extfieldsofstudy: Medicine Chemistry

text:
Effect of Cold Plasma on Meat Cholesterol and Lipid Oxidation

Cold atmospheric plasma (CAP) is a novel non-thermal technology with potential applications in inactivating microorganisms in food products. However, its impact on food quality is not yet fully understood. The aim of this research is to study the impact of in-package plasma technology on the stability of cholesterol and total lipid in four different types of meat (beef, pork, lamb and chicken breast). Additionally, any changes in the primary or secondary lipid oxidation, which is undesirable from a health perspective, is investigated. CAP was not found to have any impact on the cholesterol or lipid content. However, higher peroxide and thiobarbituric acid reactive substances (TBARS) values were found for the treated samples, indicating that plasma can induce the acceleration of primary and secondary lipid oxidation. Finally, color was not affected by the treatment supporting the suitability of the technology for meat products.

Introduction
Globally, meat consumption continues to increase. According to the latest report from the Food and Agriculture Organization [1], the production of meat has increased by 1.7% from 2017 to 2018, reaching a value of 335 million tons (36.9% poultry, 36.6% pig, 21.9% bovine and 4.6% ovine). Cholesterol is present in all these meat products and plays an important role as a structural component of the phospholipid bilayer of the plasma membrane of eukaryotic cells. Cholesterol is fitted into membrane bilayers with its long axis, preventing the crystallization of fatty acyl chains and thereby modifying the activity of membrane-bound enzymes [2]. It also has vital functions in the metabolism and function of body tissue [3]. Cholesterol is an essential precursor for the synthesis of vitamin D, bile, bile acids salts, steroids and hormones [4]. For instance, vitamin D3 is a derivative of cholesterol and is formed in the skin from 7-dehydrocholesterol. Moreover, deficiencies in cholesterol during embryogenesis and organogenesis can cause severe abnormalities in the fetus [5].
In contrast, high concentrations of cholesterol in cells can be cytotoxic and pro-inflammatory [3]. Furthermore, high levels in cholesterol can lead to the development of diseases through atherogenesis, the agglomeration of low-density lipoprotein (LDP) cholesterol on the arterial wall, creating plaques creating plaques which can obstruct blood flow leading to cardiovascular diseases [6]. In addition, a disturbance on the cholesterol metabolism can also cause numerous chronic diseases including cancer, as well as disorders of metabolic and neurological tissues [6]. However, some authors suggest that only oxidized cholesterol can contribute to the pathophysiology of human diseases such as carcinogenic, cytotoxic, mutagenic, atherogenic and neurodegenerative diseases [2]. The formation of cholesterol oxidation products in food are initiated by free radicals, which can be generated by auto-oxidation, photo-oxidation and thermo-oxidation [5], leading to a chain reaction mechanism [7].
Cholesterol (5α-cholesten-3β-ol) is a lipid which belongs to the family of sterols (Figure 1). It is characterized by its reactive behavior, especially on C7, C20 and C25, leading to the formation of oxysterols which have similar structures but with the addition of other functional groups such as hydroxyl, hydroperoxide, ketone and epoxide [8,9]. The most common oxidation products from C7 are 7-α-hydroxycholesterol, 7-ketocholesterol and 7-β-hydroxycholesterol, while 20hydroxycholesterol and 25-hydroxicholesterol are formed when the oxidation occurs on C20 and C25, respectively [8]. These compounds belong to the group of oxysterols, a class of compounds reported to be involved in several neurodegenerative diseases including Huntington's, Parkinson's and Alzheimer's disease [10]. Cold atmospheric plasma (CAP) is a novel technology with several uses in various industries. A plasma atmosphere is made up of different free radical molecules, electrons, UV-photons, positive and negative ions, ozone as well as carbon and nitrogen oxides, which vary according to the gas applied [11]. Recently, the technology has been proposed as a decontamination step for the preservation and safety assurance of foods [12][13][14]. Wang et al. [15] described a dielectric barrier discharge operating at 80 kV for 180 s which was applied to chicken breasts packaged in food trays in both atmospheric air and modified atmosphere gas (65% O2, 30% CO2 and 5% N2). While no significant reduction of microbial populations was found for samples packaged using atmospheric air, they reported that the treatment was effective for chicken packaged under modified atmosphere, suggesting that CAP treatment could increase shelf-life from 7 days to at least 14 days. Furthermore, Yong et al. [12] treated beef jerky at 15 kV for 2.5, 5, and 10 min using a dielectric barrier discharge system which resulted in a reduction of Listeria monocytogenes, Escherichia coli, Salmonella typhimurium and Aspergillus flavus for all treatment times. Significant reductions of Listeria monocytogenes and Escherichia coli were also found for pork loin treated by a dielectric barrier discharge (DBD) plasma operating at 3 kV with a 30 kHz bipolar square wave for treatment times of 5 and 10 min [13].
However, it is shown that these radical species present in the plasma may modify food components, leading to their oxidation [14]. Lipid oxidation has long been considered a radical chain reaction triggered by hydrogen abstractions with hydrogen peroxides being considered the first stable products. However, following their decomposition, hydrogen peroxides may generate secondary lipid oxidation products [16]. When investigating lipid oxidation, simultaneous pathways Cold atmospheric plasma (CAP) is a novel technology with several uses in various industries. A plasma atmosphere is made up of different free radical molecules, electrons, UV-photons, positive and negative ions, ozone as well as carbon and nitrogen oxides, which vary according to the gas applied [11]. Recently, the technology has been proposed as a decontamination step for the preservation and safety assurance of foods [12][13][14]. Wang et al. [15] described a dielectric barrier discharge operating at 80 kV for 180 s which was applied to chicken breasts packaged in food trays in both atmospheric air and modified atmosphere gas (65% O 2 , 30% CO 2 and 5% N 2 ). While no significant reduction of microbial populations was found for samples packaged using atmospheric air, they reported that the treatment was effective for chicken packaged under modified atmosphere, suggesting that CAP treatment could increase shelf-life from 7 days to at least 14 days. Furthermore, Yong et al. [12] treated beef jerky at 15 kV for 2.5, 5, and 10 min using a dielectric barrier discharge system which resulted in a reduction of Listeria monocytogenes, Escherichia coli, Salmonella typhimurium and Aspergillus flavus for all treatment times. Significant reductions of Listeria monocytogenes and Escherichia coli were also found for pork loin treated by a dielectric barrier discharge (DBD) plasma operating at 3 kV with a 30 kHz bipolar square wave for treatment times of 5 and 10 min [13].
However, it is shown that these radical species present in the plasma may modify food components, leading to their oxidation [14]. Lipid oxidation has long been considered a radical chain reaction triggered by hydrogen abstractions with hydrogen peroxides being considered the first stable products. However, following their decomposition, hydrogen peroxides may generate secondary lipid oxidation products [16]. When investigating lipid oxidation, simultaneous pathways for generation of secondary lipid oxidation products should be considered; various reactions of addition, rearrangement or dismutation of lipid peroxyl radicals (LOO) can lead to further formation of dimers, epoxides, aldehydes or ketones in parallel to hydroperoxides. Therefore, to fully assess lipid oxidation along with the determination of lipid peroxides, it is important to analyze a complex mix of secondary lipid Foods 2020, 9, 1786 3 of 13 oxidation products [16,17]. The thiobarbituric acid reactive substances (TBARS) assay is a commonly used method to assess secondary lipid oxidation by measuring the content of a secondary degradation product, namely malonaldehyde. While heated in an acidic medium, malonaldehyde reacts with thiobarbituric acid (TBA) to form a pink Schiff base adduct with an absorption maximum in the region of 532-535 nm [18]. However, the TBARS method also determines a complex mixture of various other secondary oxidation products including alkanals, alkenals, alkadienals and others which react with TBA. Nevertheless, it is widely used as an indicator of lipid oxidation, particularly in meat products [16].
Using TBARS to detect lipid oxidation, it has been reported that cold atmospheric plasma can accelerate the production of peroxides, as well as lipid and protein oxidation in pork during storage [19]. A flexible thin-layer dielectric barrier discharge plasma also oxidized lipids in beef jerky [12], as well as pork butt and beef loin [20]. In another study, it was reported that TBARS values were significantly higher in dry-cured beef, "bresaola" after plasma treatment [14]. Furthermore, Kim et al. [21] also found an increase in lipid oxidation products in bacon after cold atmospheric plasma treatment by deploying the TBARS assay. Finally, CAP not only had an impact on the quality of meat products; it also accelerated the oxidation of proteins in mackerel fillets [22].
There is a need for more research on the effects of plasma on meat chemistry before adoption of this technology by the industry. It is important to evaluate how plasma may affect all the components present in the matrix, and to determine if CAP could increase the shelf-life of the products without affecting their quality or safety or exposing the consumers to any health risk.
Hence, the objective of this study was to investigate if CAP could cause any undesirable effects on lipids such as the oxidation or degradation of cholesterol leading to the formation of oxidized compounds. To investigate the effects, we first applied CAP directly to a cholesterol standard. The results were then compared to the effects observed for four different meat minces, namely beef, lamb, pork and chicken, where the complexity of the matrix may display protective properties. In addition, total lipid oxidation as well as possibly changes in color of the meats were investigated following CAP treatment.

