PLoS ONE
Public Library of Science
Nutritional and physicochemical characteristics of purple sweet corn juice before and after boiling
Volume: 15, Issue: 5
DOI 10.1371/journal.pone.0233094
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### Notes

Abstract

Sweet corn juice is becoming increasingly popular in China. In order to provide valuable health-related information to consumers, the nutritional and physicochemical characteristics of raw and boiled purple sweet corn juices were herein investigated. Sugars, antinutrients, total free phenols, anthocyanins, and antioxidant activity were analyzed by conventional chemical methods. The viscosity and stability of juices were determined by Ubbelohde viscosity meter and centrifugation, respectively. Boiling process could elevate viscosity, stability and sugar content, and reduce antinutrients, total free phenols, anthocyanins, and antioxidant activity in corn juice. In addition, short time boiling efficiently reduced the degradation of anthocyanins during subsequent refrigeration. The content of amino acids, vitamin B1/B2 and E were detected by High Performance Liquid Chromatography. Gas Chromatography Mass Spectrometry was used for the analysis of fatty acids and aroma compounds. Several aroma compounds not previously reported in corn were identified, including 1-heptanol, 2-methyl-2-butenal, (Z)-3-nonen-1-ol, 3-ethyl-2-methyl-1,3-hexadiene, and 2,4-bis(1,1-dimethylethyl)phenol. Interestingly, the boiling process had no apparent effect on the amino acids profile, but it caused a 45.8% loss of fatty acids in the juice by promoting the retention of fatty acids in the corn residue. These results provide detailed information that could be used for increasing consumers’ knowledge of sweet corn juice, further development of sweet corn juice by food producers, and maize breeding programs.

Feng, Pan, Wang, Liao, Wang, Zhang, Guo, Hu, Li, Xu, Wu, Lu, and Matsakas: Nutritional and physicochemical characteristics of purple sweet corn juice before and after boiling

## Introduction

Sweet corns differ from normal field corns in the mutation of starch synthesis genes, e.g., Sugary (Su), Shrunken (Sh), and Brittle (Bt ), which result in a higher accumulation of sugar and water-soluble polysaccharides at the milk stage than in normal field corns, and provide the sweet taste and creamy texture of sweet corns [1]. Consequently, sweet corns have a slightly different metabolic change in their carbohydrates, and there is a different nutritional profile compared with normal field corns. Generally, sweet corns have higher lipid and amino acid contents, especially the limiting amino acid lysine, than normal field corns at the milk stage [24].

In addition to good taste, sweet corn is rich in nutrients and bioactive compounds, including amino acids, unsaturated fats, dietary fiber, vitamins, minerals, phenols, and other phytochemicals [5]. Corn has the highest content of phenols among common grains, including rice, wheat, and oats [6]. The phenol content is directly related to total antioxidant activity, such that corn has the highest total antioxidant activity, which is beneficial for human health [6,7]. It was reported that consumption of corn and its derived products helps reduce the risk of chronic diseases like cardiovascular disease, type II diabetes, and obesity [5,8,9]. Therefore, the consumption of corn is advocated and has been increasing annually worldwide [7,10].

Sweet corns are one of the most popular vegetables in North America and China and are becoming increasingly popular in the rest of the world as well [5]. Canned-, frozen-, and fresh- sweet corn are major traditional forms of its consumption, and sweet corn ranks third among vegetables consumed in the United States [5]. Consumption of the fresh juice of sweet corn has increased rapidly in China in recent years because of its pleasant taste and rich nutritional content. Sweet corn juice can optionally be boiled before drinking. Boiling enhances the stability of corn juice by starch gelatinization, improves the fragrance via the release of aroma compounds, and can kill pathogenic bacteria to better meet food safety requirements. However, cooked foods have been widely considered to have lower nutritional value, such as lower phenols, anthocyanins, antioxidant, and vitamin contents than the corresponding fresh commodities [1116]. Although heating is usually associated with nutrition loss in most foods, thermal processing has been reported to elevate the total antioxidant activity and total phenols contents in sweet corn, kale, white cabbage, and onion [11,14,15]. Boiling and roasting have been found to increase the total amino acid contents in four marine fishes [17], and microwaving increased the vitamin A and vitamin E contents in African catfish [18]. In general, boiling can effectively reduce anti-nutritional factors and improve nutritional quality, such as enhancing the true ileal digestibility of egg protein and improving tocopherol content [4,16,19,20]. Anti-nutritional factors, such as trypsin inhibitors, tannin, and phytic acid, have inhibitory effects on digesting enzymes and can retard mineral absorption, thus limiting the bioavailability of nutrients [16].

Along with the increasing consumption of sweet corn juice in China and popularity of anthocyanin-enriched foods, a purple sweet corn, which typically has higher anthocyanins than yellow or white sweet corns, was selected for comparing the nutritional and physicochemical characteristics of juices before and after boiling. Collectively, several major results were listed here. 1. The boiling process could elevate viscosity, stability, and sugar content, and reduce antinutrients, total free phenols, anthocyanins, and antioxidant activity in corn juice. 2. The boiling process caused a 45.8% loss of fatty acids in the juice by promoting the retention of fatty acids in the corn residue. 3. Heat blanching for a short time (boiling at 95°C for 5 min) suppressed the degradation of phenols and anthocyanins, probably through an enzymatic pathway. 4. Several aroma compounds previously unreported in corn were identified in this purple sweet corn. The results of this study could provide more information for increasing consumers’ knowledge of sweet corn juice, further development of sweet corn juice by the food producers, and maize breeding programs.

## Materials and methods

### Chemicals

Sodium chloride, methanol, ethanol, amino acid standards, acetonitrile, phenyl isothiocyanate, gallic acid, triethylamine, n-hexane, boron trifluoride-methanol, dichloromethane, formic acid, diethyl ether, petroleum ether, sodium hydroxide, potassium hydroxide, hydrochloric acid, and perchloric acid were purchased from ANPEL Laboratory Technologies (Shanghai) Inc. Vitamin B1/B2 and vitamin E were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Folin-Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), mixed standards of fatty acids, cyanidin 3-O-glucoside, anthranone, 1,2,3-trihydroxybenzene, benzoyl-dl-arginine p-nitroanilide hydrochloride, dimethyl sulfoxide, sulfosalicylic acid, trypsin, and phytic acid were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The purity of chemicals used for High-Performance Liquid Chromatography (HPLC) and Gas Chromatography Mass Spectroscopy (GCMS) complied with the requirement of relevant procedures. All other chemicals were of analytical grade.

### Sample preparation

The purple sweet corn (SICAU76, bred by Sichuan Agricultural University, China) used herein was cultured at a farm in Chongzhou in the summer of 2019, and four biological replicates were prepared. Fresh ears were harvested 23 days after pollination. Three biological replicates and six ears of each replicate were dehusked, and the kernels were removed from the cobs and mixed together. Then, 400 g of kernels (water content = 69.5%) were weighed and homogenized with 1200 mL water using a juice extractor at 10,000 rpm for 1 min. The homogenate was divided into two equal parts. One part was designated as the raw sample. The other part was sealed in a bottle and heated at 95°C for 5 min to prepare the boiled sample. The raw and boiled homogenates were filtered through two layers of medical gauze (cotton fabrics with 1 mm aperture size). After filtration, a mean of 5.428 g (5.217, 5.465, and 5.602 g for each replicate) and 4.571 g (4.41, 4.521, and 4.782 g for each replicate) dry residues were obtained from the raw (residue ratio, 8.90%) and boiled (residue ratio, 7.49%) homogenates, respectively. Samples were stored at −80°C and thawed at room temperature before use. The thermal processing used for the volatile components assay is different and is described in the corresponding section.

### Statistical analysis

All of the experiments were repeated more than three times and each time with three technical repetitions. Values are displayed as the Mean ± SD (standard deviation). Differences between the raw and boiled juice were determined by paired t-test using GraphPad Prism 7, and by two-way ANOVA using Excel 2010. Significance was determined at two levels: P < 0.05 and P < 0.01.

### Amino acid profile assay

The corn juice samples (3 mL) were hydrolyzed in a closed vessel using 6 mol/L HCl at 110°C for 24 h. After hydrolysis, 1 mL of supernatant was evaporated to dryness at 85°C in a water bath, and then 1 mL of water was added to the dried hydrolysates, and the mixture was evaporated to dryness again. HCl (0.02 mol/L, 10 mL) was mixed with the hydrolysates. For the derivative reaction, 500 μL of the hydrolysates was reconstituted with 250 μL phenyl isothiocyanate (0.1 mol/L in acetonitrile) and 250 μL triethylamine (1 mol/L in acetonitrile) for approximately 1 h. n-Hexane (2 mL) was added to the derivatives, followed by vibrating and allowing to stand until the layers separated. After stratification, the bottom layer was filtered through cellulose membrane (pore size = 0.45 μm) syringe filters.

Amino acid analysis was performed using an Agilent-1260 HPLC with a C18 chromatographic column (Shiseido, 4.6 mm × 250 mm × 5 μm). The process used was modified from the China national standard (GB/T 30987–2014). The injection volume was 10 μL, the column temperature was 40°C, and the mobile phase consisted of solvent A (97 vol% sodium acetate solution (0.1 mol/L) and 3 vol% acetonitrile, pH 6.5) and solvent B (80 vol% acetonitrile and 20 vol% water). The elution conditions were as follows: 0–14 min, solvent A was reduced from 100 to 85%; 14–29 min, solvent A was reduced from 85 to 66%; 29–30 min, solvent A was decreased to 0%; 30–37 min, solvent B was held at 100%; 37–38 min, solvent B was decreased to 0%; 38–45 min, solvent A was held at 100%. All the gradients were linear, and the flow rate was 1 mL/min.

The total protein content of the juice was estimated from the total amino acids content. The predicted protein efficiency ratio (P-PER) was calculated using the following equations developed by Alsmeyer, Cunningham, and Happich (1974), as cited by Mohapatra et al. [16].

