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Analysis of reducing sugars, organic acids and minerals in 15 cultivars of jujube (Ziziphus jujuba mill.) fruits in China
Accepted Manuscript Title: Analysis of Reducing Sugars, Organic Acids and Minerals in 15 Cultivars of Jujube (Ziziphus jujuba Mill.) Fruits in China Authors: Lina Wang, Haiyan Fu, Weizheng Wang, Yaqi Wang, Fuping Zheng, Hui Ni, Feng Chen PII: DOI: Reference:
S0889-1575(18)30573-8 https://doi.org/10.1016/j.jfca.2018.07.008 YJFCA 3117
To appear in: Received date: Revised date: Accepted date:
12-2-2018 24-6-2018 17-7-2018
Please cite this article as: Wang L, Fu H, Wang W, Wang Y, Zheng F, Ni H, Chen F, Analysis of Reducing Sugars, Organic Acids and Minerals in 15 Cultivars of Jujube (Ziziphus jujuba Mill.) Fruits in China, Journal of Food Composition and Analysis (2018), https://doi.org/10.1016/j.jfca.2018.07.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Analysis of Reducing Sugars, Organic Acids and Minerals in 15 Cultivars of Jujube (Ziziphus jujuba Mill.) Fruits in China Lina Wang a,b, Haiyan Fu b,c, Weizheng Wang a, Yaqi Wang a, Fuping Zheng a,b, Hui Ni b,d, Feng Chen a,b *
a. Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing
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Technology and Business University, Beijing 100048, China
b. Department of Food, Nutrition and Packaging Sciences, Clemson University, Clemson, SC
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29634, USA；
c. School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan 430074, China
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d: College of Food and Biology Engineering, Jimei University, Xiamen, Fujian Province
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361021, China
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*Corresponding author:
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Dr. Feng Chen Professor
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Department of Food, Nutrition and Packaging Sciences Clemson University, Clemson, SC 29634, USA Tel: 864-656-5702
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Email: [email protected]
Highlights:
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Different cultivars of Chinese jujube were collected from Shanxi Province, China
Compositions of reducing sugars and organic acids were determined by HPLC-UV
Composition of minerals was determined by ICP-OES
Principal component analysis and hierarchical cluster analysis were used for classification 1
ABSTRACT
Ziziphus jujuba Mill. has been used as both an edible fruit and Chinese medicine for a long time. In this study, the chemical profiles in terms of reducing sugars, organic acids, and minerals of 15 cultivars of jujube were analyzed. The reducing sugars and organic acids were
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measured by HPLC-UV (high performance liquid chromatography with ultraviolet detection).
Minerals were detected by ICP-OES (inductively coupled plasma-optical emission
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spectrometry). Glucose (85.9‒1005 mg/100 g FW), malic acid (120‒509 mg/100 g FW), citric acid (29.4‒181 mg/100 g FW), iron (5.27‒12.5 mg/100 g DW), calcium (16.2‒30.2 mg/100 g DW) and magnesium (51.2‒70.0 mg/100 g DW) were found to be the major components in
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the fruits. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were
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used, in order to classify these cultivars based on the aforementioned chemical profiles.
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According to the PCA, classification based on the content of reducing sugars is more
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categorized into 6 groups.
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reasonable and reliable than other parameters. In this classification, 15 cultivars were
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Keywords: Jujube, Reducing Sugars, Organic Acids, Minerals, Classification, Quantification,
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HPLC-UV, ICP-OES, Food analysis, Food composition
1.
Introduction
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Ziziphus jujuba Mill. is a common fruit, which has been cultivated in Asia for more
than one thousand years. It was reported that there are more than 700 varieties and cultivars of the fruits in China (Chen et al., 2013), which are distributed in different regions, including Henan, Shanxi, Shandong, Shaanxi, Hebei, Gansu provinces, and Xinjiang autonomous region, in the Peoples’ Republic of China (see the supplemental Fig. S1). These jujube fruits 2
have been found to have quite different chemical profiles because of the influence of various environmental conditions, in terms of location, climate, soil, precipitation, etc.. Jujube has a long history of being used as a herbal medicine in ancient China, and is considered as a functional food, due to its rich amounts of nutrients, including sugars, fatty acids, amino acids, minerals, vitamins, polyphenols and other antioxidants (Li et al., 2007). In addition, the
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essential oil extracted from the seeds of jujube was reported to possess anti-inflammatory activity (Al-Reza et al., 2010); the polysaccharides from jujube have hepatoprotective activity
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(Wang et al., 2012) and immunomodulatory activity (Zhang et al., 2013; Zhao et al., 2008); betulinic acid from sour jujube fruits was reported to be able to inhibit breast cancer cells (Sun et al., 2013). In addition, jujube fruits were found to have antioxidant capacity (Xue et
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al., 2009).