Cholesterol Standard Preparation
A 1 mg/mL solution of cholesterol standard in methanol was prepared. An aliquot of 200 µL of this solution was dispensed into a glass petri dish (2.5 cm radius × 1.5 cm height), and the solvent was let to evaporate. Then, the petri dish was sealed using Parafilm.

Cholesterol Standard Plasma Treatment
A petri dish containing cholesterol standard as previously described was treated using an in-house dielectric barrier discharge atmospheric plasma system, which was described previously [23]. The atmosphere used to fill the petri dish in all our experiments was standard laboratory air. This petri dish was placed between two circular aluminum electrodes (outer diameter = 158 mm) separated by two polypropylene (PP) dielectric layers (2 mm thickness) as per Figure 2. The distance between the The atmosphere used to fill the petri dish in all our experiments was standard laboratory air. This petri dish was placed between two circular aluminum electrodes (outer diameter = 158 mm) separated by two polypropylene (PP) dielectric layers (2 mm thickness) as per Figure 2. The distance between the dielectric layers was the height of the petri dish (1.5 cm). Two different voltages, 60 kV and 80 kV RMS (root mean square) were applied for two different durations, 5 and 10 min, in triplicate; these conditions were previously shown to control microbial growth in mackerel [24]. After the treatment, control (n = 3) and treated samples (n = 3) were kept at 4 °C for 24 h to ensure the induced plasma components can interact with the cholesterol as suggested by Ziuzina et al. [25]. Following this storage, 200 µL of the internal standard (5α-cholestan-3β-ol; 50 mg/mL in dichloromethane) was added to the petri dish and left to stand until full evaporation of the dichloromethane. Once the dichloromethane was evaporated, the residues of both, the cholesterol and internal, standards were reconstituted in 10 mL of dichloromethane and kept at −80 °C until the day of analysis.

Meat Sample Preparation
Fresh meat minces from four different species (beef, lamb, pork and chicken breast) were purchased at a local butcher in Dublin in November 2018. Neither the type of muscle used to produce the mince, nor the origin of the meat were identified at this stage. Using a spoon, a homogenous portion of 100 g of each was added to a black amorphous polyethylene terephthalate (APET/PE) tray (195 × 155 × 30 mm), sealed under atmospheric air conditions using a low oxygen permeable barrier polyvinyl-chloride film (3 cm 3 /m 2 /24 h at Standard Temperature and Pressure (STP); Versatile Packaging, Silverstream, Co. Monaghan, Ireland), and packaged individually (Ilpra Foodpack VG 400 Packaging Machine, Mortara, Italy) to mirror common commercial practice. Samples of each meat were divided in four different batches: control and treated with 24 h of storage post-treatment as well as control and treated with 7 days of storage post-treatment. All conditions were prepared in triplicate.

Meat Sample Plasma Treatment
The same dielectric barrier discharge system as described in Section 2.3 was used for this study. The samples were treated with an in-package mode, where the plasma was induced inside the gas contained inside the sealed package. Each packed sample was placed between the two electrodes separated by 3 cm, i.e., the height of the tray. Ten minutes of treatment was performed at a discharge voltage of 80 kV RMS; these settings have previously been shown to control microbial growth in mackerel [24]. Similar to the cholesterol standard study, once the CAP treatment was finished both the control and plasma samples were kept at 4 °C for either 24 h or 7 days. Following this storage, i.e., either 24 h or 7 days, all samples were individually vacuum-packed and stored at −80 °C until the day of the analysis. All conditions were prepared in triplicate.

Meat Sample Preparation
Fresh meat minces from four different species (beef, lamb, pork and chicken breast) were purchased at a local butcher in Dublin in November 2018. Neither the type of muscle used to produce the mince, nor the origin of the meat were identified at this stage. Using a spoon, a homogenous portion of 100 g of each was added to a black amorphous polyethylene terephthalate (APET/PE) tray (195 × 155 × 30 mm), sealed under atmospheric air conditions using a low oxygen permeable barrier polyvinyl-chloride film (3 cm 3 /m 2 /24 h at Standard Temperature and Pressure (STP); Versatile Packaging, Silverstream, Co. Monaghan, Ireland), and packaged individually (Ilpra Foodpack VG 400 Packaging Machine, Mortara, Italy) to mirror common commercial practice. Samples of each meat were divided in four different batches: control and treated with 24 h of storage post-treatment as well as control and treated with 7 days of storage post-treatment. All conditions were prepared in triplicate.

Meat Sample Plasma Treatment
The same dielectric barrier discharge system as described in Section 2.3 was used for this study. The samples were treated with an in-package mode, where the plasma was induced inside the gas contained inside the sealed package. Each packed sample was placed between the two electrodes separated by 3 cm, i.e., the height of the tray. Ten minutes of treatment was performed at a discharge voltage of 80 kV RMS; these settings have previously been shown to control microbial growth in mackerel [24]. Similar to the cholesterol standard study, once the CAP treatment was finished both the control and plasma samples were kept at 4 • C for either 24 h or 7 days. Following this storage, i.e., either 24 h or 7 days, all samples were individually vacuum-packed and stored at −80 • C until the day of the analysis. All conditions were prepared in triplicate.

Cholesterol Extraction
Cholesterol was quantified according to [26]. This method has previously been fully validated for the determination of cholesterol in turkey meat products. Briefly, approximately 0.5 g of chilled meat mince sample, 200 µL of internal standard (5α-cholestan-3β-ol, concentration: 50 mg/mL) and 30 mL of 4 M KOH in methanol were mixed and homogenized using an Ultraturrax homogenizer (Labortechnik, Staufen, Germany) at 13,500 rpm for 30 s in a 50 mL tube. Saponification was carried out by placing the sample into a water bath at 60 • C for one hour. After cooling down, a liquid-liquid extraction was performed by adding 5 mL of Milli Q water and 5 mL of heptane; phase separation was achieved by centrifuging the sample at 4000 rpm (2500× g) for 10 min. The organic phase was transferred to another tube and the extraction was repeated twice more by adding 5 mL of heptane each time. All organic layers were combined and dried using a sample concentrator and reconstituted into 10 mL of dichloromethane. Derivatization of cholesterol was performed by mixing 0.5 mL of this extract, 200 µL of BSTFA-TMCS, 100 µL of pyridine and leaving them in a water bath for 15 min at 70 • C. After cooling down, the solution was diluted to 10 mL with dichloromethane and an aliquot was transfer to the vial to be injected into the gas chromatography system.