$\mathrm{P}\mathrm{E}\mathrm{R}=\mathrm{‐}0.684+0.456\left(\mathrm{L}\mathrm{e}\mathrm{u}\right)-0.047\left(\mathrm{P}\mathrm{r}\mathrm{o}\right)$
$\mathrm{P}\mathrm{E}\mathrm{R}=\mathrm{‐}0.468+0.454\left(\mathrm{L}\mathrm{e}\mathrm{u}\right)-0.105\left(\mathrm{T}\mathrm{y}\mathrm{r}\right)$

### Fatty acid profile assay

The hydrolysis was performed as follows: 3 mL of corn juice or 1 g corn residue, 2 mL 95% ethyl alcohol, 100 mg pyrogallic acid, and several zeolites were added to a flask. Then, 10 mL HCl (6 mol/L) was added and the temperature was maintained at 80°C for 40 min. The lipid extraction was performed as follows: ethyl alcohol (95%, 10 mL) was mixed with the hydrolysate and the mixture was transferred to a separatory funnel. Then, 50 mL ether-petroleum ether (50:50 vol%) was used to rinse the flask and subsequently transferred to the separatory funnel. The mixture was vibrated for 5 min in the separatory funnel and allowed to stand for 10 min. The organic layer was collected in a new flask and the process was repeated three times. Finally, the mixture was evaporated in a water bath and dried in an oven at 100°C for 2 h.

Esterification was performed as follows: 2 mL 2% NaOH/methyl alcohol solution (2 g NaOH dissolved in 100 mL methyl alcohol) was added to the dried extraction and kept at 85°C for 30 min. Next, 3 mL 14% boron trifluoride (BF3)/methyl alcohol solution (14 g boron trifluoride dissolved in 100 mL methyl alcohol) was added and maintained at 85°C for 30 min. Then, 1 mL n-hexane was added, the mixture was vibrated for 2 min, and then allowed to stand for 1 h. A 100-μL aliquot of the supernatant liquid was collected and diluted to 1 mL using n-hexane. Finally, the solution was filtered through cellulose membrane (pore size = 0.45 μm) syringe filters.

The fatty acid profile was determined by GCMS using a Thermo Trace1310/ISQ instrument with a TG-5MS (30 m × 0.25 mm × 0.25 μm) chromatographic column. The applied process was modified from the China national standard (GB 5009.168–2016). The temperature program was as follows: 80°C for 1 min and heating to 200°C by 10°C/min; 5°C/min to 250°C, 2°C/min to 270°C, and finally holding at 270°C for 3 min. The instrument parameters were: inlet temperature = 290°C; carrier gas flow rate = 1.2 mL/min; split ratio = unsplit; ion source temperature = 280°C; transfer line temperature = 280°C; solvent delay time = 5 min; scanned area = 30–400 amu; ion source = EI, 70 eV.

### Identification of volatile components

Raw corn juice (1 mL) was added to a headspace bottle and mixed with 3 mL of saturated NaCl. The volatile components isolated after 30 min at 37°C and 95°C were considered to come from the raw and boiled juices, respectively.

The volatile components were detected by GCMS using an Agilent 6890N-5975B with a solid-phase 100 μL polydimethylsiloxane microextraction fiber and an HP-5MS (30 m × 0.25 mm × 0.25 μm) chromatographic column with a carrier gas flow rate of 1 mL/min. The applied process was modified from the China national standard (GB/T 35862–2018). The temperature program was as follows: 45°C for 4 min, heating to 200°C by 6°C/min and holding at 200°C for 5 min, heating at 10°C/min to 250°C, and holding at 250°C for 5 min. The instrument parameters were the following: split ratio = unsplit; inlet temperature = 250°C; scanning mode = full scan; ion source temperature = 230°C; quadrupole temperature = 180°C; ion source = EI, 70 eV. The relative contents of each component were analyzed by the area normalization method.

### Determination of vitamins B1, B2, and E

The process used for the vitamin B (VB) assay was modified from the China national standard (GB 5009.84–2016). Corn juice (5 mL) was added to a centrifuge tube and dried at 60°C under nitrogen air flow. Then, 4 mL of 2% formic acid was added and the mixture was ultrasonicated for 20 min. Next, 3 mL dichloromethane was added to mixture and vibrated for 2 min. The mixture was centrifuged at 5000 × g for 10 min, and 1 mL supernatant was transferred to a C18 solid-phase extraction column and eluted with 2% ammonia in methanol. The eluent was dried by blowing nitrogen over the sample. The mobile phase consisted of solvent A (acetic acid/sodium acetate (1.8%/0.98%) solution) and solvent B (methyl alcohol). The elution conditions were as follows: 0–8 min, 90% solvent A; 8–15 min, solvent A was reduced from 90% to 65%; 15–25 min, solvent A was reduced from 65% to 50%; 25–25.1 min, solvent A was increased from 50% to 90%; 25.1–35 min, solvent A was held at 90%. All the gradients were linear and the flow rate was 1 mL/min. Finally, the dried compounds were redissolved in 1 mL water. Analysis was performed on an Agilent 1260 Infinity LC with a C18 (Shiseido, 4.6 mm × 250 mm × 5 μm) chromatographic column, DAD detector, 30°C column temperature, 10 μL injection volume, and 270 nm test wavelength.

For vitamin E (VE) assay, the applied process was modified from the China national standard (GB/T 17812–2008). Corn juice (5 mL) was mixed with 100 mL ascorbic acid/ethyl alcohol solution (2 g ascorbic acid dissolved in 10 mL water and mixed with 90 mL ethyl alcohol) and 25 mL KOH solution (50 g KOH dissolved in 50 mL water). After 30 min, 50 mL petroleum ether was used to extract vitamin E. Water was then mixed with petroleum ether and then separated to remove the acid. The petroleum ether was dried successively by anhydrous sodium sulfate, rotary evaporation, and nitrogen flow. Finally, the dried compounds were dissolved in 10 mL methyl alcohol and filtered through a 0.22 μm membrane before analysis using a Thermo-U3000 HPLC with a C18 chromatographic column (Agilent, 4.6 mm × 150 mm × 5 μm). The instrument parameters were as follows: detector = DAD-FLD; column temperature = 20°C; injection volume = 10 μL; flow rate = 1 mL/min; excitation wavelength = 294 nm; emission wavelength = 328 nm. The mobile phase was 98% methyl alcohol and 2% water.

### Total soluble sugar, free phenols, anthocyanins, and radical scavenging activity determination

Total soluble sugar content was determined using anthrone reagent with glucose as the standard [13]. Total free phenol content and anthocyanin content was determined using the Folin–Ciocalteau colorimetric method [21] and the pH differential method [22], respectively. Gallic acid and cyanidin 3-O-glucoside were used as the standards in phenols and anthocyanin determination, respectively.

DPPH scavenging activity was performed according to Mohapatra’s method [16], and 1,2,3-trihydroxybenzene was used to determinate free radical scavenging activity by following Li’s method [23].

### Anti-nutritional factors

Trypsin inhibition activity and phytic acid content were determined by following Dwivedi’s method [24]. For trypsin inhibition activity, benzoyl-dl-arginine p-nitroanilide hydrochloride (BAPA) was used to detect trypsin activity by measuring absorbance at 410 nm using a UV-Vis spectrophotometer, and the trypsin inhibition activity was calculated by the difference between the sample and the control. For phytic acid content, Wade reagent (0.03% solution of FeCl3·6H2O containing 0.3% sulfosalicylic acid in water) was used, and absorbance was measured at 500 nm using a UV-Vis spectrophotometer. The final content was calculated based on the standard curve.

### Viscosity and stability

Fresh corn juice was diluted five-fold its volume, and half the quantity was then boiled. The raw and boiled homogenates were filtered through two layers of medical gauze (cotton fabrics with 1 mm aperture size), and the filtrate was used for viscosity and stability determination. The viscosity of the juice was determined using an Ubbelohde viscosity meter. The stability of the juice was determined from the change in turbidity after centrifugation for 10 min at 2000 × g. The transmittance of the corn juice was measured at 660 nm before (T0) and after (T10) centrifugation. The stability was calculated as T0/(T10-T0); the larger this ratio, the more stable the juice.

## Results and discussion

### Amino acid composition and total soluble sugar

The total amino acid and sugar contents are two important quality characteristics of sweet corn. At the milk stage, sweet corns have remarkably higher sugar and amino acid contents, particularly that of lysine, than normal field corns [25]. The amino acid composition and total soluble sugar contents of the raw and boiled sweet corn juices are presented in Table 1 and Fig 1, respectively. The total amino acid (TAA) for the two juices was comparable and the boiled juice contained more sugars. Generally, cooking processes cause a reduction of amino acids and soluble sugar in solid foods. This is particularly true for the boiling process because free amino acids and soluble sugars will leach into the surrounding water [13,17]. In the present study, the comparable TAA contents between boiled and raw juices implied that a short boiling time had no effect on the amino acid content. The proportion of total essential amino acids (TEAA) to TAA is an important indicator of protein quality. TEAA/TAA values of 39%, 26%, and 11% are considered adequate for infants, children, and adults, respectively [25]. The raw and boiled sweet corn juices, having TEAA/TAA values of 42.7–43.39%, were adequate for infants and comparable to wheat flour (44.7–47.3%) [26] and some fish (43.6–46.49%) [17], and were superior to sorghum grain (36.8–38.06%) [16].

Fig 1
Asterisk (*) represents that the difference between raw and boiled juices was significant. * P<0.05.The content of soluble sugar, vitamins B1 (VB1), B2 (VB2) and E (VE) in raw and boiled juices.
Table 1
Amino acid composition in raw and boiled purple sweet corn juice.
RawBoiled
Mean+SD(mg/L)%TotalMean+SD(mg/L)%Total
Asp612.53+78.065.58571.07+19.755.48
Glu1419.05+262.6612.931319.70+75.9112.66
Cys1189.15+42.3210.841359.27+100.3713.04
Ser449.79+82.134.10399.61+10.253.83
Gly399.73+42.273.64382.47+10.403.67
His#354.17+32.933.23338.97+4.103.25
Arg#574.89+176.685.24538.52+86.445.17
Thr#268.63+61.522.45248.43+14.322.38
Ala722.05+77.976.58676.43+22.686.49
Pro1418.52+58.1812.931262.57+103.3712.11
Tyr#429.78+57.243.92393.24+7.443.77
Val#510.82+61.714.66476.82+12.034.58
Met#297.62+25.642.71298.96+15.802.87
Ile#406.21+33.653.70372.52+14.183.57
Leu#899.95+143.988.20822.24+34.337.89
Phe#477.30+64.834.35443.53+10.004.26
Lys#540.50+47.424.93517.29+12.454.96
TEAA4759.8843.394450.5242.70
TNEAA6210.8256.615971.1257.30
TAA10970.710010421.64100
RawBoiled
PER⑴2.452.34
PER⑵2.842.72
TEAA: total essential amino acids. TNEAA: total non-essential amino acids. TAA: total amino acids PER: protein efficiency ratio. Details were stated in Materials and methods. Superscript (#) represents essential and semi-essential amino acids for adults and infants.