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Primary metabolites, such as carbohydrates, proteins, lipids, and amino acids play
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important roles in growth and development of plants, animals and humans (Azmir et al.,
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2013). Sugars such as glucose, fructose and sucrose are the major sugars in fruits, such as
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papaya (Kelebek et al., 2015), berries, peach, apple, watermelon, and cherry, (Ma et al., 2014) etc.. They can be measured by HPLC with detectors like UV-Vis and fluorescence detector
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(FD) after sugar derivatization, for which 1-phenyl-3-methyl-5-pyrazolone (PMP) is a popular chemical reagent (Bai et al., 2015; Dai et al., 2010; Lv et al., 2009; Myron et al., 2015; Zhang
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et al., 2009). This method is used for its high recovery, lack of isomerization, high sensitivity and better accuracy for detection by UV or DAD detectors. In previous studies, fructose,
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glucose and sucrose were found to be the main sugars in jujube fruits; while rhamnose could not be found in most cultivars. In addition, it was found that the sugars in jujube fruits increased as their maturity increased (Guo et al., 2015). However, other reports on reducing sugars in jujube fruits are limited. Organic acids are very important for fruit quality in terms of taste acidity, color, 3
texture, and flavor (de Jesús Ornelas-Paz et al., 2013). In many fruits, such as melon, peach (Flores et al., 2012), pomegranate (Gundogdu and Yilmaz, 2012), strawberry (de Jesús Ornelas-Paz et al., 2013), malic and citric acids are the most common and major acids. It is well known that environmental factors can significantly affect the contents of organic acids in fruits (Arena et al., 2013; Sweetman et al., 2014). Gao et al. (2012b) analyzed organic acids in
could not be detected in the cultivars Zaowangzao and Junchangyihao.
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10 jujube cultivars, and found that malic acid was the major organic acid, while succinic acid
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Minerals often serve as important cofactors of enzymes and/or are involved in biological reactions. Inductively coupled plasma-optical emission spectrometry (ICP-OES) is an efficient instrument to determine mineral content. It can simultaneously analyze the
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micro-minerals (e. g., manganese, zinc, iron, copper, magnesium, selenium, iodine, chromium)
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and macro-minerals (e. g., chloride, sodium, potassium, calcium, phosphorus) in foods
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(Danbaba et al., 2015; Gülfen and Özdemir, 2016). Minerals are also important in commercial
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products such as dried jujube, jujube jelly, etc. (Kao and Chen, 2014). Since the
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overwhelming majority of minerals in plants are from the soil, their contents and relevant fruit quality can be affected by plantation locations. Therefore, minerals could be used for possible
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classification of jujubes.
Ziziphus jujuba Mill. has been widely planted in China. Its plantation area was
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increased from 2.95 million acres to 3.78 million acres from 2004 to 2011. Based on the data from the Chinese Jujube Market Competition and Development (Wang and Hu, 2016), the
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yield of jujube in China approached 7.4 million tons in 2014. Various jujube products, such as fresh jujube, dried jujube, jujube beverage, wine, and vinegar, as well as jujube extracts, have been consumed and/or used as food additives in the world market. Since jujube fruits have high economic value, accurate determination of the jujube composition and their authenticity is very important for customers and the industry. In this study, nutritional compounds of 15 4
cultivars of jujube that were collected from the same farm in Shanxi province of China were analyzed. Reducing sugar-PMP derivatives, organic acids, as well as lactic acid and acetic acid, were analyzed by HPLC/UV; 12 minerals including heavy metals were analyzed by ICP-OES. In general, the objectives of this research were to: (1) obtain the chemical profiles of
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the aforementioned jujube cultivars, and identify their compositional differences which can
help customers to choose the correct fruits for different uses; (2) use principal component
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analysis (PCA) and hierarchical cluster analysis (HCA), based on the aforementioned
nutritional chemical data, to classify the jujube cultivars so as to facilitate the potential
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development of authentic jujube products.
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2. Materials and methods
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2.1 Jujube sample collection
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All the jujube samples were collected from the same farm in Shanxi province, China
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in October, 2015. The specified site of the farm is shown in Supplemental Fig. S2. Intact fruits of similar shape and size were collected, without any visible blemishes. Samples were
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transported to the lab and frozen at ‒-80 °C. All the fruits were peeled to remove the seeds to just keep the pulp for analysis. The analyzed cultivars of Ziziphus jujuba Mill. included cv.