Gas Chromatography-Flame Ionization Detector Analysis
Separation was carried out using a Clarus 580 Gas Chromatograph (Perkin Elmer, Waltham, MA, USA) fitted with a flame ionization detector (GC-FID) set at 260 • C according to [26]. These authors previously showed the method to have a limit of quantitation of 0.1 µg/mL (equivalent to 0.4 mg cholesterol per g meat); a limit of detection was not reported. The separation and quantification of cholesterol was carried out employing a ZB-5 capillary column (Phenomenex, Torrance, CA, USA) with a film thickness of 0.25 µm and a length of 30 m × 0.25 mm. The injection volume was 0.5 µL and the inlet temperature was set to 200 • C. Hydrogen was flushed at a constant flow of 2.0 mL/min, and the split ratio was set at 5:1. The oven temperature started at 180 • C with an initial temperature ramp of 8.0 • C/min to 260 • C followed be a second temperature ramp of 2 • C/min to 280 • C which was held for 10 min.
The content of cholesterol was calculated using following equation (Equation (1)) according to Grasso et al. [26].
where, IS Purity is the purity of the internal standard as given on the certificate of analysis, RRF is the relative response factor for cholesterol (namely 1.001), and 20 is the dilution factor.

Lipid Content
The total lipid content was determined using the Bligh & Dyer (B & D) method which applies a mixture of chloroform, methanol and Milli Q water for the extraction of lipids from the muscle tissue [27]. The extraction was performed in duplicate. Briefly, experimental meat samples were minced with a kitchen blender (Bosch MSM87140, Frankfurt, Germany) and 10 g of the obtained mince was transferred into centrifuge tubes. The centrifuge tubes were kept on ice during the whole procedure. Then, distilled water (10 mL), chloroform (20 mL) and cold methanol (40 mL) were added to each tube. The mixture was homogenized using an Ultraturrax (IKA T18, Staufen, Germany) for 2 min at 9000 rpm. Additional amount of chloroform (20 mL) and distilled water (20 mL) was added separately, and the mixture was homogenized again using the Ultraturrax for 30 s after each addition. After the homogenization, the tubes were centrifuged (Hettich Universal 16A Centrifuge, Berlin, Germany) for 10 min at 11,800 rpm. An aliquot of the chloroform phase (2 mL) was collected from the bottom of each of the tubes and transferred into a pre-weighed Kimax glass tube (10 mL). The Kimax glass tube with chloroform phase was placed in an evaporation unit and kept at 60 • C with N 2 -stream for 1 h. After the evaporation, the tubes were corked, cooled down to room temperature and weighed again. The results are expressed as total lipid (average ± standard deviation) in percentage of wet weight meat sample [27].
The remaining chloroform phase in the centrifuge tubes was collected and transferred to plastic tubes resistant to chloroform, flushed with N 2 -gas and stored at −80 • C prior to analysis of peroxide values (PV) and TBARS.

Peroxide Value
PV was determined by using the iodometric titration method described by [28]. The end point of titration was assessed potentiometrically with an automatic titrator (TitroLine 7800, Xylem Analytics, Mainz, Germany) fitted with a platinum electrode (Pt 62). In line with the AOCS method, the analysis was performed in duplicate and the results were expressed in meq active oxygen/kg lipids as an average ± SD.

TBARS
The TBARS assay assesses secondary lipid oxidation products through a reaction between malondialdehyde and thiobarbituric acid (TBA). It is a good indicator of the general oxidative status in fish products because TBA reacts with a wide range of aldehydes and oxidized molecules derived from lipids and proteins. In complex food matrices such as meat and fish, lipid oxidation may take place via complex pathways due to co-oxidation reactions between lipid radicals, secondary oxidation products, pro-oxidants such as transition metals, blood or myoglobin, as well as other system components such as proteins [17]. Co-oxidation reactions result in the oxidation of other food molecules such as proteins with involvement of lipid oxidation intermediates and products [16,18]. Therefore, it is very important to apply a method such as the TBARS assay that can assess the general status of secondary lipid oxidation.
For this study, secondary lipid oxidation was investigated using TBARS determined in the chloroform phase according to the method of Ke and Woyewoda [29]. 1,1,3,3-tetraethoxypropane (T 9889) was used as a standard. The analysis was performed in triplicate and the results were expressed as average ± standard deviation in µMol TBARS/g lipid.

Color
Color characteristics of plasma-treated and untreated meat samples were measured instrumentally using a Minolta Chroma meter CR-400 (Konica-Minolta, Osaka, Japan). For this, meat samples with a thickness of 1 cm were prepared. Before starting the analysis, the instrument was calibrated with a standard white plate. The data was recorded in color coordinates of L* (lightness, black = 0, white = 100), a* (redness > 0, greenness < 0), and b* (yellowness > 0, blueness < 0) according to the Commission Internationale de l'Éclairage (CIE) Lab scale [30]. Three measurements were performed on each of the meat samples, and the average with standard deviation was determined.

Statistical Analysis
Analysis of variance (ANOVA) of dependent variables was carried out using Minitab 17.1.0 (Minitab Inc, State College, PA, USA). Statistics were calculated using a general lineal model (GLM) considering the entire variable as fixed factors. When differences were observed (p < 0.05), a Tukey's multiple comparison was calculated to study the effect of the plasma treatment on the cholesterol. Experiments were performed in triplicate and analyses were carried out in triplicate.

Cholesterol Content
Cholesterol is an important food nutrient due to its role in the biosynthesis of vitamin D, bile acids and steroid hormones such as gonadal (testosterone, estrogens, progesterone) and adrenal (aldosterone, cortisol) [31]. Moreover, it is an important constituent of the cell membrane. For this reason, it is important to study if a novel non-thermal technology such as CAP could have any impact on this micronutrient despite previous research showing that CAP may not be able to significantly penetrate solids [14].

Cholesterol Standard
Cholesterol standard was exposed to CAP at two voltages (60 and 80 kV) for two different durations, namely 5 or 10 min. Overall, results show that CAP has a significant effect (p < 0.05) on the cholesterol content of the treated standard with CAP treatment significantly reducing the amount of cholesterol recovered from the petri-dishes. As seen in Figure 3, the cholesterol content decreased by around 35% for all the different treatments, suggesting that this could be because plasma can lead to oxidation and/or degradation of cholesterol. As was mentioned previously, oxidation of cholesterol can be initiated by free radical species, consequently, many of the different radical species which are presented in the plasma bulk could cause this reaction to happen [32]. However, there were no significant differences between the different treatments, i.e., oxidation and/or degradation was not dependent on voltage or duration of treatment. (aldosterone, cortisol) [31]. Moreover, it is an important constituent of the cell membrane. For this reason, it is important to study if a novel non-thermal technology such as CAP could have any impact on this micronutrient despite previous research showing that CAP may not be able to significantly penetrate solids [14].