The P-PER is another important indicator of protein quality, reflecting the potential efficiency of bioconversion of the tested protein into body weight. The experimentally determined PER usually ranges from 0 for a very poor protein to just over 4 for a high-quality protein [25]. Casein, a dairy protein and the standard reference for the PER, has a PER of 2.5 [25]. For the purple sweet corn used herein, the P-PER ranged from 2.34 to 2.84, comparable to those of wheat flour (2.19–2.46) [26] and some fish (2.29–2.60) [17], and superior to those of the sorghum grain (0.02–0.64) [16] and millet (1.32–1.66) [26].

The increased sugar content in the boiled juice may be due to the release of sugar from the sweet corn residue by thermal processing and/or hydrolysis of starch by amylase. It was consistent with prior reports, which stated that boiling promoted the leaching of sugars from broccoli into the water [13] and promoted the hydrolysis of starch into reducing sugars in sweet potatoes [27]. Another explanation is that large amounts of glycosyls were released into solution during the boiling-induced degradation of anthocyanins since purple corns contain high amounts of glycosylated anthocyanins [28].

### Vitamins B1, B2, and E

Vitamins are very important trophic factors in food. The contents of thiamin (vitamin B1), riboflavin (vitamin B2), vitamin B6, and vitamin E in yellow and white sweet corns are significantly lower than those in normal yellow and white field corns [5]. In this study, vitamins B1, B2, and E were determined in purple sweet corn juices. As shown in Fig 1, the contents of vitamins B1, B2, and E in the raw corn juice were 1.83, 0.62, and 0.41 mg/L, respectively. When the vitamin contents in the fresh corn kernels were calculated from the juice values, the contents of the vitamins B1, B2, and E were 5.22, 1.78, and 1.16 mg/kg, respectively. The vitamin B1 content in the purple sweet corn was higher than that reported in other sweet corns (white: 2 mg/kg, yellow: 1.55 mg/kg) and normal field corns (white: 3.85 mg/kg, yellow: 3.85 mg/kg) [5]. Moreover, the vitamin B2 and E contents in the purple sweet corn were higher than those reported in white (B2: 0.6 mg/kg, E: 0.7 mg/kg) and yellow (B2: 0.55 mg/kg, E: 0.7 mg/kg) sweet corns [5].

Vitamin B1 is water-soluble and thermally sensitive under moist conditions [18]. Therefore, there is a large loss of vitamin B1 upon boiling and steaming, but a trivial loss from brief microwave heating [29]. In contrast, vitamins B2 and E are more heat-stable than vitamin B1 under moist conditions, and only slightly affected by thermal processing. However, vitamin B2 is easily degraded by light, and vitamin E is susceptible to chemical modifications in the presence of reactive oxygen species [4,18,30]. The results from this study, however, were puzzling. After 5 min of boiling, the content of vitamin B1 increased from 1.83 to 3.02 mg/L, instead of decreasing as expected. In contrast, the content of vitamin B2, a heat-stable vitamin, decreased from 0.62 to 0.34 mg/L. Vitamin E content did not show any significant change. The increase of vitamin B1 content in the purple sweet corn juice after boiling was difficult to explain. Further research is needed to understand this phenomenon.

### Fatty acid composition

Fatty acid composition is an important indicator of food quality. Corn is reported to have high fatty acid content in the germ, with a fraction of unsaturated fatty acid (UFA) being as high as 80% [3,5]. Thus, corn oil is a high-quality edible oil. In addition, sweet corns have been reported to have higher fatty acid content at the milk stage than normal field corns [2,3].

As shown in Table 2, 35 different fatty acids were identified (fatty acids with a content less than 0.5 mg/L are showed as n). The amounts of the vast majority of fatty acids in the sweet corn juice decreased after boiling, with the largest reduction being ~57.6% for oleic acid. Among several other major components, the loss was 35.8% for linoleic acid, 55.3% for hexadecanoic acid, and 50.4% for stearic acid. However, the erucic acid content did not decrease after boiling. Considering the presence of aroma compounds (Table 3), it was initially speculated that boiling enhanced fatty acid loss by promoting their evaporation. This is because large amounts of oleic acid, linoleic acid, and hexadecanoic acid were identified in the aroma compounds of the boiling juice, but only a trace amount of that were found in the raw juice. However, it is not believed that evaporation of boiling water makes such a big difference in the content of long-chain fatty acids. Thus, the residues from corn juices were analyzed. As expected, most fatty acids showed significantly higher content in the boiled corn residue than in the raw corn residue, except for erucic acid (Table 2). The contents of oleic acid, linoleic acid, hexadecanoic acid, and stearic acid increased by 99.78%, 103.78%, 76.51%, and 95.70%, respectively. Based on these results, it is proposed that boiling process greatly increases the retention of fatty acids in corn residues, potentially by forming covalent linkages or by binding with cell wall components, as fatty acids are known to react with cellulose via esterification [31].