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Banzao (BZ), Dabailing (DB), Cang county Jinsixiaozao (JS), Huping (HP), Lingbao (LB), Yuanling (YL), Jidan (JD), Lizao (LZ), Baode Youzao (YZ), Bin county Jinzao (BJ), Junzao
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(JB), Pingshun Junzao (PB), Xiangzao (XZ), Pozao (PZ), and Neihuangbianhesuan (NP).
2.2 Chemicals Chemical standards, including oxalic acid, malic acid, citric acid, fumaric acid, succinic acid, lactic acid, tartaric acid, acetic acid, rhamnose, mannose, glucose, galactose, 5
xylose, and arabinose, were bought from J. T. Baker Chemical Co. (Fair Lawn, NJ); 1-Phenyl-3-methyl-5-pyrazolone (PMP) (99%) was purchased from Sigma-Aldrich (St Louis, MO). HPLC grade methanol and acetonitrile were bought from Thermo Fisher Scientific (Waltham, PA). 2.3 Analysis of reducing sugars
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One half gram of each individual cultivar sample was placed in a 50-mL test tube with
10 mL of water and shaken well. Then the tube was kept in an 80 °C water bath for 1 hour
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before the samples were centrifuged at 5000 rpm for 15 minutes at room temperature
(Johnson et al., 2013). The supernatant was collected and diluted to 25 mL. One milliliter of the diluted solution was dried by mild nitrogen gas purge. The dried sample was reacted with
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0.5 mol/L of the PMP-methanol reagent and 0.3 mol/L of NaOH in a 70 °C water bath for 60
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minutes. After the sample was cooled to room temperature, 0.3 mL of 0.3 mol/L HCl were
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added. Then, 1 mL of chloroform was added to extract the excess PMP (Lv et al., 2009). The
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aqueous phase was filtered through a 0.22-μm membrane prior to HPLC analysis.
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A Thermo U3000 (Thermo Fisher Scientific, Waltham, MA) series HPLC-UV was used to detect the sugars, for which a Thermo Hypersil Gold C18 column (4.6 × 250 mm, 5
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μm) was used to separate the different sugar-PMP derivatives under isocratic conditions. The mobile phase was composed of 0.1 mol/L phosphate solution: acetonitrile (82:18, v/v) at pH
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7.0. The flow rate was 1 mL/min; the column temperature was controlled at 25 °C; injection volume was 15 μL, and the detected wavelength was 245 nm.
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2.4 Analysis of organic acids This method was based on a previous study (Gao et al., 2012a) with some
modifications. An amount of 1 gram of jujube pulp sample was mixed with 30 mL of deionized water and extracted under ultrasonic treatment for 30 minutes at room temperature. The solution was centrifuged at 5000 rpm for 10 minutes under room temperature to collect 6
the supernatant. The residue was re-extracted three times using the same procedure and under the same conditions. After four extractions, the supernatants were combined, and then evaporated at 65 ºC. The supernatant was filtered through a 0.45-μm nylon filter (Macherey-Nagel Inc., Bethlehem, PA) before HPLC analysis. Concentrations were expressed as mg/100 g fresh weight (FW).
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Organic acids were also analyzed using Thermo U3000 series HPLC-UV with the aforementioned reverse phase Hypersil Gold column and detected at 210 nm. Mobile phase
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was 0.5% NH4H2PO4 (pH 2.6); the flow rate was 0.8 mL/min. The concentration (expressed
in mg per 100 g FW) of the different compounds was calculated based on the external standard method.
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2.5 Analysis of minerals
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This method was based on Hong et al. (2016) with some modifications. The pulps of
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jujube fruits were dried using a conventional oven at 65 °C. Then, 1 g of the dried sample was
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placed in a digestion tube, mixed with 10 mL HNO3 and 2 mL HClO4, which was kept at
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room temperature overnight. Then, the tube was moved into a digestion oven at 280 °C until the solution became clear. After that, distilled water was added to make a final volume of 50
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mL. The mineral contents in the jujube samples were measured by ICP-OES (Perkin Elmer Inc., Waltham, MA). The operating power of the instrument was 1.20 kW, the plasma flow
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rate was 15.0 L/min, the auxiliary flow rate was 1.50 L/min, and the nebulizer pressure was 200 kPa.