Cholesterol Standard
Cholesterol standard was exposed to CAP at two voltages (60 and 80 kV) for two different durations, namely 5 or 10 min. Overall, results show that CAP has a significant effect (p < 0.05) on the cholesterol content of the treated standard with CAP treatment significantly reducing the amount of cholesterol recovered from the petri-dishes. As seen in Figure 3, the cholesterol content decreased by around 35% for all the different treatments, suggesting that this could be because plasma can lead to oxidation and/or degradation of cholesterol. As was mentioned previously, oxidation of cholesterol can be initiated by free radical species, consequently, many of the different radical species which are presented in the plasma bulk could cause this reaction to happen [32]. However, there were no significant differences between the different treatments, i.e., oxidation and/or degradation was not dependent on voltage or duration of treatment. Although the effect of cold plasma on isolated nutrients has already been reported, to the best of our knowledge, this is the first time that the effect of CAP on cholesterol is reported. However, as the present study used GC-FID for the quantification of cholesterol, it was not possible to identify the breakdown products produced by the plasma. Further research using e.g., a GC coupled to a (tandem) mass spectrometer may give further insight into the processes involved and may help in identifying which carbons in the cholesterol molecule are more prone to oxidation/degradation than others.
Previously, several food protein powders (gelatin, hemoglobin and lung protein extract) were treated at 80 kV (RMS) for 15 min using a dielectric barrier discharge plasma system [33]. Authors observed that the direct treatment of cold plasma on these three protein powders affected their native structure, leading to a significant change in their functional, rheological and gelling properties. Similar results were observed by Ji et al. [34] after treating peanut isolated protein with a different dielectric barrier discharge at 35 kV for 1, 2, 3 or 4 min. Although the effect of cold plasma on isolated nutrients has already been reported, to the best of our knowledge, this is the first time that the effect of CAP on cholesterol is reported. However, as the present study used GC-FID for the quantification of cholesterol, it was not possible to identify the breakdown products produced by the plasma. Further research using e.g., a GC coupled to a (tandem) mass spectrometer may give further insight into the processes involved and may help in identifying which carbons in the cholesterol molecule are more prone to oxidation/degradation than others.
Previously, several food protein powders (gelatin, hemoglobin and lung protein extract) were treated at 80 kV (RMS) for 15 min using a dielectric barrier discharge plasma system [33]. Authors observed that the direct treatment of cold plasma on these three protein powders affected their native structure, leading to a significant change in their functional, rheological and gelling properties. Similar results were observed by Ji et al. [34] after treating peanut isolated protein with a different dielectric barrier discharge at 35 kV for 1, 2, 3 or 4 min.

Cholesterol in Meat Products
Since the impact of CAP on fatty acids has been described previously (e.g., [22,24]), cholesterol was chosen as a model to investigate possible oxidation caused by CAP for the present study.
CAP did not have a significant impact on the cholesterol content in any of the four different meat mince samples (p > 0.05) as can be seen in Figure 4. This may be because plasma only affects the surface of the meat and hence cannot penetrate into the meat product [14]. On the other hand, the complexity of the matrix and components inside the food could protect the cholesterol from the potential impact of cold plasma. For instance, other lipids present in the meat such as triglycerides in general and polyunsaturated fatty acids more specifically, could be more susceptible to oxidation and/or reaction with the plasma thus protecting cholesterol from interaction with the radical species resulting in cholesterol oxidation and/or degradation. In addition, the presence of naturally occurring antioxidants such as carnosine, anserine, carnitine or taurine, in meat could not only prevent the formation of undesirable oxidation products in general [21] but more specifically the oxidation of cholesterol. Moreover, there are also some antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase), vitamins with antioxidant properties (ascorbic acid and α-tocopherol) and minerals like zinc or selenium [35] which could give extra protection against the potential impact of the reactive species present in the plasma atmosphere. Nevertheless, given that the samples were minced, offering a large exposure area, were exposed to extended treatment times and retained in an atmosphere containing induced plasma species for up to 7 days due to the use of the in-package technology, the data indicated no observable effects on the cholesterol contents of the tested meats. mince samples (p > 0.05) as can be seen in Figure 4. This may be because plasma only affects the surface of the meat and hence cannot penetrate into the meat product [14]. On the other hand, the complexity of the matrix and components inside the food could protect the cholesterol from the potential impact of cold plasma. For instance, other lipids present in the meat such as triglycerides in general and polyunsaturated fatty acids more specifically, could be more susceptible to oxidation and/or reaction with the plasma thus protecting cholesterol from interaction with the radical species resulting in cholesterol oxidation and/or degradation. In addition, the presence of naturally occurring antioxidants such as carnosine, anserine, carnitine or taurine, in meat could not only prevent the formation of undesirable oxidation products in general [21] but more specifically the oxidation of cholesterol. Moreover, there are also some antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase), vitamins with antioxidant properties (ascorbic acid and α-tocopherol) and minerals like zinc or selenium [35] which could give extra protection against the potential impact of the reactive species present in the plasma atmosphere. Nevertheless, given that the samples were minced, offering a large exposure area, were exposed to extended treatment times and retained in an atmosphere containing induced plasma species for up to 7 days due to the use of the in-package technology, the data indicated no observable effects on the cholesterol contents of the tested meats.

Lipid Content
Total lipid content in meat samples varied from 1.7 ± 0.7% for chicken (white meat) to 30.6 ± 1.9% for lamb (red meat; Figure 5). However, no significant variation in the total lipid content was found between plasma-treated and untreated samples during the storage period for any of the meats investigated.

Lipid Content
Total lipid content in meat samples varied from 1.7 ± 0.7% for chicken (white meat) to 30.6 ± 1.9% for lamb (red meat; Figure 5). However, no significant variation in the total lipid content was found between plasma-treated and untreated samples during the storage period for any of the meats investigated.

Peroxide Value
As the official method used [28] has a detection limit of 0.05 mL lipid per 12 mL chloroform (equivalent to a lipid content of about 5-6%), it was not possible to measure peroxide value in chicken samples due to their very low lipid content (<2% w/w). However, novel methods such as the one recently published by [36] may give further information on peroxide values of low-lipid samples.
The rest of the samples with higher lipid content in the tissue, i.e., those with more than 5% lipid content, showed increased peroxide values in all plasma-treated samples compared to non-treated

Peroxide Value
As the official method used [28] has a detection limit of 0.05 mL lipid per 12 mL chloroform (equivalent to a lipid content of about 5-6%), it was not possible to measure peroxide value in chicken samples due to their very low lipid content (<2% w/w). However, novel methods such as the one recently published by [36] may give further information on peroxide values of low-lipid samples.
The rest of the samples with higher lipid content in the tissue, i.e., those with more than 5% lipid content, showed increased peroxide values in all plasma-treated samples compared to non-treated (control) samples (Figure 6). At the same time, only plasma-treated pork and beef samples exceeded the limit for PV established by the CODEX STAN (10 meq O 2 /kg lipid [37]) on day 7 of chilled storage ( Figure 5).

Peroxide Value
As the official method used [28] has a detection limit of 0.05 mL lipid per 12 mL chloroform (equivalent to a lipid content of about 5-6%), it was not possible to measure peroxide value in chicken samples due to their very low lipid content (<2% w/w). However, novel methods such as the one recently published by [36] may give further information on peroxide values of low-lipid samples.
The rest of the samples with higher lipid content in the tissue, i.e., those with more than 5% lipid content, showed increased peroxide values in all plasma-treated samples compared to non-treated (control) samples (Figure 6). At the same time, only plasma-treated pork and beef samples exceeded the limit for PV established by the CODEX STAN (10 meq O2/kg lipid [37]) on day 7 of chilled storage ( Figure 5). Plasma-treatment could result in the accumulation of reactive species accelerating lipid oxidation [38]. This suggestion is supported by the study of Albertos et al. [24] who previously used a non-thermal plasma (NTP) system to treat fresh mackerel fillets. They observed a similar trend for PV results after the treatment of mackerel samples with NTP. The treatment resulted in an over 5fold increase in peroxide values compared to control samples, with both exposure time and voltage affecting the rate of oxidation [24]. Furthermore, our observations are in line with those made by Plasma-treatment could result in the accumulation of reactive species accelerating lipid oxidation [38]. This suggestion is supported by the study of Albertos et al. [24] who previously used a non-thermal plasma (NTP) system to treat fresh mackerel fillets. They observed a similar trend for PV results after the treatment of mackerel samples with NTP. The treatment resulted in an over 5-fold increase in peroxide values compared to control samples, with both exposure time and voltage affecting the rate of oxidation [24]. Furthermore, our observations are in line with those made by Yong et al. [12] who reported a significant increase in the PV of beef jerky following flexible thin-layer plasma treatment for 10 min. However, this group only found a significant increase after 10 min of treatment while beef jerky treated for 2.5 or 5 min was not significantly different from the control.

