queries and suggestions. In our opinion, the soil characteristics in different regions within the same province may differ significantly due to the vast area, resulting in different appropriate levels of nitrogen fertilizer for different areas. The field experiment in our study was conducted in Panjin area of the Liaoning Province, located in the Liaohe River Basin, which is one of the main areas for producing rice in China. The nitrogen application rate was based on the local traditional conventional nitrogen application rate of 260 N kg/ha (100%). According to the design principle of threshold experiment, 160 N kg/ha was set as the low nitrogen group (60%), 420 kg N/ha was the high nitrogen group (160%), and no nitrogen treatment acted as the control group (0%). We have added the following statements in the "Materials and methods" section to indicate this: "The following treatment groups were set up for our studies: a control (CK) group without nitrogen treatment (0 kg N/ha), and three treatment groups with insufficient (IN; 160 kg N/ha), adequate (AN; 260 kg N/ha), and excessive (EN; 420 kg N/ha) nitrogen, based on the typical nitrogen fertilization of 260 kg N/ha used by local farmers." at the Line 81 & 84.

For cooking time normally and the best way to do with a relatively better precise data you should use a mattson cooker.
We express our deepest thanks to the reviewer for their advice. The Mattson cooker is indeed a very good instrument for measuring cooking time. We will consider using this instrument in future experiments. Although we did not use a Mattson cooker to determine the cooking time of rice, our method of estimation was based on a number of previous reports, such as Gujral and Kumar [20], Ziegler et al. [21], and Cui et al. [22]. We also hope to compare our conclusion with other studies.
• Results and discussion 5. Results are not very well presented and structured, Results presented and the discussion are not balanced. It will better for the authors to present the results and discussion in two separate sections, We have now rewritten this part according to the reviewer's suggestion. Although we did not divide the results and discussion into two parts, we restructured this part to reorganize the narrative logic of the results, focusing on the integrity of the chain of proof.