Table 2
Fatty acid composition of raw and boiled purple sweet corn juice.
Corn JuiceCorn Residues
RawBoiledRawBoiled
Mean+SD (mg/L)%TotalMean+SD (mg/L)%TotalMean+SD (mg/kg Dry base)%TotalMean+SD (mg/kg Dry base)%Total
C8.01.56+0.120.01nn16.90+2.540.0214.62+2.060.01
C10.0nnnn1.74+0.660.003.28+2.080.00
C11.0nnnnnnnn
C12.00.87+0.100.011.41+0.540.0233.14+5.030.0453.38+36.490.04
C13.0nnnnnnnn
C14.07.12+0.870.073.69+0.39 **0.0694.73+5.520.12133.10+39.880.09
C14.1nnnnnnnn
C15.01.77+0.340.020.98+0.097 *0.0220.10+0.740.0327.42+3.93 *0.02
C15.11.39+0.100.011.36+0.250.02nnnn
C16.02609.80+455.1724.111165.54+121.31 *19.8818325.03+544.2923.6232344.69+6565.03 *21.30
C16.113.87+3.250.137.13+2.010.12182.97+8.120.24326.19+88.97 *0.21
C17.010.77+2.580.104.62+0.58 *0.08100.61+1.170.13172.85+28.79 *0.11
C17.1nnnn30.13+1.710.0464.95+8.83 **0.04
C18.0392.98+54.043.63194.77+33.97 *3.321122.61+67.111.452196.95+355.13 *1.45
C18.1N9C3329.52+779.2930.761412.51+157.12 *24.0916913.46+1168.6621.8033789.96+6922.85 *22.26
C18.1N9T88.50+19.070.8250.28+14.420.86344.69+6.380.44604.01+30.10 **0.40
C18.2N6C3476.72+866.6632.122231.04+206.66 *38.0538997.97+2098.4650.2679470.83+13970.82 *52.34
C18.2N6Tnnnn6.44+1.360.0122.62+3.20 **0.01
C18.3N354.42+14.790.5045.24+8.630.77387.20+29.540.501267.48+152.41 **0.83
C18.3N6nnnnnnnn
C20.069.05+13.250.6427.93+5.81 *0.48207.01+5.240.27342.70+51.19 *0.23
C20.129.48+4.090.2713.08+1.50 **0.22111.49+7.460.14192.06+34.98 *0.13
C20.22.85+0.480.031.57+0.23 *0.0311.71+0.560.0222.33+3.88 *0.01
C20.3N3nnnnnnnn
C20.3N61.50+0.060.011.22+0.11 *0.02nnnn
C20.4N6nnnnnnnn
C20.5N31.62+0.370.010.98+0.160.02nnnn
C21.01.52+0.250.011.02+0.160.023.56+0.490.005.01+0.830.00
C22.034.34+5.880.3219.78+3.13 *0.34119.98+17.250.15131.66+36.120.09
C22.1N9619.55+81.375.72650.80+84.6111.10493.21+26.050.64554.79+58.790.37
C22.213.20+2.310.1214.21+2.640.2411.55+1.000.0112.73+2.060.01
C22.6N3nnnnnnnn
C23.02.62+0.640.021.40+0.360.024.88+0.030.016.97+1.17 *0.00
C24.047.26+10.610.4420.08+2.12 *0.3444.05+4.360.0660.92+16.600.04
C24.112.40+5.640.119.01+0.930.15nnnn
TSFA3179.6629.371441.2224.5820094.3525.9035493.5523.38
TMUFA4094.7137.832144.1736.5718075.9623.3035531.9623.40
TPUFA3550.3132.802278.4838.8539414.8850.8080796.0053.22
TFA10824.685863.8777585.19151821.51
Total FA of dry kernels74.47 mg/g
TSFA: total saturated fatty acid; TMUFA: total monounsaturated fatty acid; TPUFA: total polyunsaturated fatty acid; TFA: total fatty acid. Asterisk (*) represents difference between raw and boiled was significant.
* P<0.05
** P<0.01.
Table 3
Volatile compounds of raw and boiling purple sweet corn juice.
RawBoiled
123123
Alcohols, Aldehydes and Ketones
1,3-Propanediol, 2-(hydroxymethyl)-2-nitro-nnnn0.21n
1-Heptanol0.780.880.66nn0.28
1-Hexanol2.5922.620.54n0.39
1H-Inden-1-one, octahydro-7a-hydroxy-nn1.55nnn
1-Octanol0.730.570.490.29n0.31
1-Octen-3-ol5.154.114.54nn0.72
1-Pentanol1.68n1.89nnn
2,3-Octanedionennnn0.23n
2-Butenal0.47nnnnn
2-Butenal, 2-methyl-n0.951.21.56n1.53
2-Cyclohexen-1-one, 3-methyl-0.51nnnnn
2-Cyclopenten-1-onenn0.35nnn
2-Ethylidenecyclohexanonenn24.03n1.48n
2-Furancarboxaldehyde, 5-(hydroxymethyl)-nnnn0.25n
2-Furanmethanol, tetrahydro-nnnn0.21n
2-Heptanone2.142.53.35nnn
2-Hepten-4-oln1.59nnnn
2-Heptenal, (Z)-nnn0.390.30.39
2-Nonenal, (E)-3.552.421.091.320.690.83
2-Octanonen3.81n0.671.021.06
2-Octen-1-ol, (E)-nnnnn0.24
2-Octenal, (E)-1.631.471.50.650.380.5
3-Nonen-1-ol, (Z)-n0.290.140.60.310.52
3-Octanol, 2-methyl-n0.31nnnn
3-Octen-2-one0.30.48nnnn
4-Methyl-2,5-dimethoxybenzaldehydennn0.570.82n
5-Methyl-5-hexen-3-yn-2-oln0.18nnnn
6-Dodecanolnnnnn0.22
Cis-3-methylpent-3-ene-5-ol2.25nnnnn
Cyclohexanone1.36nnnnn
Cyclohexanone, 2-butyl-nnn0.73n0.7
Cyclohexanone, 2-methyl-5-(1-methylethyl)-nnnn0.59n
Decanal0.260.11n0.33nn
Ethanone, 1-(2-hydroxy-4,6-dimethoxyphenyl)-nnn0.47nn
Ethanone, 1-(3-methoxyphenyl)-nnn0.97nn
Heptanal11.499.879.881.471.011.13
Heptanolnnnn0.45n
Hexanal18.9223.1921.380.590.570.51
Nonanal1.76n1.621.110.530.81
Octanaln1.05nn0.36n
Silanediol, dimethyl-4.74nnnn0.96
Tridecanalnnn0.27nn
Vanillinnnnn0.2n
Aliphatic Acids and Lactones
1,2-Benzenedicarboxylic acid, butyl 2-methylpropyl esternnn0.3nn
1,2-Benzenedicarboxylic acid, butyl octyl ester0.44nnnnn
11,14-Eicosadienoic acid, methyl ester0.19nnnnn
2,4-Hexadienoic acid, ethyl esternn0.12nnn
2-Propenoic acid, 3-(dimethylamino)-, methyl estern1.87nnnn
3-Nonenoic acidnnnn0.290.26
Acetic acid2.943.38n0.36nn
Benzoic acid, 2,4-bis[(trimethylsilyl)oxy]-, trimethylsilyl esternn0.13nnn
Butyric acid, 1-propylpentyl esternn1.870.54nn
Carbamic acid, methyl esternnnn3.89n
Dodecanoic acidnnn1.640.480.4
Egtazic Acidnnnn0.21n
Formic acid, pentyl estern1.21nnnn
Heptanoic acidn0.51n0.350.230.29
Hexanoic acidnn1.710.89nn
Nonanoic acidnnn0.841.40.59
Octanoic Acid0.960.680.50.850.360.47
Oleic Acid0.59nn16.08nn
Aliphatic hydrocarbon
1-Nonen-3-olnnnn0.58n
1-Pentene, 3-ethyl-3-methyl-n1.45nnnn
2-Pentene, 3,4-dimethyl-,(Z)-nn0.36nnn
2-Pentene, 4,4-dimethyl-,(E)-0.38nnnnn
2-Pentene, 4-methyl-,(Z)-n0.1nnnn
8-Oxabicyclo[5.1.0]octanen0.15nnnn
Cyclodecene, (E)-nnn0.4nn
Cyclooctanenn1.15nnn
Aliphatic hydrocarbon
Cyclooctene, 3-methyl-0.46nnnnn
Cyclopentane, 1,1,3-trimethyl-n1.8nnnn
Cyclopentane, 1-ethyl-2-methyl-, cis-1.58nnnnn
Cyclopentene, 1-butyl-nnn2.67nn
Heneicosanennnn0.48n
Spiro[4.4]nonane-1,6-dionen0.35nnnn
Triallylvinylsilanenn0.1nnn
Nitrogen, Furans, Pyrans, and Aromatic Compounds
1,2-Benzenediol, 3,5-bis(1,1-dimethylethyl)-nn0.34nnn
1,2-Bis(trimethylsilyl)benzene2.190.59nnnn
1,3-Propanediamine, N-methyl-nn0.14nnn
1-Butanamine, N-butylidene-nnnn2.06n
1H-Indole, 1-methyl-2-phenyl-n1.98n3.34nn
1-Methyl-4-[nitromethyl]-4-piperidinolnn0.09nnn
2(3H)-Furanone, dihydro-5-pentyl-0.881.090.981.912.060.86
2,3,5,6-Tetrafluoroanisolennnnn0.66
2-Amino-6-methylbenzoic acidnnnnn0.91
2-Methoxy-4-vinylphenolnnnn1.281.3
2-Nonenenitrilen0.12nn0.210.23
2-Propanaminenn8.2nnn
3,3'-Iminobispropylaminenn0.06nnn
4-Amino-6-hydroxypyrimidine0.81n0.95nnn
5H-Naphtho[2,3-b]carbazolenn0.11nnn
5-Methyl-2-phenylindolizinenn0.38nnn
6H-Pyrazolo[1,2-a][1,2,4,5]tetrazine, hexahydro-2,3-dimethyl-nn0.1nnn
7H-Dibenzo[b,g]carbazole, 7-methyl-nnnnn0.32
Auramine o0.190.15nnnn
Benzaldehydennnn0.2n
Benzaldehyde, 2,5-bis[(trimethylsilyl)oxy]-0.47nnnnn
Benzene, 1-methoxy-4-(1-propenyl)-0.19nn0.770.25n
Benzeneacetaldehyden0.1nnnn
Benzofuran, 2,3-dihydro-nnnn0.19n
Furan, 2-(dichloromethyl)-tetrahydro-n2.14nnnn
Furan, 2-pentyl-3.62n4.15nnn
Hexahydropyridine, 1-methyl-4-[4,5-dihydroxyphenyl]-nn0.11nnn
Indolizinennn0.34nn
Phenol, 2-(1-methylpropyl)-nnnnn0.36
Phenol, 2,4-bis(1,1-dimethylethyl)-nnn1.260.781.39
Phenol, 2,5-bis(1,1-dimethylethyl)-nn0.08nnn
Phenol, 4-(1-methylpropyl)-nnn0.350.46n
Propanenitrile, 3-(methylamino)-nn0.21nnn
Nitrogen, Furans, Pyrans, and Aromatic Compounds
Pyridine, 4-methoxy-n0.09nnnn
Silane,[[4-[1,2-bis[(trimethylsilyl)oxy]ethyl]-1,2-phenylene]bis(oxy)]bis[trimethyl-0.27nnnnn
s-Triazole, 3-acetamido-1.07nnnnn
trans-4-Dimethylamino-4'-methoxychalconennnnn0.28
The compounds with gray background had been reported in previous studies.

On a dry weight basis, the total fatty acid content in this purple sweet corn was ~7%, which is lower than that in high-oil corn (~10%) and higher than that in most normal field corns (~5%) [3,32,33]. However, the content (~24%) of hexadecanoic acid, the main component of saturated fatty acids, in the purple sweet corn was notably higher than those reported for white and yellow corn (less than 10%) [3,32]. Thus, the proportion of UFA in the purple sweet corn was remarkably lower than that in normal field corns. There is a potential possibility that the biosynthesis of anthocyanin may have adverse effects on the accumulation of UFA, and further research is needed for verification.

### Aroma compounds

The relative amounts of the aroma compounds in three biological replicates of raw and boiled juices are individually shown in Table 3. A total of 136 compounds, a noticeably larger quantity than the 43–87 compounds reported in previous papers, were identified from six samples. Among these compounds, only 46 had been reported previously in sweet corns, popcorns, corn tortillas, and related products [3436]. The identification of a large number of new compounds in this study may be attributed to advances in identification techniques and sufficient biological replicates. The presence of most compounds varied greatly between three biological replicates used in this study, and most of the new compounds were only sporadically identified in the three replicates, suggesting they occurred in trace amounts. In addition, 20 compounds, of which 17 had been previously reported, could be consistently identified in all three replicates of the raw or boiled juice. The major components identified in the purple sweet corn were hexanal, 1-octen-3-ol, 1-hexanol, 2-heptanone, and (E )-2-nonenal, which is consistent with the major components previously identified in corns. Moreover, some previously reported major compounds of corn, such as 1-hydroxy-2-propanone, 3-hydroxy-2-butanone, 2,3-butanediol, and acetaldehyde, were not identified in this study [3436].

Five of the newly identified compounds, 1-heptanol, 2-methyl-2-butenal, (Z )-3-nonen-1-ol, 3-ethyl-2-methyl-1,3-hexadiene, and 2,4-bis(1,1-dimethylethyl)phenol, were consistently detected in the biological replicates. In particular, 3-ethyl-2-methyl-1,3-hexadiene accounted for 20% of the volatile compounds and was approximately equal in amount to the major component hexanal, which has been consistently reported to occur [3436]. Furthermore, in a previous study, heptanal accounted for a very small proportion of the aroma compounds [3436], whereas in the present work it was found to be a major component (~10%) of the aroma compounds. Collectively, there is a big difference in the aroma compound composition for the purple sweet corn investigated in the present work and those of previously reported corns, which may be the cause of different aromas between normal field corn and sweet corn [3436].

Boiling promotes the release of aroma compounds from corn juice, thus enhancing properties related to desirable odors. In addition, boiling changes the composition of the volatile compounds through Maillard reactions [37]. Consequently, forty-nine compounds were identified exclusively in the boiling corn juice, such as (Z)-2-heptenal, dodecanoic acid, nonanoic acid and 2,4-bis(1,1-dimethylethyl)-phenol. Fifty-four compounds, such as 1-pentanol, 2-heptanone, (E,E)-3,5-octadien-2-one, and 2-pentyl-furan, were identified exclusively from the raw corn juice. Thus, a difference in aroma due to the boiling of purple sweet corn juice can be expected.