Thiobarbituric Acid Reactive Substances (TBARS)
Lipid oxidation leads to the formation of a very wide range of different oxidation products, making the determination of lipid oxidation challenging. The determination of TBARS is one of the oldest and the most commonly used methods for assessing secondary lipid oxidation status by measuring one of the end product of polyunsaturated fatty acid (PUFA) peroxidation-malondialdehyde [39][40][41][42][43][44][45], but it will also determine other aldehydes [43].
Except for beef samples after 7 days of storage and all the chicken samples, plasma-treated meat samples were characterized with significantly higher TBARS-values compared to the untreated ones ( Figure 7). This effect can be explained by the radical-initiating mechanism of plasma treatment as mentioned previously [24]. However, the highest TBARS-values were surprisingly found in chicken samples which contained the lowest lipid content. This may possibly be explained by the typically higher content of PUFAs in chicken as compared to beef or pork (e.g., [46,47]). However, research has also shown that PUFAs vary depending on diet [48]. As the samples for the present study were purchased at a commercial butcher, no information on dietary history or muscle used to produce the mince could be obtained. This information, along with the determination of the fatty acid profile of the samples used in the present study may have assisted in explaining the differences in TBARS found for the different meats.

Color
No significant difference between control and plasma-treated meat samples (for each category of meat) was observed, suggesting that plasma treatment did not have notable effects on the color of the meat tissue. These findings are in line with those reported by Jayasena et al. [20] for beef and pork. This group also reported no significant changes in L* values of beef loin and pork butt while also no significant changes were observed for b* for pork butt. In contrast, however, the same group reported significantly lower a* values for plasma-treated beef loin and pork butt along with significantly higher b* values for beef loin. Finally, Yong et al. [12] reported significantly lower L* values for plasma-treated beef jerky treated for 10 min when compared to the control sample while reporting significantly higher b* values. These authors did not find any significant effect on a* values caused by plasma treatment, in line with the observations made in this study.

Conclusions
Our results show that the induced reactive species can degrade cholesterol in its pure form. However, the cholesterol content in meat samples was not affected by the treatment, suggesting that the matrix effect plays an important role in protecting this food micronutrient from degradation. In addition, higher peroxide values and TBARS were found for the treated samples as compared to the control samples, indicating that plasma can induce the acceleration of primary and secondary lipid oxidation without affecting absolute lipid content. Finally, color was not affected by the treatment, supporting the suitability of the technology for meat products.
However, further research is required on the impact of this technology on the quality of food products prior to its approval and adoption by regulators and industry, respectively. There is a need to understand the chemical reactions associated with plasma species to avoid quality deterioration. This is particularly true when trying to understand the impact of CAP on lipid fractions and as such, future research should focus on investigating several lipid fractions in parallel such as cholesterol as well as fatty acids to get a clearer understanding of the order in which lipid fractions get oxidized. As our research shows, plasma studies need to be performed on real food products and not only model solutions in order to determine which operational conditions suit the vast array of potential food products that could be treated. Optimization of plasma control parameters, in particular the inducer gas employed, need to be investigated in depth to identify the conditions which can provide the required product safety and yet retain key quality attributes. While the length of this study was chosen to represent shelf-life in a retail setting, experimental duration has recently been extended by several authors in order to investigate long-term impact of plasma treatment on the food matrix. For instance, studies on pork [19] and chicken [49] investigated the impact of plasma for 12 and 21 days, respectively. While [19] reported a significant increase in lipid oxidation on days 4, 8 and 12 for plasma-treated samples as compared to day 0 as well as the control samples, no such observations were made in chicken, even after 21 days of cold storage [49].
Nevertheless, our findings are in contrast to the findings reported by Jayasena et al. [20] who reported a significant increase of TBARS values in beef loin following treatment with flexible thin-layer dielectric barrier discharge plasma for 10 min. Furthermore, these authors only report a significant increase after 10 min treatment while samples treated for 2.5 or 5 min were not significantly different from the control. In addition, the same authors also reported TBARS for pork butt treated for the same durations as the beef loin (i.e., 2.5, 5 and 10 min). However, unlike the beef loin, pork butt samples did not display significant increases in TBARS when compared to the control. In another study [50], ham was packed in three different gas mixtures, namely 20% O 2 , 40% N 2 and 40% CO 2 (treatment 1), 50% CO 2 and 50% N 2 (treatment 2) or 100% CO 2 (treatment 3) and treated at 30 kV for either 5 or 10 min using a dielectric barrier discharge (DBD) plasma system. The authors reported that TBARS values were significantly higher for the treated samples compared with the control. A dielectric barrier discharge system was also used to treat chicken breast at 100 kV for 1, 3, and 5 min durations [51]. TBARS for treated samples were significantly higher than for untreated ones. More studies should be performed in this direction to investigate these opposing findings reported thus far.

Color
No significant difference between control and plasma-treated meat samples (for each category of meat) was observed, suggesting that plasma treatment did not have notable effects on the color of the meat tissue. These findings are in line with those reported by Jayasena et al. [20] for beef and pork. This group also reported no significant changes in L* values of beef loin and pork butt while also no significant changes were observed for b* for pork butt. In contrast, however, the same group reported significantly lower a* values for plasma-treated beef loin and pork butt along with significantly higher b* values for beef loin. Finally, Yong et al. [12] reported significantly lower L* values for plasma-treated beef jerky treated for 10 min when compared to the control sample while reporting significantly higher b* values. These authors did not find any significant effect on a* values caused by plasma treatment, in line with the observations made in this study.

Conclusions
Our results show that the induced reactive species can degrade cholesterol in its pure form. However, the cholesterol content in meat samples was not affected by the treatment, suggesting that the matrix effect plays an important role in protecting this food micronutrient from degradation.
In addition, higher peroxide values and TBARS were found for the treated samples as compared to the control samples, indicating that plasma can induce the acceleration of primary and secondary lipid oxidation without affecting absolute lipid content. Finally, color was not affected by the treatment, supporting the suitability of the technology for meat products.
However, further research is required on the impact of this technology on the quality of food products prior to its approval and adoption by regulators and industry, respectively. There is a need to understand the chemical reactions associated with plasma species to avoid quality deterioration. This is particularly true when trying to understand the impact of CAP on lipid fractions and as such, future research should focus on investigating several lipid fractions in parallel such as cholesterol as well as fatty acids to get a clearer understanding of the order in which lipid fractions get oxidized. As our research shows, plasma studies need to be performed on real food products and not only model solutions in order to determine which operational conditions suit the vast array of potential food products that could be treated. Optimization of plasma control parameters, in particular the inducer gas employed, need to be investigated in depth to identify the conditions which can provide the required product safety and yet retain key quality attributes.