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2.6 Statistics
All the samples were analyzed in triplicate. Evaluation of the significant difference
level was by one-way analysis of variance (ANOVA) using JMP Pro 12.2.0 (SAS Institute Inc., Cary, NC). Tukey test was used to evaluate the significance level (p < 0.05). Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were conducted using 7
Matlab (MathWorks, Inc., Natick, MA).
3 Results and discussion 3.1 Analysis of reducing sugars Previous researchers have demonstrated that the environmental conditions are a major
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factor affecting sugar synthesis (Rosa et al., 2009; Ruan et al., 2010; Nguyen-Huu et al., 2015; Paul and Pellny, 2003).. In many previous studies, sucrose, fructose and glucose were
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reported as the major sugars in jujube fruits (Gao et al., 2012a; Gao et al., 2012b; Guo et al., 2015; Li et al., 2007), but information about other reducing sugars is limited. It was first
reported by Honda et al. (1989) that reducing sugars could react with the PMP reagent to form
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sugar-PMP derivatives, which can be easily detected by UV detectors due to their strong UV
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absorbance. Among the jujube cultivars, significantly different concentrations of their
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reducing sugars (see Table 1) were observed. For example, mannose was only detected in the
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cultivars of JB, LB, LZ, NP and XZ. Although there was no significant difference (p < 0.05)
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among the cultivars of JB, LB and XZ based on their mannose content, there were significant differences between the cultivars of LZ and NP and the aforementioned three cultivars (p <
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0.05). Xylose was only detected in cultivars JB, LB, XZ and YZ. The contents of xylose in the cultivars JB and YZ were not significantly different (p < 0.05); but there was a significant
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difference in the contents (p < 0.05) of LB and XZ.
The content of rhamnose was within a
range of 1.42 to 5.48 mg/100 g FW, which was lower than that in a previous report (Gao et al.,
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2012b). The content of glucose was measured in a wide range from 85.9 to 1005 mg/100 g FW, for which the cultivar YZ showed the lowest content while the cultivar PZ contained the highest amount. Arabinose was also measured in jujube; the content varied from 2.30 to 16.8 mg/100 g FW. 3.2 Analysis of organic acids 8
Organic acids contribute to the acidity of fruits and are important to the fruit texture and flavor. Malic, citric, and succinic acids have been found in jujube (Gao et al., 2012a; Gao et al., 2012b). In this study, oxalic, tartaric, malic, lactic, acetic, citric, fumaric and succinic acids were also determined (Table 2). Similar to the contents of reducing sugars, the contents of organic acids had a significant difference in different jujube cultivars (p < 0.05).
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Particularly, lactic and succinic acids were not detected in some cultivars, such as DB, HP and LB. The predominant organic acid was malic acid, of which the amount (120 to 509 mg/100 g
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FW) was the highest among the organic acids in all the tested cultivars. Ziziphus jujuba Mill.
cv. JB was determined to contain 279 mg of malic acid/100 g FW, which was similar to another cultivated JB (294 mg/100 g FW), which is grown in Yulin county in China (Gao et
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al., 2012b); Strawberry was reported to contain around 200 mg/100 g FW of malic acid (de
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Jesús Ornelas-Paz et al., 2013), which was similar to most cultivars of jujube fruits; grapes
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(1095 mg/100 g FW) and peaches (2183 mg/100 g FW) were reported to contain higher malic
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acid content than jujube, while lemons (228 mg/100 g FW) and oranges (131 mg/100 g FW)
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have similar contents (Flores et al., 2012). Ziziphus jujuba Mill. cv. JS contained the lowest amount of oxalic acid (12.1 mg/100 g FW), while the cultivar LB contained the highest
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amount (62.7 mg/100 g FW). The content of tartaric acid varied from 73.7 to 147 mg/100 g FW. Lactic acid could not be detected in the DB, LB, PZ and HP cultivars but showed
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significant difference (p < 0.05) from 8.41 to 31.5 mg/100 g FW in the other cultivars. Acetic acid in the LB and JD cultivars was below 3 mg/100 g FW, but its value was above 10 mg/100
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g in the other cultivars. Its highest content (111 mg/100 g FW) was found in the cultivar XZ. The content of citric acid (29.4 to 181 mg/100 g FW) was higher than other acids in most cultivars. This result is similar to that in a previous report (Gao et al., 2012b). Fumaric acid was detected in all cultivars. Its lowest content was only 0.548 mg/100 g FW in JB, while its highest content was 163 mg/100 g FW in PB. Succinic acid was not detected in some cultivars 9
(i.e., BZ, DB, LB, HP, BJ, JS). In the cultivar YL, its amount was only 2.27 mg/100 g FW, while it was 163 mg/100g FW in the cultivar NP. 3.3 Analysis of minerals Contents of minerals of 15 jujube cultivars are listed in Table 3. The data indicated that calcium, magnesium, and iron were the major minerals in these jujube fruits. According
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to Table 3-1, lead was only detected in Ziziphus jujuba Mill. cv. BZ, DB, HP, LB, LZ, NP, and
PB, of which the content was in a range from 0.023 to 0.130 mg/100g DW. The contents of
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nickel (0.219 to 0.295 mg/100g DW), aluminum (13.8 to 16.8 mg/100 g DW), boron (1.59 to
3.59 mg/100 g DW), titanium (0.194 to 0.309 mg/100 g DW), and chromium (0.366 to 0.991 mg/100 g DW) were not significantly different among most cultivars (p < 0.05). The detected
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ranged from 4.63 to 6.53 mg/100 g (San et al., 2009).