### Phenols, anthocyanins, and antioxidant activity

The total free phenols content and antioxidant activity of the purple sweet corn juice decreased after boiling (Table 4; Frozen juice). This conflicts with Dewanto’s report that thermal processing in sealed cans at 100, 115, and 121°C for 25 min remarkably elevated the total free phenols content and antioxidant activity of yellow sweet corn kernels [11]. In the cited work, severe heat treatment was believed to promote the release of bound phenols via an autohydrolysis reaction. However, a shorter heating time and lower temperature (95°C for 5 min) was applied in the present work. The investigated process conditions may have been insufficient for the release of bound phenols from corn residues. The change in anthocyanins content also differed from what is reported in previous literature, as anthocyanins are generally heat-sensitive and readily degrade during thermal processing [22,38]. However, the present work found that boiling for 5 min increased the total anthocyanin content in purple sweet corn juice (Table 4; Frozen juice).

Table 4
Free phenols, anthocyanins and antioxidant activity of raw and boiled purple sweet corn juice.
Frozen juice (Mean+SD)Fresh juice (Mean+SD)
RawBoiledRawBoiled
Phenols0.292+0.011Bb0.236+0.011Cc0.341+0.010Aa0.248+0.013Cc
Anthocyanins0.038+0.005Bc0.048+0.007ABbc0.062+0.008Aa0.049+0.005ABb
Antioxidant-DPPH33.697+1.679Ab21.655+0.834Bc38.886+1.040Aa23.527+3.099Bc
Antioxidant-Trihydroxybenzene28.348+0.935Ab23.437+0.765Bc31.847+0.966Aa24.854+0.843Bc
Phenols and anthocyanins content were expressed as mg gallic acid equivalent per mL of the juice and mg cyanidin 3-O-glucoside equivalent per mL of the juice, respectively. Antioxidant activity was expressed as free radical scavenging percentage. Superscripts on numerical values are notes of significance test of different treatments, lowercase letters: P < 0.05; uppercase letters: P < 0.01.

Enzymatic degradation of anthocyanins is ubiquitous in tissue homogenate [38]. Heat blanching of a purple corn cob for 4 min and 10 min reduced peroxidase activity by 99% and 100%, respectively, and hence, elevated the stability of the anthocyanins [38]. Thus, we speculated that a short period of heat blanching may inactivate enzymes, such as anthocyanase, polyphenol oxidase, and peroxidase, and consequently reduce the degradation of anthocyanins during storage (approximately 20 days at -80°C). Therefore, fresh (unrefrigerated) juice was used to determine the total free phenols content, anthocyanins content, and antioxidant activity. As shown in Table 4 (Fresh juice), the phenols and anthocyanins content decreased by 27.3% and 21.0%, respectively, after boiling when using fresh juice. Accordingly, the antioxidant activity of boiled fresh juice decreased by 39.5% and 22.0%, as determined by the DPPH and trihydroxybenzene methods, respectively. However, in freeze-preserved juice, the phenol content decreased by 19.2% and the anthocyanin content increased by 26.3%. Consequently, the antioxidant capacity decreased after boiling by 35.7% and 17.3% as determined by the DPPH and trihydroxybenzene methods, respectively. In addition to that of anthocyanins, the degradation of phenols during refrigerated storage is also reduced by boiling process. However, boiling itself has an adverse impact on phenols, anthocyanins, and antioxidant activity in corn juice. This confirmed that the degradation of anthocyanins and phenols happened during refrigeration in the raw juice but not the boiled juice, and a boiling process is necessary for a purple corn juice which needs long-term refrigeration (Table 4 and S1 Table).

### Anti-nutritional factors

Trypsin inhibitors and phytic acid are two primary anti-nutritional factors in corn [39]. They have inhibitory effects on digestive enzymes and can retard mineral absorption, thus limiting the bioavailability of nutrients [16]. The trypsin inhibition activity and phytic acid content in the raw and boiled juices are presented in Fig 2. Trypsin inhibition activity and phytic acid content were decreased by 50% and 55%, respectively, for boiled corn juice. These results are consistent with previous reports that thermal processing effectively reduces anti-nutritional factors [16]. Therefore, the nutrients in boiled corn juice are better absorbed by the body.

Fig 2
Asterisk (*) indicates significant difference between raw and boiled samples. * P<0.05, ** P<0.01.The content of two anti-nutritional factors, viscosity and stability of juices.

### Viscosity and stability

Thermal processing at 95°C for 5 min promotes the gelatinization of starch in corn juice [40]. Gelatinized starch will change the physical properties of the juice, such as viscosity and stability. Therefore, the viscosity and stability of the purple sweet corn juice were tested. As shown in Fig 2, boiling process slightly elevated both viscosity (from 1.29 to 1.52 mm2/s) and stability (from 0.40 to 0.61) of the purple sweet corn juice. These physical changes are beneficial because the appearance of the beverage is more homogenous and stable.

## Conclusions

This study offers a comprehensive understanding of the advantages and disadvantages of boiling purple sweet corn juice. Boiling improved the stability of the juice, and potentially favored enhanced odor by releasing aroma compounds from the juice into the air. Moreover, several aroma compounds unreported in previous studies were identified in the purple sweet corn, such as 1-heptanol, 2-methyl-2-butenal, (Z)-3-nonen-1-ol, 3-ethyl-2-methyl-1,3-hexadiene, and 2,4-bis(1,1-dimethylethyl)-phenol. The sugar content was elevated, and anti-nutritional factors were to a large extent removed by boiling. Unexpectedly, the fatty acid content in the boiled juice was remarkably lower than that in the raw juice because boiling process greatly increased the retention of fatty acids in the corn residues. In purple sweet corn juice, the total free phenols and anthocyanins contents, along with the antioxidant activity, were reduced after boiling. Phenols and anthocyanins in the raw juice were degraded during refrigerated storage. However, heat blanching for a short time significantly suppressed the degradation process and had no apparent effect on the amino acid composition and vitamin E content of the corn juice.

## Acknowledgements

We thank Dr. Xuejun Hua, Ioannis Dogaris, Leonidas Matsakas, and two anonymous reviewers for their suggestions and language polishing.

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31 Mar 2020

PONE-D-20-06814

Nutritional and physicochemical characteristics of purple sweet corn juice before and after boiling

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

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Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comment 1: Please upload all underlying data as attachments to the PLOS ONE submission site.

L24-44: The abstract should not include methodological details. Please rephrase/remove. See author guidelines.

L25-28: Text: In order to contribute to the increased knowledge available for consumers on sweet corn juice, the nutritional and physicochemical characteristics of raw and boiled purple sweet juice was herein investigated.

L33-34: Refrigerated samples? Is this the freezed or raw samples? Both of those samples reach similar levels on anthocyanin after boiling. Please rephrase.

L40: Text: The boiling process had no … However, the content of fatty acids were …

L42-44: Text: These results provide detailed information relevant for further development of sweet corn juice …

L77: Text: Boiling and roasting has been found to…

L92: Is heat-blanching investigated in the present work? If not remove/rephrase.

L100-109: CAS numbers for the products and their purity/quality is missing.

L117: What type of juice extractor? Can I expect the same homogenization if I use my extractor at 10 000 rpm for 1 min?

L120: Medical gauze? Details? Pore-size? Material? This will affect the degree of filtration.

L126-129: Level of significance used (alpha value(s))? As mentioned in the guidelines, this should be specified.

L132: How is degradation and racemization of amino-acids accounted for in the applied method? Such effects will affect the estimated nutritional values of the juice.

L132-134: 1 mL of water was added to the dried/evaporated 1 mL aliquot, then added 1 mL of water before a second evaporation to dryness? This should be written clearer.

L134: What is the hydrolysate? The original liquid from which 1 mL of supernatant was withdrawn? Or the aliquot eventually evaporated? I guess both are hydrolysates and the difference/similarity should be stated clearer.

L140: What type of filter paper? Technical details?

L154: Equations should be numbered, referred to in the text, and described in detail.

L164: Ether-petroleum ether mixture? Diethyl ether mixed with petroleum ether? Which ratio?

L171: Added to the extraction and staying at 85 °C? Added to the separated and dried organic phase and kept at 85 °C for 30 min? Please clarify.

L172: Text: … and kept at 85 …

L177: Membrane material?

L181: Text: Heating to 200 °C…

L194: Text: Heating to …

L196: Text: Split ratio = unsplit; inlet temperature = 250 °C; ….

L219: Text: … to remove the acid.

L224: Text: Detector = DAD-FLD; column temperature = …

L239-240: Please give a short summary of the applied method.

L243: Technical details for medical gauze is missing.

L244: What are the units for the obtained viscosity?

L247: Why was absorbance used and why 660 nm? Which component in the juice absorbs light at this wavelength and why is this related to the stability of the juice?

L271: Table 1: Please clarify (with details, or correct) how there is a statistical difference (significance level of 0.05) between for example Raw vs. Boiled samples and their Methionine content considering the concentration in the juice is given as 297.62 ± 25.64 g/L vs. 298.96 ± 15.80 g/L, respectively. The same holds for Thr, Lys, Phe, Leu, …

L280: Text: Casein, a dairy protein…

L596: Figure 1 contain abbreviations not described in the text (VB1, VB2 and VE) and Figure text is missing for both Figure 1 and 2. Caption for Figure 2 is on L447, while caption for Figure 1 is missing. Please follow instructions given in the guidelines.

L301-303: I guess the values obtained for sweet and normal field corns are obtained from reference [5]? Anyways, please include the reference subsequent the states values, or at the end of L303.

L308: Text: … loss of vitamin B1 upon boiling and steaming…

L310: Text: … and only slightly affected by thermal processing.

L311: Text: … is easily degraded by light…

L312: Text: … is susceptible for chemical modifications in …

L321: Text: … in the germ, with a fraction of... being as high as…

L315: Text: … 35 different fatty acids were identified (fatty acids with a content less than 0.5 mg/L are not included)…

L330-333: Text: Considering the presence of aromatic compounds (Table 3), it was initially speculated that boiling enhanced fatty acid volatility, promoting their evaporation. This because …

L335-337: Please avoid personal pronouns. Text: However, it is believed that… What is the volatility of long chained fatty acids? Boiling points or vapour pressure for pure components? Text: Thus, the residues from … were also analysed.