Conflicts of Interest:
The authors declare no conflict of interest.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
N-Use Efﬁciency and Yield of Cotton ( G. hirsutumn L.) Are Improved through the Combination of N-Fertilizer Reduction and N-Efﬁcient Cultivar

: Nitrogen (N) fertilizer plays a vital role in increasing cotton yield, but its excessive application leads to lower yield, lower nitrogen use efﬁciency (NUE), and environmental pollution. The main objective of this study was to ﬁnd an effective method to enhance the NUE in cotton production. A two-year ﬁeld experiment was conducted by using a split plot design with N rates (N0, 0 kg N ha − 1 ; N1, 112.5 kg N ha − 1 ; N2, 225 kg N ha − 1 , N3, 337.5 kg N ha − 1 ), and cotton cultivars (CRI 69; ZZM 1017; ZZM GD89 and XLZ 30) to evaluate both their individual effect and their interactions on cotton yield and NUE. The results showed that the biomass and N accumulation of four cultivars increased with an increase in N rate, whereas the NUE decreased. Cotton yield increased ﬁrst and then decreased for CRI 69 and ZZM 1017, while kept increasing from N0 to N3 for ZZM GD89 and XLZ 30. Compared with ZZM GD89 and XLZ 30, CRI 69, and ZZM 1017 showed higher yield, resulted from higher biomass, bolls per plant, and boll weight, especially under low N level. In addition, the CRI 69 and ZZM 1017 had stronger N absorption and transformation capabilities, and showed higher NUE than those of ZZM GD89 and XLZ 30 under the same N rate. The results indicated that CRI 69 and ZZM 1017 show advantages over ZZM GD89 and XLZ 30 in yield, and NUE, especially under low N rate.

Introduction
Cotton (G. hirsutumn L.) is the most important fiber crop [1], and China is the largest cotton producer as well as consumer globally [2,3]. Cotton growth is influenced by several factors including genotype, environmental conditions, and management practices. Fertilizer is one of the major inputs in cotton production, especially N, which is one of the limiting factors for yield and quality and is required more than other nutrients [4]. Thus, farmers tend to apply large amounts of N fertilizers to improve growth and productivity and ensure high yield [5]. However, excessive N application results in not only excessive cotton vegetative growth, delayed maturity, and reduction of yield and quality, but also increase of N release and environmental pollution [6].
Appropriate increase of nitrogen fertilizer could increase the accumulation of dry matter and content chlorophyll, while an overdose of nitrogen could lead to an imbalance of carbon and nitrogen metabolism, excessive vegetative growth, late maturity [7,8], and decrease the yield and NUE [9][10][11]. Previous studies found that nitrogen application within bounds can improve the lint yield and the fiber quality by increasing the dry matter accumulation and photosynthesis [12,13]. Furthermore, other studies indicated that reducing the application of nitrogen without sacrificing cotton yield under reasonable management is feasible and can improve the NUE [5,14]. The same results were found in other crops [15][16][17][18]. Besides, stalk recycling to the field is one of the main approaches to avoid the further increase in fertilizers during production [19][20][21]. However, the excessive application of nitrogen fertilizer in cotton production has not been ameliorated. Therefore, an in-depth analysis of reducing the use of nitrogen fertilizer effects on N use efficiency is still important for cotton production.
In addition to N availability, the genotype is also a key factor determining the growth rates and grain yields of crops. Therefore, it is feasible for the farmer to give up the concept of high-N fertilizer levels and utilize more environmentally friendly genotypes with low-N fertilizer requirements. Since Harvey first reported the differences in nitrogen uptake and utilization among different maize cultivars in 1939 [22], a lot of work on genotype differences of nitrogen efficiency has made great achievements. So far, numerous studies have found that various crops with different genotypes display variance in nitrogen absorption and utilization of nitrogen [23]. The dry weight, photosynthetic efficiency, and yield of nitrogen efficient cultivars were higher than that of nitrogen inefficient cultivars under the same conditions, such as maize [24][25][26][27], rice [28][29][30][31], wheat [32][33][34], and other crops [4,35]. In cotton, predecessors have established the screening and evaluation system of different nitrogen efficiency cultivars at the seedling stage [36]. These studies have laid a certain foundation for the efficient utilization of crop nutrient resources and proved to be a feasible way to improve the utilization efficiency of nutrient resources by using nutrient efficient cultivars. Thus, making full use of the plant's own nutrition genetic characteristics for NUE, economize manure prolific breeding, and reasonable application of nitrogen fertilizer are effective way to improve the efficiency of plant nutrients.
Cultivars of CRI 69 and ZZM 1017 had more dry matter and N accumulation in seedling stage than those of ZZM GD89 and XLZ 30 under either N deficiency or sufficiency through hydroponic experiments [37]. We assume that cultivars of CRI 69 and ZZM 1017 may also show their own advantages in the field, making full use of their own advantage can reduce N input, improve N fertilizer utilization. Therefore, a field experiment was conducted on cotton cultivars with varying NUE under various N levels. The aims of this research is to reveal the differences of different cultivars. The purpose of this research is to reveal the differences of various cultivars through the dry matter, N absorption, and distribution as well as yield, and NUE at four N levels. This study is expected to give more insights on the NUE under field conditions with the aim of optimizing N fertilizer utilization and yield.

Plant Materials, Experimental Design, and Field Management
Four cotton cultivars (G. hirsutumn L.) were used (CRI 69 and ZZM 1017 were the high NUE cultivars, HNUEC; ZZM GD89 and XLZ 30were the low NUE cultivars, LNUEC) according to our previous work [37].
Field experiments were conducted at the experimental farm of the Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang, Henan province, China (36 • 06 N, 114 • 21 E) during the cotton growing seasons of 2018 and 2019. The experimental field has been used for cotton planting in the past 5 years, and cotton straw was returned to the soil after harvest. The chemical properties of the field soil were measured for two years ( Table 1). The meteorological data are shown in the attached Figure 1. There was no significant difference in monthly average temperature between two years. However, there was a large difference in rainfall between the two years, especially during the boll period. The total monthly rainfall in the growing season was 12.9 mm, 56 mm, and 45 mm in 2018, 2019 and the last 5 years, respectively. boll period. The total monthly rainfall in the growing season was 12.9 mm, 56 mm, and 45 mm in 2018, 2019 and the last 5 years, respectively.  The two-year experiment was conducted under a split block experimental design with three replications where the main plot were four N levels (N0, 0 kg N ha −1 ; N1, 112.5 kg N ha −1 ; N2, 225 kg N ha −1 ; N3, 337.5 kg N ha −1 ), and the subplot was cotton cultivar. As for N treatment, half of the target N was used as basal fertilizer and the other half was applied at the full blooming stage by using urea (46.4% N) as N source. In addition, P and K were applied as a basal fertilizer at the rate of 120 kg P ha −1 using triple superphosphate (46% P2O5) and 150 kg K ha −1 using potassium sulphate (50% K2O). Experiment plot was 6.4 m wide × 8 m long with 8 rows each spaced 0.8 m apart. A uniform cotton planting density of approximately 52,500 plants per hectare was maintained. The cultivars were planted on 28 April in 2018 and 3 May in 2019, respectively. The cultivation management methods were consistent.

Data Collection
During the growing seasons, plants in the three inner rows of each plot were used for biomass accumulation and N content measurement. At the end of the growing season, the other five rows were used for measuring yield and its components.

Yield and Yield Components
Seed cotton of each experimental plot was hand-picked two times (8 and 28 October in 2018, 4 and 31 October in 2019) and weighed after drying to calculate the seed cotton yield. Before harvest, 10 consecutive cotton plants from the inner row were used to determine the yield components. Open bolls were counted for boll number per plant and harvested to calculate the average boll weight. Seed cotton of the 10 plants was ginned to measure lint percentage. The two-year experiment was conducted under a split block experimental design with three replications where the main plot were four N levels (N0, 0 kg N ha −1 ; N1, 112.5 kg N ha −1 ; N2, 225 kg N ha −1 ; N3, 337.5 kg N ha −1 ), and the subplot was cotton cultivar. As for N treatment, half of the target N was used as basal fertilizer and the other half was applied at the full blooming stage by using urea (46.4% N) as N source. In addition, P and K were applied as a basal fertilizer at the rate of 120 kg P ha −1 using triple superphosphate (46% P 2 O 5 ) and 150 kg K ha −1 using potassium sulphate (50% K 2 O). Experiment plot was 6.4 m wide × 8 m long with 8 rows each spaced 0.8 m apart. A uniform cotton planting density of approximately 52,500 plants per hectare was maintained. The cultivars were planted on 28 April in 2018 and 3 May in 2019, respectively. The cultivation management methods were consistent.