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content of boron in our test cultivars was less than that in the jujubes of Turkey, where values
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Iron is an essential mineral for humans. The iron content in these jujube cultivars was
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from 5.27 to 12.5 mg/100 g DW (see Table 3-2). Most cultivars in this study contained similar
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amounts of iron to those in five cultivars of jujube fruits grown in Jinan in China (Li et al., 2007), which included Yazao, Jianzao and Sanbianhong, as well as Junzao (JB) and
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Jinsixiaozao (JS) that were also analyzed in this study,. However, Ziziphus jujuba Mill. cv. BZ, PZ, YL contained higher content of iron (8.56 to 12.5 mg/100g DW) than those five cultivars
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of jujube (4.68 to 7.90 mg/100 g DW). In this study, the content of iron in the cultivars JS (6.40 mg/100g) and JB (8.38 mg/100g) was higher than that in the same cultivars (4.68 and
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7.90 mg/100 g DW) reported in the previous study (Li et al., 2007). The content of iron in all the cultivars in this study was higher than that (0.67 to 1.43 mg/100 g DW) in the Turkey cultivars (San et al., 2009). The average content of iron (131.9 μg/g) in jujube fruits that were planted in Xinjiang autonomous region of China (Zhu et al., 2014) was higher than the iron content of jujube planted in Shanxi province. This difference could be attributed to the quite 10
different climate and soil conditions in these two regions. Copper is a co-factor of many antioxidants (Gropper and Smith, 2012). In Table 3-2, the content of copper was determined in a range of 0.381 to 0.812 mg/100 g DW. This value was higher than the amount in Li’s report (0.19 to 0.42 mg/100 g), but similar to that in the jujubes planted in Bayikuleng region (4.62 to 8.28 μg/g) in Xinjiang autonomous region (Zhu
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et al., 2014).
Zinc is important for many enzymes, such as carbonic anhydrase, alkaline phosphatase,
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etc. (Gropper and Smith, 2012). Ziziphus jujuba Mill. cv. LB contained the lowest content of
zinc (1.12 mg/ 100 g DW), while Ziziphus jujuba Mill. cv. JD had the highest value (1.76 mg/100 g). The content of zinc in the cultivars in this study was higher than that in Li’s report
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(0.63 mg/100 g compared with 0.35 mg/100 g; (Li et al., 2007)) and San’s report
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(0.53mg/100g to 1.27 mg/100g) (San et al., 2009). However, our result was in agreement with
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the value of zinc in jujubes that were grown in the Xinjiang autonomous region (Zhu et al.,
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2014)
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Calcium is well known to be essential for growth of bones and teeth. Table 3-2 shows that the lowest content of calcium was 16.2 mg/100 g DW found in Ziziphus jujuba Mill. cv.
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YL, while the highest content was 30.2 mg/100 g DW in Ziziphus jujuba Mill. cv. DB. However, its content in all the cultivars was found to be lower than that in both Li’ report
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(45.6 to 118 mg/100g) (Li et al., 2007) and San’s report (79.33 to 121.33 mg/100g) (San et al., 2009).
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Based on the data shown in Table 3-2, magnesium was obviously a primary mineral
because its content was significantly higher than that of all the other minerals. Ziziphus jujuba Mill. cv. HP had the lowest content (51.2 mg/100 g DW), and Ziziphus jujuba Mill. cv. DB contained the highest content (70.0 mg/100 g DW). These values were higher than those of jujube (15.8 to 20.8 mg/100 g) found in Turkey (San et al., 2009). 11
The content of manganese of jujube in this study varied from 0.479 to 1.07 mg/100 g DW. All values were higher than those in the jujube fruits from Turkey (0.10 mg/100 g to 0.20 mg/100 g), and similar to the jujubes in the Xinjiang autonomous region in China . Overall, the contents of minerals in the different cultivars were significantly different (p < 0.05). However, regarding the same mineral, most of the cultivars did not have
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significant differences, which is attributed to the main factor that all of the cultivars were grown and collected from the same farm, so the environmental factors, such as climate and
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soil, were excluded from the major factors that can significantly affect the minerals’ absorbance. In this context, the aforementioned differences among the cultivars are considered to be caused, to a large degree, by the genotype of jujube, though it needs more
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investigations.