L341: Avoid ‘we’.

L341-344: Very interesting!

L345: Text: On a dry weight basis, …

L358: Three biological replicates of what? Of raw and boiled purple sweet corn juice? Clarify.

L364: Text: The presence of most compounds varied…

L366-367: Text: …, suggesting they occurred in trace amounts.

L369: Text: … in the purple sweet …

L375: Table 3: Units are missing. Which internal standard was used during GC-MS for fatty acids and volatile components? Please include this information in the appropriate sections within the Materials and methods.

L382: Text: … which has been reported to occur persistently [References].

L384: Text: … whereas in the present work it was found to be a major component (~10%) within the aromatic compounds.

L386: References relating to the statement regarding aromatic compound composition is missing. Text: … for the purple sweet corn investigated in the present work and those of previously reported corns, …

L388-389: Text: …, thus enhancing properties related to desirable smells. In addition, boiling changes the composition of the volatile compounds through …

L393: In the raw corn juice, 54 compounds were exclusively identified, such as …

L395-396: Did you measure the overall aroma from the juices? If not I would change the text: Thus, a difference of aroma due to the boiling of purple sweet corn juice can be expected.

L399: Text: … (Table 4; Frozen juice)…

L402: Text: In the cited work, severe heat …

L404: Text: … lower temperature (95 °C for 5 min) was applied in the present work. The investigated process conditions could have been insufficient for the release of …

L405-406: Text: The change in anthocyanin content was also in contrast to what is reported in literature, as in general, anthocyanin’s are …

L407-409: Text: However, the present work found that boiling for 5 min increased the total anthocyanin content in purple sweet corn juice (Table 4; Frozen juice).

L415: Table 4: Regarding the anthocyanin content in frozen vs. fresh juice – what are possible explanations for the contradicting results apparently occurring due to the freezing process? How was the samples thawed after freezing (specify in Materials and methods)? The content of the boiled samples seem consistent (thus not being affected by the freezing) while the raw samples seem more ‘sensitive’ to the freezing process. Interesting!

L418-423: Even though enzymatic degradation may occur, thus explain any decrease of anthocyanin’s, the results suggest that freezing (which can be expected to correlate to low enzymatic activities) decrease the anthocyanin content more than that of raw samples. How long, and at which temperatures where the ‘raw’ and ‘frozen’ samples stored for/at? This should be specified in the Materials and Methods.

L426: Text: … contents decreased by …

L428: Text: … fresh juice decreased by …

L430: Text: … phenol content decreased by …

L431: Text: … content increased with …

L431: And consequently antioxidant capacity was decreased by 35.7 and 17.3% after boiling? Is antioxidant activity related the anthocyanin content? It was previously mentioned that the antioxidant content is related to the content of phenols. Please rewrite or provide references or data linking antioxidant activity with anthocyanin content, or compare the chemistry of phenol’s and anthocyanin’s.

L432-433: … after boiling as determined by the DPPH and …

L433: The improvement of antioxidant capacity of fresh raw juice as compared to what? The samples frozen and/or boiled? Which antioxidant capacity? Table 5 states that (P value of 0.012) the freezing not significantly affects (assuming a limit of significance equal to 0.05) the DPPH radical scavenging activity of the samples. I would rewrite L433 to something like: “The high antioxidant capacity observed for fresh raw juice samples (Table 4) seem to correlate with the elevated contents of phenols and anthocyanins”. However, the fact that frozen samples present phenol/anthocyanin content developments where their respective contents either decrease or increase due to the boiling treatment, while for raw samples both contents respond on boiling with a decrease does not justify the degradation statement on L434-436. Please rewrite.

L433: Text: … boiled juices are presented in Fig 2…

L444-445: Text: These results are consistent with previous…

L446: Regarding references: Please be consistent with the format used for the references.

L447: Place figure texts in accordance with author guidelines.

L448: Text: Asterix (*) indicates significant difference between raw and boiled samples…

L451: Please provide reference regarding the statement for gelatinization unless this was confirmed directly by any of the results in the present work.

L456-457: Rewrite the final sentence. Why is it desirable with elevated viscosity and stability? Is it beneficial that the appearance of beverages is homogenous? What about mouthfeel?

L459: Text: This study offer a comprehensive understanding on the…

L461: Was starch gelatinization measured directly? If not rewrite.

L461: Rewrite the statement on fragrance and aromatic compounds. The volatile components identified in the present work is given with a relative content, also, boiling only potentially enhance fragrance related properties as this not has been measured directly. Thus, a concluding sentence on the changes of volatile/aromatic potentially favouring enhanced smell-properties would seem more accurate.

L467-470: Was enzymatic activity in the raw material and the response to heat-blanching tested in the present work? If not remove this from the conclusion (could be moved to discussion).

L471-474: This can be moved to the part covering the fragrance/aromatic section occurring earlier in the conclusion.

Reviewer #2: The study presents the results of original research. The authors investigated the nutritional and physicochemical characteristics of purple sweet corn juice for human consumption, before and after boiling. The results reported do not seem to have been published elsewhere. Experiments, statistics, and other analyses were performed to a high technical standard and the methods were described in sufficient detailed or referenced when already published elsewhere. All data were provided in the manuscript and supported the conclusions. Conclusions were a summary of the main experimental data. One suggestion is that the authors also discuss possible implications for their results in the Conclusions section, especially if boiling of sweet corn juice is beneficial or not to human consumption and nutrition, which seems to be the main goal of this study. The article is presented in an intelligible fashion, is well-structured, and is written in standard English with very few grammatical errors. Please find below some minor corrections and suggestions.

Line, comment/suggestion

36, replace “analyzation” with “analysis”

37, replace “has” with “have”

59, replace “benefit” with “beneficial”

88, insert “of” after “because”

256, insert “, respectively” after Fig. 1

269, replace “B1, B2 and E” with “B1 (VB1), B2 (VB2), and E (VE)” to match the abbreviations in the figure.

271, Table 1, “TAA” line, total% should be 100 not 1.

318, One hypothesis could be that B1 was released from the residue during boiling.

350, delete the parenthesis “)” after “[3,32]”

375, Table 3, units are missing

446, replace “(Mohapatra, Patel, Kar, Deshpande, & Tripathi, 2019)” with correct reference number [16]

130, Are there any references for the following methods or have been developed/modified in house? If reported elsewhere please add the reference and if needed briefly describe the important steps or any modifications. Amino acid profile assay, Fatty acid profile assay, Identification of volatile components, Determination of vitamins B1, B2 and E.

Reviewer #3: General comment: The authors reported the effect of boiling on nutritional and physicochemical properties of purple sweet corn juice. The investigation was appropriate, and the authors did a thorough job in discussing the results (for most part). However, several issues need to be addressed before the manuscript is acceptable for publication.

Line 28: Change all “antinutrition” to “antinutrient” or “antinutrients”.

Line 30: “stability” is not specific. Stability of what?

Line 30: “Ubbelohde”.

Line 36: Change “analyzation” to “analyses”.

Line 37: “have”.

Line 59: “beneficial”.

Line 69: What does “stability” refer to?

Lines 88-97: Results do not belong here. Write objectives and underlying hypothesis here.

Line 111: “bred”.

Lines 126-129: There should be a separate section for statistical analysis.

Line 232: The Folin-Ciocaulteau method lacks specificity. Why did not the authors analyze anthocyanins and phenolic compounds using an HPLC?

Lines 239-240: Provide detailed experimental procedures.

Line 311: “easily”.

Line 312: “reactive oxygen species”.

Line 333: These fatty acids have much higher boiling point that the tested temperature.

Lines 351-353: Why? Please provide references to support this speculation.

Line 375: What is the unit?

Line 376: “gray background”.

Line 392: “boiled”.

Line 417: Please indicate which comparisons were labeled with lowercase letters for statistical significance and which ones with uppercase letters.

Line 437: This table is unnecessary and should only be included as an appendix.

Lines 440-457: Discussion is very limited in these two sections and should be expanded.

Figure 1: It does not make sense to group sugar and vitamins in one figure.

Figure 2: The antinutrients results can be in one figure, and the physical properties in another.

**********

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Reviewer #1: No

Reviewer #2: Yes: Ioannis Dogaris

Reviewer #3: No

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7 Apr 2020

Reviewer #1

L24-44: The abstract should not include methodological details. Please rephrase/remove.

Thanks. It has been revised. As the guideline required, we explained how the study was done without methodological detail in the abstract.

L25-28: Text: In order to contribute to the increased knowledge available for consumers on sweet corn juice, the nutritional and physicochemical characteristics of raw and boiled purple sweet juice was herein investigated.

Thanks. It has been revised. Line27-29

L33-34: Refrigerated samples? Is this the freezed or raw samples? Both of those samples reach similar levels on anthocyanin after boiling. Please rephrase.

Thanks. It has been revised. Line35

L40: Text: The boiling process had no … However, the content of fatty acids were …

Thanks. It has been revised. Line41-43

L42-44: Text: These results provide detailed information relevant for further development of sweet corn juice …

Thanks. It has been revised. Line44-45

L77: Text: Boiling and roasting has been found to…

Thanks. It has been revised. Line78

L92: Is heat-blanching investigated in the present work? If not remove/rephrase.

Heat-blanching is another expression of boiling processing at 95oC for 5 min. It has been revised. Line92-94

L100-109: CAS numbers for the products and their purity/quality is missing.

Thanks. There are so many reagents, like amino acids. We don't think it is a good choice to list the CAS number. It will make text verbose. Information of purity has been added in text. Line111-113

L117: What type of juice extractor? Can I expect the same homogenization if I use my extractor at 10 000 rpm for 1 min?

Yes, you can. It is a normal household juicer.

L120: Medical gauze? Details? Pore-size? Material? This will affect the degree of filtration.

Details have been added. Line124-125

L126-129: Level of significance used (alpha value(s))? As mentioned in the guidelines, this should be specified.

Details have been added. Line134-136

L132: How is degradation and racemization of amino-acids accounted for in the applied method? Such effects will affect the estimated nutritional values of the juice.