Data Collection
During the growing seasons, plants in the three inner rows of each plot were used for biomass accumulation and N content measurement. At the end of the growing season, the other five rows were used for measuring yield and its components.

Yield and Yield Components
Seed cotton of each experimental plot was hand-picked two times (8 and 28 October in 2018, 4 and 31 October in 2019) and weighed after drying to calculate the seed cotton yield. Before harvest, 10 consecutive cotton plants from the inner row were used to determine the yield components. Open bolls were counted for boll number per plant and harvested to calculate the average boll weight. Seed cotton of the 10 plants was ginned to measure lint percentage.

Biomass Accumulation and Partitioning, N Uptake, and N Use Efficiency
The biomass accumulations were measured at 40, 69, 98, and 120 days after emergence (DAE) in 2018 and at 41, 63, 90, and 112 DAE in 2019. Three randomly selected plants from each plot were sampled by uprooting them slowly, and partitioned into vegetative (root, stem (including branches), and leaves), and reproductive organs (buds, flowers, and bolls), and then put in the paper bag. Samples were placed an oven for cell killing at 105 • C for 30 min and then drying at 80 • C to a constant weight before weighing. Biomass partitioning was measured at the boll stage as using the biomass ratio of different plant organs.
With respect to total N uptake, samples of each plant part were milled and screened through a 0.5-mm sieve. The total N concentration was determined using the micro-flow Auto Analyzer 3 (AA3 SEAL, Germany) by the H 2 O 2 -H 2 SO 4 digestion method and was expressed on a dry weight basis. N accumulation and N utilization-related index were calculated using the following equations [38,39].  (7) where NBE stands for N biological efficiency, NAR is short for N apparent recovery efficiency, NUpE stands for N uptake efficiency; NUtE represents for N utilization efficiency; NUE represents for N use efficiency and NHI is short for N harvest index.

Statistical Analysis
All data were presented as the means of the replicates. Statistical analysis was conducted using SAS 8.1 software (SAS Institute, Cary, NC, USA, 1989). Analysis of variance (ANOVA) was used to test the significance of treatment, group and their interactions using a general linear model. Means were separated using the least significance difference (LSD) tests at the 5% probability level. The maximum yield and nitrogen application amount were calculated through regression analysis of yield and nitrogen fertilizer.

Effects of N and Cotton Cultivar on Biomass Accumulation and Partitioning
As shown in Table 2, the total dry weight increased as plants grew, following a normal growth curve after emergence. The growth rate of 2019 was faster than that of 2018, especially at the peak bloom stage and boll opening stage, which possibly due to more rainfall 2019 ( Figure 1 and Table 2). Averaged across cultivars, the biomass of N1, N2, and N3 treatments were 6%, 15%, and 24% in 2018, and 13%, 30%, and 36% higher in 2019 than N0 treatment at the boll opening stage, respectively. N rate and cotton cultivar have significant effects on biomass accumulation of all cotton growing stages ( Table 2). Biomass accumulation significantly increased with an increase in N rate from N0 to N2 (0−225 kg N ha −1 ) at the boll setting and opening stage. However, there was no apparent difference between N2 and N3 treatments. In addition, the difference of dry matter accumulation among different N rates increased gradually with the advance of cotton growth period. As refers to cotton cultivars, high NUE cultivars CRI 69 and ZZM 1017 accumulated more biomass than that of low NUE cultivars ZZM GD89 and XLZ 30 at any growth stage. Moreover, the biomass difference between the two cotton types increased as plants grew (Table 2).

Effects of N and Cotton Cultivar on Yield and Yield Components
The yield and yield components varied significantly across years (except boll numbers), cultivars and N fertilizers. More importantly, the N by cultivar interactions effects on yield were significant (Table 3) (Table 3). Boll weight varied among different N rates and cotton cultivars. The interaction effect of year by N and cultivar on boll weight were significant ( Table 3). As the N rate increased, the boll weight was increased first and stable, which were indicating that appropriate increase of nitrogen fertilizer could increase the boll weight. However, when the nitrogen application exceeded the threshold, the boll weight would not continue to increase. Averaged across cultivars, the boll weight of N1, N2, and N3 treatments were higher 4%, 6%, and 4% in 2018, and 6%, 10%, and 10% in 2019 than N0 treatment, respectively. Additionally, the mean boll weight of CRI 69 and ZZM 1017 was higher 7%, 8%, 6%, and 5% in 2018 and 13%, 14%, 13%, and 12% in 2019 than that of ZZM GD89 and XLZ 30 at N rates (N0-N3), respectively. This result indicated that the cultivars of CRI 69 and ZZM 1017 could get more boll weight especially at the low N level, and the difference between two different NUE cotton types can be reduced by applying more N (Table 3).
A significant interaction was observed between N rates and cotton cultivars for seed cotton yield during both years. Additionally, after comparing and analyzing the yield component, it was found that the yield difference mainly comes from the boll weight (Table 3). In this study, the yield of two different NUE cotton types responds differently to nitrogen. The yield of ZZM GD89 and XLZ 30 were the highest under the highest N rate (N3), however, the yield of CRI 69 and ZZM 1017 were the highest under the middle N rate (N2). Furthermore, the yield of CRI 69 and ZZM 1017 were significantly higher than that of ZZM GD89 and XLZ 30, and the difference between two cotton types were reduced as the N increase. Averaged across N rate, the yield of HNUEC cotton was 19% and 18% higher than that of LNUEC in 2018 and in 2019. This result indicated that the LNUEC plants need more N fertilizers to gain an equivalent higher yield compare to HNUEC plants. As expected, as shown in Figure 2, the CRI 69 and ZZM 1017 can obtain the highest yield (4694 kg hm −2 and 4789 kg hm −2 ) at the N level of 345 kg N hm −2 and 27,070 kg N hm −2 , while the ZZM GD89 and XLZ 30 needs more N (623 kg N hm −2 and 4319 kg N hm −2 , respectively) to achieve their highest yields (4659 kg hm −2 and 8886 6 kg hm −2 ).

Effect of N and Cotton Cultivars on N Accumulation and N Utilization Related Traits
A significant difference was observed between two cotton types in some parameters of N accumulation and N utilization. With the increase of N rate, the N content in reproductive organs and total N accumulation of CRI 69 and ZZM 1017 showed a stable trend after increasing, whereas the relevant traits of ZZM GD89 and XLZ 30 were still increasing ( Figure 3). Furthermore, CRI 69 and ZZM 1017 had more N accumulation than that of ZZM GD89 and XLZ 30, the mean N accumulation of CRI 69 and ZZM 1017 was higher 36%, 38%, 41%, and 36% in 2018 and 40%, 48%, 47%, and 39% in 2019 than that of ZZM GD89 and XLZ 30 at N rates (N0-N3), respectively (Figure 3).   As shown in Table 4, NBE, NAR, NUpE, NUtE, as well as NUE varied significantly across years, cultivars and N fertilizers (except NHI). Additionally, the interaction effect of cultivar by N on N utilization were significant (except NBE, NUtE, and NHI). All of N utilization decreased as nitrogen increases, (except NHI). In comparison with N1 treatment, the NUE of N2 and N3 was reduced by 48% and 64% in 2018, and 46% and 63% in 2019, respectively. The mean NUE of CRI 69 and ZZM 1017 was higher 21%, 17%, and 14% in 2018 and 18%, 16%, and 11% in 2019 than that of ZZM GD89 and XLZ 30 at N rates (N1-N3), respectively (Figure 3), indicating that the NUE of CRI 69 and ZZM 1017 were significantly higher than that of ZZM GD89 and XLZ 30, and the difference between two different NUE cotton types can be reduced by applying more N.