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3.4 Hierarchical cluster analysis and principal component analysis
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Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were
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used to categorize the cultivars into subgroups in an effort to explore the existing differences
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among the groups. The former statistical method indicates the similarity among the different cultivars, while the latter method indicates the significant differences among the cultivars. For
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the HCA method, cultivars in the same group are more similar than the cultivars in the other group. In this study, the classifications of HCA were based on the distance equal to 39. In
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contrast, all the ellipses of the constant distance of the PCA method were calculated with a 95% confidence interval. For example, Fig. 1 profiles the dendrogram of all the cultivars based on
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the contents of reducing sugars. According to the mean values of the components, the studied jujube cultivars were categorized into six groups. Ziziphus jujuba Mill. cv. BZ, PZ, DB, NP were in the same group; HP, JD, JS, BJ, PB were classified into another group; YZ and LB were clustered together into the third group, and Ziziphus jujuba Mill. cv. XZ and JB were clustered together into the fourth group. Moreover, the remaining two cultivars YL and LZ 12
were separated into two independent different groups. Fig. 2 shows the score plot of PCA for reducing sugars. After reducing the variable dimensions, the first two PCs (i.e., PC 1 and PC 2) could explain 85.6% of total data variability. As shown in Fig. 2, group A which included DB and PZ was in quadrant I of the coordinate; group D including YL, HP, JS, PB and JD, as well as group E (BZ) and group F (DB), were located in quadrant II; group C including YZ, XZ,
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JB and LB was in quadrant III; and group B (NP and LZ) was observed in quadrant IV. Table 4 lists the ellipse data obtained from the PCA method, and Fig. 2 profiles the corresponding
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ellipse plots for the different groups. If there is no intersection between two ellipses (groups), it means those two groups of clusters have significant differences. In this case, group A, B, C and D had significant differences between each other, and they were all different from group
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E and F. However, since the group E and F had an intersection between them, they could not
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be distinguished from each other. That means there was not a significant difference between
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groups E and F.
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Based on the results of PCA and HCA of the reducing sugars, organic acids, and
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minerals, different jujube cultivars were categorized into different clusters. However, compared with the PCA of organic acids and minerals, only the PCA of reducing sugars might
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be reliable, since it could explain more than 80% of the data variance (See Table 5). In other words, the classified subgroups from the PCA and HCA based on the concentrations of
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organic acids and minerals might not be accurate and convincing enough because of insufficient variability between the samples. Nevertheless, the classification according to the
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reducing sugars was more reliable in comparison to other components.
4. Conclusions In this study, reducing sugars, organic acids, and minerals of 15 cultivars of jujube fruits were analyzed. As a result, the contents of reducing sugars and organic acids were found 13
to be significantly different (p<0.05) among the cultivars. According to PCA, only the reducing sugars could be used as a reliable parameter to classify the cultivars because its first two principal components could explain more than 80% of data variance. In contrast, other jujube nutritional components like the organic acids and minerals were not convincingly reliable for the jujube classification by PCA. Nevertheless, the obtained data of the
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components in the jujube fruits have provided insights into the nutritional value of jujube. In addition, it is a useful attempt to classify different jujube cultivars by PCA and HCA based on
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the aforementioned data, in an effort to improve processing quality, and avoid adulteration of the final products. Finally, these systematical analyses of jujube fruits can increase the
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utilization of the jujube as a functional food.
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Declaration of interest: None. This research did not receive any specific grant from funding
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agencies in the public, commercial, or not-for-profit sectors.