Maize kernels contain few D-Amino acids. Thus, we do not care about racemization. Hydrolysed amino acids were determined after derivative reaction and quantified by the standard curve. So, the degraded portion could be rectified by the standard curve except for tryptophan which was thoroughly degraded.

L132-134: 1 mL of water was added to the dried/evaporated 1 mL aliquot, then added 1 mL of water before a second evaporation to dryness? This should be written clearer.

Thanks. It has been revised. Line140

L134: What is the hydrolysate? The original liquid from which 1 mL of supernatant was withdrawn? Or the aliquot eventually evaporated? I guess both are hydrolysates and the difference/similarity should be stated clearer.

Thanks. It has been revised. Line40

L140: What type of filter paper? Technical details?

Thanks. It has been revised. Line146

L154: Equations should be numbered, referred to in the text, and described in detail.

Thanks. It has been revised. Line163-164

L164: Ether-petroleum ether mixture? Diethyl ether mixed with petroleum ether? Which ratio?

Details have been added. Line171-172

L171: Added to the extraction and staying at 85 °C? Added to the separated and dried organic phase and kept at 85 °C for 30 min? Please clarify.

Yes. Details have been added. Line 178

L172: Text: … and kept at 85 …

Thanks. It has been revised. Line 179

L177: Membrane material?

Details have been added. Line184-185

L181: Text: Heating to 200 °C…

Thanks. It has been revised.

L194: Text: Heating to …

Thanks. It has been revised. Line190

L196: Text: Split ratio = unsplit; inlet temperature = 250 °C; ….

Thanks. It has been revised.

L219: Text: … to remove the acid.

Thanks. It has been revised. Line233

L224: Text: Detector = DAD-FLD; column temperature = …

Thanks. It has been revised. Line 238-240

L239-240: Please give a short summary of the applied method.

Summary has been added. Line254-260

L243: Technical details for medical gauze is missing.

Details have been added. Line263-264

L244: What are the units for the obtained viscosity?

Unit is mm2/s, and it was showed in result.

L247: Why was absorbance used and why 660 nm? Which component in the juice absorbs light at this wavelength and why is this related to the stability of the juice?

Both absorbance and transmissivity can meet our requirement. For better understanding, absorbance has been replaced by transmissivity in text. Transmissivity at 660 nm was widely used to measure the density of bacterium or small particles in culture medium because the light of this wavelength was blocked mainly by particles in liquid. Before our experiment, long time and high speed centrifuged corn juice was tested under 660 nm, and there was no absorption peak. However, non-centrifuged juice can block the vast majority of light. The more unstable the liquid system, the more rapid the particles deposited during centrifugation. Thus, a moderate speed and time for centrifugation was chose and stability was expressed as a ratio of transmissivity between post- and pre- centrifugation. Line267-269

L271: Table 1: Please clarify (with details, or correct) how there is a statistical difference (significance level of 0.05) between for example Raw vs. Boiled samples and their Methionine content considering the concentration in the juice is given as 297.62 ± 25.64 g/L vs. 298.96 ± 15.80 g/L, respectively. The same holds for Thr, Lys, Phe, Leu, …

I am sorry. I think that you may confuse the information of Fig1 and Table1. There is no difference between raw and boiled sample in Table1.

L280: Text: Casein, a dairy protein…

Thanks. It has been revised. Line302

L596: Figure 1 contain abbreviations not described in the text (VB1, VB2 and VE) and Figure text is missing for both Figure 1 and 2. Caption for Figure 2 is on L447, while caption for Figure 1 is missing. Please follow instructions given in the guidelines.

I am sorry. I think that you may miss it. It was there before Table1.

L301-303: I guess the values obtained for sweet and normal field corns are obtained from reference [5]? Anyways, please include the reference subsequent the states values, or at the end of L303.

Thanks. It has been revised. Line326

L308: Text: … loss of vitamin B1 upon boiling and steaming…

Thanks. It has been revised. Line330

L310: Text: … and only slightly affected by thermal processing.

Thanks. It has been revised. Line332-333

L311: Text: … is easily degraded by light…

Thanks. It has been revised. Line333

L312: Text: … is susceptible for chemical modifications in …

Thanks. It has been revised. Line334

L321: Text: … in the germ, with a fraction of... being as high as…

Thanks. It has been revised. Line343

L325: Text: … 35 different fatty acids were identified (fatty acids with a content less than 0.5 mg/L are not included)…

Thanks. It has been revised. Line347-348

L330-333: Text: Considering the presence of aromatic compounds (Table 3), it was initially speculated that boiling enhanced fatty acid volatility, promoting their evaporation. This because …

Thanks. It has been revised. Line352-354

L335-337: Please avoid personal pronouns. Text: However, it is believed that… What is the volatility of long chained fatty acids? Boiling points or vapour pressure for pure components? Text: Thus, the residues from … were also analysed.

Thanks. It has been revised. Line358-358

L341: Avoid ‘we’.

Thanks. It has been revised. Line362

L341-344: Very interesting!

Thanks.

L345: Text: On a dry weight basis, …

Thanks. It has been revised. Line366

L358: Three biological replicates of what? Of raw and boiled purple sweet corn juice? Clarify.

Thanks. It has been revised. Line380

L364: Text: The presence of most compounds varied…

Thanks. It has been revised. Line385-386

L366-367: Text: …, suggesting they occurred in trace amounts.

Thanks. It has been revised. Line388

L369: Text: … in the purple sweet …

Thanks. It has been revised. Line390

L375: Table 3: Units are missing. Which internal standard was used during GC-MS for fatty acids and volatile components? Please include this information in the appropriate sections within the Materials and methods.

I am sorry. It is not quantitative data here. It is relative content of each component analyzed by area normalization method. Details have been added to the Materials and methods. Line208-209

L382: Text: … which has been reported to occur persistently [References].

Thanks. It has been revised. Line401

L384: Text: … whereas in the present work it was found to be a major component (~10%) within the aromatic compounds.

Thanks. It has been revised. Line403-404

L386: References relating to the statement regarding aromatic compound composition is missing. Text: … for the purple sweet corn investigated in the present work and those of previously reported corns, …

Thanks. It has been revised. Line405-406

L388-389: Text: …, thus enhancing properties related to desirable smells. In addition, boiling changes the composition of the volatile compounds through …

Thanks. It has been revised. Line409-410

L393: In the raw corn juice, 54 compounds were exclusively identified, such as …

Thanks. It has been revised. Line413-414

L395-396: Did you measure the overall aroma from the juices? If not I would change the text: Thus, a difference of aroma due to the boiling of purple sweet corn juice can be expected.

Thank you. This change is better. Line415-416

L399: Text: … (Table 4; Frozen juice)…

Thanks. It has been revised. Line421

L402: Text: In the cited work, severe heat …

Thanks. It has been revised. Line424

L404: Text: … lower temperature (95 °C for 5 min) was applied in the present work. The investigated process conditions could have been insufficient for the release of …

Thanks. It has been revised. Line426-427

L405-406: Text: The change in anthocyanin content was also in contrast to what is reported in literature, as in general, anthocyanin’s are …

Thanks. It has been revised. Line428-429

L407-409: Text: However, the present work found that boiling for 5 min increased the total anthocyanin content in purple sweet corn juice (Table 4; Frozen juice).

Thanks. It has been revised. Line430-432

L415: Table 4: Regarding the anthocyanin content in frozen vs. fresh juice – what are possible explanations for the contradicting results apparently occurring due to the freezing process? How was the samples thawed after freezing (specify in Materials and methods)? The content of the boiled samples seem consistent (thus not being affected by the freezing) while the raw samples seem more ‘sensitive’ to the freezing process. Interesting!

Yes. It can explain the contradicting results. Details about thawing have been added. Line128-129

L418-423: Even though enzymatic degradation may occur, thus explain any decrease of anthocyanin’s, the results suggest that freezing (which can be expected to correlate to low enzymatic activities) decrease the anthocyanin content more than that of raw samples. How long, and at which temperatures where the ‘raw’ and ‘frozen’ samples stored for/at? This should be specified in the Materials and Methods.

It is not convenient to point out the storing time, because samples for different experiments were stored for different length of time. The samples used in this experiment were specified in result. Line438-439

L426: Text: … contents decreased by …

Thanks. It has been revised. Line441

L428: Text: … fresh juice decreased by …

Thanks. It has been revised. Line443

L430: Text: … phenol content decreased by …

Thanks. It has been revised. Line445

L431: Text: … content increased with …

Thanks. It has been revised. Line445

L431: And consequently antioxidant capacity was decreased by 35.7 and 17.3% after boiling? Is antioxidant activity related the anthocyanin content? It was previously mentioned that the antioxidant content is related to the content of phenols. Please rewrite or provide references or data linking antioxidant activity with anthocyanin content, or compare the chemistry of phenol’s and anthocyanin’s.

Yes. Previous reports cited in introduction were about yellow or white corn, so the content of phenols mainly determined the antioxidant activity. In the present study, purple corn was used, a large amount of anthocyanins will display big effect on the antioxidant activity.

L432-433: … after boiling as determined by the DPPH and …

Thanks. It has been revised. Line446

L433: The improvement of antioxidant capacity of fresh raw juice as compared to what? The samples frozen and/or boiled? Which antioxidant capacity? Table 5 states that (P value of 0.012) the freezing not significantly affects (assuming a limit of significance equal to 0.05) the DPPH radical scavenging activity of the samples. I would rewrite L433 to something like: “The high antioxidant capacity observed for fresh raw juice samples (Table 4) seem to correlate with the elevated contents of phenols and anthocyanins”. However, the fact that frozen samples present phenol/anthocyanin content developments where their respective contents either decrease or increase due to the boiling treatment, while for raw samples both contents respond on boiling with a decrease does not justify the degradation statement on L434-436. Please rewrite.

Here, P value of 0.012 is small than 0.05, so the difference is significant. This part has been rewritten. Line447-453

L443: Text: … boiled juices are presented in Fig 2…

Thanks. It has been revised. Line463

L444-445: Text: These results are consistent with previous…

Thanks. It has been revised. Line465

L446: Regarding references: Please be consistent with the format used for the references.

Thanks. It has been revised. Line466

L447: Place figure texts in accordance with author guidelines.

Thanks. It has been revised.