Effect of N on Cotton Yield, Yield Components, and NUE
Cotton yield is the result of the coordinated development of yield components such as bolls per ground area, boll weight, and lint percentage. Nitrogen (N) is one of the most important nutrients needed in large amounts for better crop production. Application of optimum N improves various physiological and metabolic processes such as photosynthesis, carbon, and nitrogen metabolism, which is an important limiting factor of high yield and high quality of cotton. Therefore, the application of N fertilizer is one of the important means to increase cotton yield [8]. Many studies have confirmed that a reasonable supply of N nutrition can increase the dry matter and growth rate of cotton at all stages [7]. Additionally, it also improves the dry matter distribution ratio [34,40,41], increases the accumulation and translocation of photosynthetic products [42], and promotes the production [43].
Consistent with previous studies [5,12], increased N rate (N0-N2) significantly improved yield and boll weight. The results indicated that the increased yield was thus attributed to increased boll weight and nitrogen plays an important role in the formation of boll weight and is the main factor affecting yield. However, the biomass and the N accumulation in plants were highest under the N3 treatment. The studies showed that the increase of nitrogen fertilizer can increase cotton nitrogen absorption, which is beneficial to plant photosynthesis and carbohydrate accumulation [12,34], thereby increasing the biomass, whereas the boll weight was not the highest under the N3. These results proved that excessive use of nitrogen broke the balance between the growth of reproductive and vegetative, leading to a preference for vegetative growth. Besides, we found that the proportion of reproductive organs was the highest under N2 treatment, which was consistent with the research results of Chen et al. [41] and Liu et al. [34] that suitable nitrogen input can promote the transfer of carbohydrates to the reproductive organs and increase economic output. In general, there is positive correlation between yield and biomass, in a certain range. Therefore, yield stabilization can be achieved by maintaining a moderate biological yield and relatively more assimilate distribution on the reproductive organs under the condition of reducing the amount of N fertilizer.
It is well known that the input of N fertilizer increases the N concentration and N accumulation in plants [44][45][46]. However, NBE, NAR, NUpE, NUtE, and NUE were decreased with an increase in N application [47][48][49]. We also found that the N content of the reproductive organs increased first and then stable or decreased, but the N content in the vegetative organs N increased, as the N rate increasing, which were proved that the distribution of N assimilate to the reproductive organs reduced under the high N rate or low N rate, it was not conducive to the formation of boll weight and yield. Additionally, the interaction effect of year by N on NUE were significant, proving that the input of nitrogen fertilizer directly affects the nitrogen use efficiency of crops. However, the NUE were much lower than in developed countries such as Europe and America (50-70%), even lower than the average NUE in China (30-35%) [50]. According to China's national conditions, we can only pursue a reasonable NUE with a high yield, instead of blindly pursuing a high NUE to reduce yield. Therefore, it is urgent to find a balance point between nitrogen fertilizer usage, yield, and NUE. In the present study, we found that optimizing the nitrogen allocation ratio of economic organs is a feasible research direction.

Effect of Cotton Cultivars on Yield, Yield Components, and NUE
Many studies on corn and other crops found that the use of N-efficient cultivars can maintains a moderate biological yield and relatively more assimilation distribution on the reproductive organs, to maintain a stable yield, and increase NUE under the conditions of reduced N fertilization. It is may be a way to solve N pollution and is also beneficial to the sustainable development of cotton agriculture. Combining high yield with high NUE is currently challenging. In our environment, poor N use efficiency is found in the cotton.
As discussed earlier that we have identified cotton genotypes with differing NUE and biomass potential [37,51,52]. However, most of these studies focused on the seedling stage plant biomass, photosynthetic activity, and C/N metabolism, and little is known about the genotypic variations in the yield and yield components and NUE. In our study, CRI 69 and ZZM 1017 showed a strong adaptability, especially under low N conditions, so the total N accumulation of CRI 69 and ZZM 1017 are significantly higher than those of ZZM GD89 and XLZ 30, which could promote leaf area development and photosynthetic efficiency [53]. Due to the difference in N uptake and photosynthesis, there was a significant difference in biomass between cultivars. More importantly, the N content of the reproductive organs in CRI 69 and ZZM 1017 were significantly higher than those of ZZM GD89 and XLZ 30 too. The results proved that CRI 69 and ZZM 1017not only have a strong ability to absorb nitrogen, but also have a strong ability to transform into reproductive organs, it could facilitate the DM accumulation and partitioning, resulting in increased yield. The findings agreed with Bange and Milroy [54] and Dai [55]: N-efficient cultivars have a strong adaptability and tend to grow in reproductive organs and were beneficial to the formation of boll number and boll weight, especially under low N conditions. Simultaneously, similar results have been observed in other crops, like, wheat [30], maize [27], corn [34], and poplar [37].
Overall, the yield differences between cultivars maybe mainly caused by nitrogen absorption capacity and nitrogen transferred to reproductive organs. Therefore, we should choose or breed varieties with strong absorption and transfer ability to reduce the N fertilization application under the premise of ensuring yield. Owing to higher N content, and yield, the NUE, NBE, NAR, and NUpE of CRI 69 and ZZM 1017 were significantly higher than those of ZZM GD89 and XLZ 30. These results were consistent with previous studies [36] as well as in with our previous study that ZZM GD89 and XLZ 30 has a poor root system, N uptake and utilization efficiency. The high N uptake and utilization in CRI 69 and ZZM 1017 can be improved photosynthesis and translocation from source to sink tissues which ultimately increase yield, yield components, and NUE [37]. From these results, we concluded that high nitrogen use efficiency varieties have strong nitrogen absorption capacity and transfer ability, especially at low and middle N rates.
Combining high yield with highly NUE is currently challenging. In agricultural production, poor NUE is found in the cotton. Through our research, we found that the N-efficient cultivars can maintain a stable yield and improve the NUE by reducing the N application moderately. At the same time, we need further research to explore the physiological mechanism of efficient and find a highly effective ways to improve the nitrogen utilization efficiency of N-efficient cultivars to improve NUE and cut the nitrogen input. It was in line with the call of the world and China to reduce nitrogen input and N pollution.

The Interaction Effect of Cultivar by N on Yield, Yield Components, and NUE
There were no significant interactions between N and cotton genotypes for biomass, boll number, boll weight, N accumulation, and NUE in both years which agree with previous findings in other crops [18,34]. However, in our study, we have found a significant interaction between N rate and cotton cultivars for yield and NUE. These different conclusions maybe likely associated with crop genetic characteristics, agricultural management and environment. To a certain extent, the results indicated that yield of cotton and the NUE can be improved through the combination of N-fertilizer reduction and N-efficient cultivar.

Conclusions
In conclusion, the cultivars of CRI 69 and ZZM 1017 (the N-efficient cotton cultivars) have a strong ability to absorb N and transfer N to reproductive organs. Therefore, they produced more the biomass and the boll weight, which were conducive to the formation of output, especially at low N rate. Thus, we should select the N-efficient cultivars to improve the NUE and reduce the N input under the premise of providing guarantee for a yield. More importantly, breeding of high NUtE and NUpE cultivars can reduce both production cost and the environmental concern.
Author Contributions: J.N. conducted the main experiment and drafted the manuscript and H.G., H.Z., Q.D., N.P., S.W., and Z.W. helped to collect data. A.I. revised the manuscript and modified the language. X.W. performed part of the statistical analysis and revised the manuscript. M.S., G.Y. and X.W. conceived of the study, participated in its design and coordination, and helped to revise the manuscript. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.