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Al-Reza, S.M., Yoon, J.I., Kim, H.J., Kim, J.S., Kang, S.C., (2010). Anti-inflammatory activity of seed essential oil from Zizyphus jujuba. Food and Chemical Toxicology 48(2), 639-643. Arena, M.E., Zuleta, A., Dyner, L., Constenla, D., Ceci, L., Curvetto, N., (2013). Berberis buxifolia fruit growth and ripening: evolution in carbohydrate and organic acid contents. Scientia Horticulturae 158, 52-58. Azmir, J., Zaidul, I., Rahman, M., Sharif, K., Mohamed, A., Sahena, F., Jahurul, M., Ghafoor, K., Norulaini, N., Omar, A., (2013). Techniques for extraction of bioactive compounds from plant materials: a review. Journal of Food Engineering 117(4), 426-436. Bai, W., Fang, X., Zhao, W., Huang, S., Zhang, H., Qian, M., (2015). Determination of oligosaccharides and monosaccharides in Hakka rice wine by precolumn derivation high-performance liquid chromatography. journal of food and drug analysis 23(4), 645-651. Chen, J., Li, Z., Maiwulanjiang, M., Zhang, W.L., Zhan, J.Y., Lam, C.T., Zhu, K.Y., Yao, P., Choi, R.C., Lau, D.T., (2013). Chemical and biological assessment of Ziziphus jujuba fruits from China: different geographical sources and developmental stages. J Agric Food Chem 61(30), 7315-7324. Dai, J., Wu, Y., Chen, S.W., Zhu, S., Yin, H.P., Wang, M., Tang, J., (2010). Sugar compositional determination of polysaccharides from Dunaliella salina by modified RP-HPLC method of precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone. Carbohydrate Polymers 82(3), 629-635. Danbaba, N., Nkama, I., Badau, M.H., (2015). Application of response surface methodology (RSM) and central composite design (CCD) to optimize minerals composition of rice-cowpea composite blends during extrusion cooking. International Journal of Food Science and Nutrition Engineering 5(1), 40-52. de Jesús Ornelas-Paz, J., Yahia, E.M., Ramírez-Bustamante, N., Pérez-Martínez, J.D., del Pilar Escalante-Minakata, M., Ibarra-Junquera, V., Acosta-Muñiz, C., Guerrero-Prieto, V., Ochoa-Reyes, E., (2013). Physical attributes and chemical composition of organic strawberry fruit (Fragaria x ananassa Duch, Cv. Albion) at six stages of ripening. Food Chem 138(1), 372-381. Flores, P., Hellín, P., Fenoll, J., (2012). Determination of organic acids in fruits and vegetables by liquid chromatography with tandem-mass spectrometry. Food Chem 132(2), 1049-1054. Gülfen, M., Özdemir, A., (2016). Analysis of dietary minerals in selected seeds and nuts by using ICP-OES and assessment based on the recommended daily intakes. Nutrition & Food Science 46(2), 282-292. Gao, Q.-H., Wu, C.S., Wang, M., Xu, B.N., Du, L.J., (2012a). Effect of drying of jujubes (Ziziphus jujuba Mill.) on the contents of sugars, organic acids, α-tocopherol, β-carotene, and phenolic compounds. J Agric Food Chem 60(38), 9642-9648. Gao, Q.H., Wu, C.S., Yu, J.G., Wang, M., Ma, Y.J., Li, C.L., (2012b). Textural characteristic, antioxidant activity, sugar, organic acid, and phenolic profiles of 10 promising jujube (Ziziphus jujuba Mill.) Selections. J Food Sci 77(11), C1218-C1225. Gropper, S., Smith, J., (2012). Advanced nutrition and human metabolism. Cengage Learning. Gundogdu, M., Yilmaz, H., (2012). Organic acid, phenolic profile and antioxidant capacities of pomegranate (Punica granatum L.) cultivars and selected genotypes. Scientia Horticulturae 143, 38-42. Guo, S., Duan, J.A., Qian, D., Tang, Y., Wu, D., Su, S., Wang, H., Zhao, Y., (2015). Content 15