L448: Text: Asterix (*) indicates significant difference between raw and boiled samples…

Thanks. It has been revised. Line 469-470

L451: Please provide reference regarding the statement for gelatinization unless this was confirmed directly by any of the results in the present work.

Reference has been added. Line474

L456-457: Rewrite the final sentence. Why is it desirable with elevated viscosity and stability? Is it beneficial that the appearance of beverages is homogenous? What about mouthfeel?

Yes. It has been revised. Line477-479

L459: Text: This study offer a comprehensive understanding on the…

Yes. It has been revised. Line481

L461: Was starch gelatinization measured directly? If not rewrite.

It has been rewritten. Line483

L461: Rewrite the statement on fragrance and aromatic compounds. The volatile components identified in the present work is given with a relative content, also, boiling only potentially enhance fragrance related properties as this not has been measured directly. Thus, a concluding sentence on the changes of volatile/aromatic potentially favouring enhanced smell-properties would seem more accurate.

It has been rewritten. Line483

L467-470: Was enzymatic activity in the raw material and the response to heat-blanching tested in the present work? If not remove this from the conclusion (could be moved to discussion).

Thanks. It has been revised.

L471-474: This can be moved to the part covering the fragrance/aromatic section occurring earlier in the conclusion.

Thanks. It has been revised. Line484-487

Reviewer #2

One suggestion is that the authors also discuss possible implications for their results in the Conclusions section, especially if boiling of sweet corn juice is beneficial or not to human consumption and nutrition, which seems to be the main goal of this study.

Boiling processing has advantageous and disadvantageous effect on purple sweet corn juice. Here we just contribute to the increased knowledge available for consumers on sweet corn juice. It is not easy to make a decision about which kind of juice is better based on these results.

36, replace “analyzation” with “analysis”

Thanks. It has been revised. Line37

37, replace “has” with “have”

Thanks. It has been revised. Line38

59, replace “benefit” with “beneficial”

Thanks. It has been revised. Line60

88, insert “of” after “because”

Thanks. It has been revised. Line89

256, insert “, respectively” after Fig. 1

Thanks. It has been revised. Line276

269, replace “B1, B2 and E” with “B1 (VB1), B2 (VB2), and E (VE)” to match the abbreviations in the figure.

Thanks. It has been revised. Line295

271, Table 1, “TAA” line, total% should be 100 not 1.

Thanks. It has been revised.

318, One hypothesis could be that B1 was released from the residue during boiling.

However, vitamin B2 decreased from 0.62 to 0.34 mg/L. Thus, I think that VB1 release from the residue during boiling is not a good explanation.

350, delete the parenthesis “)” after “[3,32]”

Thanks. It has been revised.

375, Table 3, units are missing

I am sorry. It is not quantitative data here. It is relative content of each component analyzed by area normalization method.

446, replace “(Mohapatra, Patel, Kar, Deshpande, & Tripathi, 2019)” with correct reference number [16]

Thanks. It has been revised. Line465

130, Are there any references for the following methods or have been developed/modified in house? If reported elsewhere please add the reference and if needed briefly describe the important steps or any modifications. Amino acid profile assay, Fatty acid profile assay, Identification of volatile components, Determination of vitamins B1, B2 and E.

References have been added. Line149, line188, line202, line211, line228

Reviewer #3

Line 28: Change all “antinutrition” to “antinutrient” or “antinutrients”.

Thanks. It has been revised. Line29

Line 30: “stability” is not specific. Stability of what?

Thanks. It has been revised. Line31

Line 30: “Ubbelohde”.

Thanks. It has been revised. Line31

Line 36: Change “analyzation” to “analyses”.

Thanks. It has been revised. Line37

Line 37: “have”.

Thanks. It has been revised. Line38

Line 59: “beneficial”.

Thanks. It has been revised. Line60

Line 69: What does “stability” refer to?

It means that the appearance of beverage is homogenous and sedimentation is slow.

Lines 88-97: Results do not belong here. Write objectives and underlying hypothesis here.

Thanks. We write it based on the guidelines of PlosOne. “The introduction should: Provide background that puts the manuscript into context and allows readers outside the field to understand the purpose and significance of the study; Define the problem addressed and why it is important; Include a brief review of the key literature; Note any relevant controversies or disagreements in the field; Conclude with a brief statement of the overall aim of the work and a comment about whether that aim was achieved.”

Line 111: “bred”.

Thanks. It has been revised. Line115

Lines 126-129: There should be a separate section for statistical analysis.

Thanks. It has been revised. Line131

Line 232: The Folin-Ciocaulteau method lacks specificity. Why did not the authors analyze anthocyanins and phenolic compounds using an HPLC?

In this study, we just want to determinate the total phenols and total anthocyanins. The method used here is simple, economical, and fit for the experimental needs.

Lines 239-240: Provide detailed experimental procedures.

Details have been added. Line254-260

Line 311: “easily”.

Thanks. It has been revised. Line333

Line 312: “reactive oxygen species”.

Thanks. It has been revised. Line334

Line 333: These fatty acids have much higher boiling point that the tested temperature.

It has been rewritten. We think these fatty acids were carried into air by vapour. Line352-354

Lines 351-353: Why? Please provide references to support this speculation.

It has been rewritten. Line372-374

Line 375: What is the unit?

I am sorry. It is not quantitative data here. It is relative content of each component analyzed by area normalization method.

Line 376: “gray background”.

Thanks. It has been revised. Line418

Line 392: “boiled”.

Based on the method, aromatic compounds were collected during boiling, so “boiling” was used here.

Line 417: Please indicate which comparisons were labeled with lowercase letters for statistical significance and which ones with uppercase letters.

Details have been added. Line457

Line 437: This table is unnecessary and should only be included as an appendix.

Thanks. It has been revised.

Lines 440-457: Discussion is very limited in these two sections and should be expanded.

It has been rewritten. Line459-479

Figure 1: It does not make sense to group sugar and vitamins in one figure.

Thanks. We want the picture to be more condensed. Here the vertical line between sugar and vitamin has been modified and is more visible now.

Figure 2: The antinutrients results can be in one figure, and the physical properties in another.

Thanks. We want the picture to be more condensed. We believe it does not interfere with the information transfer.

20 Apr 2020

PONE-D-20-06814R1

Nutritional and physicochemical characteristics of purple sweet corn juice before and after boiling

PLOS ONE

Dear Miss Lu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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PLOS ONE

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Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: L38: Text: “Several aromatic compounds not previously reported were identified in sweet purple corn …”.

L113: Text: “Purity level of chemicals used for High-Performance Liquid Chromatography (HPLC) and Gas Chromatography Mass Spectroscopy (GCMS) complied with the requirement of the relevant procedures. All other chemicals were of analytical grade”.

L149-150 and L188-189 and L202-203 and L211-212 and L228-229: Text: “The applied process was modified from the national …”.

Comment to earlier remark on statistics in Table 1: Yes, I misunderstood the presentation. You apply both an asterisk the represent statistical significance and essential/semi-essential amino acids. This could be misleading or confusing (evidently). Different symbols could avoid any chance of misinterpretation.

Reviewer #2: (No Response)

Reviewer #3: General comment: The authors did a thorough job revising the manuscript and addressed all my questions. I have only a few minor comments and suggestions.

Line 27: This sentence is awkward. Why would consumer need to know about the physicochemical characteristics of sweet corn juice? Promoting consumer awareness of the nutritional value or improving quality of the product would be more reasonable justifications for the study.

Lines 28-29: “sweet corn juices”.

Line 78: “have been”.

Line 113: “were of analytical grade”.

Line 135: “ANOVA”.

Line 256: “by measuring absorbance at 410 nm using a UV-Vis spectrophotometer”.

Line 111: change “transmissivity” to “transmittance”.

**********

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Reviewer #1: No

Reviewer #2: Yes: Ioannis Dogaris

Reviewer #3: No

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27 Apr 2020

Reviewer #1: L38: Text: “Several aromatic compounds not previously reported were identified in sweet purple corn …”.

Thanks. It has been revised.

L113: Text: “Purity level of chemicals used for High-Performance Liquid Chromatography (HPLC) and Gas Chromatography Mass Spectroscopy (GCMS) complied with the requirement of the relevant procedures. All other chemicals were of analytical grade”.

Thanks. It has been revised.

L149-150 and L188-189 and L202-203 and L211-212 and L228-229: Text: “The applied process was modified from the national …”.

Thanks. It has been revised.

Comment to earlier remark on statistics in Table 1: Yes, I misunderstood the presentation. You apply both an asterisk the represent statistical significance and essential/semi-essential amino acids. This could be misleading or confusing (evidently). Different symbols could avoid any chance of misinterpretation.

Thanks. A different symbol has been used in Table 1.

Reviewer #2: (No Response)

Reviewer #3: General comment: The authors did a thorough job revising the manuscript and addressed all my questions. I have only a few minor comments and suggestions.

Line 27: This sentence is awkward. Why would consumer need to know about the physicochemical characteristics of sweet corn juice? Promoting consumer awareness of the nutritional value or improving quality of the product would be more reasonable justifications for the study.

I quite agree with your opinion. In this study, our original purpose was to compare many physicochemical characteristics between raw and boiled sweet corn juices, and by which provide consumers and producers more knowledge about sweet corn juice, and help consumers to make a decision during sweet corn juice consumption. This sentence has been rewritten.

Lines 28-29: “sweet corn juices”.

Thanks. It has been revised.

Line 78: “have been”.

Thanks. It has been revised.

Line 113: “were of analytical grade”.

Thanks. It has been revised.

Line 135: “ANOVA”.

Thanks. It has been revised.

Line 256: “by measuring absorbance at 410 nm using a UV-Vis spectrophotometer”.

Thanks. It has been revised.

Line 267: change “transmissivity” to “transmittance”.

Thanks. It has been revised.

29 Apr 2020

Nutritional and physicochemical characteristics of purple sweet corn juice before and after boiling

PONE-D-20-06814R2

Dear Dr. Lu,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Leonidas Matsakas

PLOS ONE

1 May 2020

PONE-D-20-06814R2

Nutritional and physicochemical characteristics of purple sweet corn juice before and after boiling

Dear Dr. Lu:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Leonidas Matsakas