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variations of triterpenic acid, nucleoside, nucleobase, and sugar in jujube (Ziziphus jujuba) fruit during ripening. Food Chem 167, 468-474. Honda, S., Akao, E., Suzuki, S., Okuda, M., Kakehi, K., Nakamura, J., (1989). High-performance liquid chromatography of reducing carbohydrates as strongly ultraviolet-absorbing and electrochemically sensitive 1-phenyl-3-methyl5-pyrazolone derivatives. Analytical biochemistry 180(2), 351-357. Hong, J., Wang, L., Sun, Y., Zhao, L., Niu, G., Tan, W., Rico, C.M., Peralta-Videa J.R., Gardea-Torresdey, J. L. (2016). Foliar applied nanoscale and microscale CeO 2 and CuO alter cucumber (Cucumis sativus) fruit quality. Science of The Total Environment, 563, 904-911. Johnson, C.R., Combs Jr, G.F., Thavarajah, P., (2013). Lentil (Lens culinaris L.): A prebiotic-rich whole food legume. Food Research International 51(1), 107-113. Kao, T.H., Chen, B.H., (2014). Functional components in Zizyphus with emphasis on polysaccharides, Polysaccharides. Springer, pp. 1-28. Kelebek, H., Selli, S., Gubbuk, H., Gunes, E., (2015). Comparative evaluation of volatiles, phenolics, sugars, organic acids and antioxidant properties of Sel-42 and Tainung papaya varieties. Food chemistry 173, 912-919. Li, J.W., Fan, L.P., Ding, S.D., Ding, X.L., (2007). Nutritional composition of five cultivars of chinese jujube. Food Chem 103(2), 454-460. Lv, Y., Yang, X., Zhao, Y., Ruan, Y., Yang, Y., Wang, Z., (2009). Separation and quantification of component monosaccharides of the tea polysaccharides from Gynostemma pentaphyllum by HPLC with indirect UV detection. Food Chem 112(3), 742-746. Ma, C., Sun, Z., Chen, C., Zhang, L., Zhu, S., (2014). Simultaneous separation and determination of fructose, sorbitol, glucose and sucrose in fruits by HPLC–ELSD. Food Chemistry 145, 784-788. Myron, P., Siddiquee, S., Azad, S., Yong, Y., (2015). Tributylamine Facilitated Separations of Fucosylated Chondroitin Sulfate (Fucs) by High Performance Liquid Chromatography (HPLC) into its Component Using 1-Phenyl-3-Methyl-5-Pyrazolone (PMP) Derivatization. Journal of Chromatography & Separation Techniques 2015. Nguyen-Huu, T.D., Gupta, C., Ma, B., Ott, W., Josić, K., Bennett, M.R., (2015). Timing and variability of galactose metabolic gene activation depend on the rate of environmental change. PLoS computational biology 11(7), e1004399. Paul, M.J., Pellny, T.K., (2003). Carbon metabolite feedback regulation of leaf photosynthesis and development. J Exp Bot 54(382), 539-547. Rosa, M., Prado, C., Podazza, G., Interdonato, R., González, J.A., Hilal, M., Prado, F.E., (2009). Soluble sugars: Metabolism, sensing and abiotic stress: A complex network in the life of plants. Plant signaling & behavior 4(5), 388-393. Ruan, Y.-L., Jin, Y., Yang, Y.-J., Li, G.-J., Boyer, J.S., (2010). Sugar input, metabolism, and signaling mediated by invertase: roles in development, yield potential, and response to drought and heat. Molecular Plant 3(6), 942-955. San, B., Yildirim, A.N., Polat, M., Yildirim, F., (2009). Mineral composition of leaves and fruits of some promising Jujube (Zizyphus jujuba miller) genotypes. Asian J Chem 21, 2898-2902. Sun, Y.F., Song, C.K., Viernstein, H., Unger, F., Liang, Z.S., (2013). Apoptosis of human breast cancer cells induced by microencapsulated betulinic acid from sour jujube fruits through the mitochondria transduction pathway. Food Chem 138(2–3), 1998-2007. Sweetman, C., Sadras, V., Hancock, R., Soole, K., Ford, C., (2014). Metabolic effects of elevated temperature on organic acid degradation in ripening Vitis vinifera fruit. J Exp Bot 65(20), 5975-5988. Wang, D., Zhao, Y., Jiao, Y., Yu, L., Yang, S., Yang, X., (2012). Antioxidative and 16
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hepatoprotective effects of the polysaccharides from Zizyphus jujube cv. Shaanbeitanzao. Carbohydrate Polymers 88(4), 1453-1459. Wang, L., Hu, R., (2016). Technical and cost efficiency of jujube growers in Henan Province, China. CUSTOS E AGRONEGOCIO ON LINE 12(2), 279-297. Xue, Z., Feng, W., Cao, J., Cao, D., Jiang, W., (2009). Antioxidant activity and total phenolic contents in peel and pulp of Chinese jujube (Ziziphus jujuba Mill) fruits. Journal of Food Biochemistry 33(5), 613-629. Zhang, J., Chen, J., Wang, D., Hu, Y., Zhang, C., Qin, T., Liu, C., Sheng, X., Nguyen, T.L., (2013). Immune-enhancing activity comparison of sulfated ophiopogonpolysaccharide and sulfated jujube polysaccharide. International Journal of Biological Macromolecules 52, 212-217. Zhang, J., Zhang, Q., Wang, J., Shi, X., Zhang, Z., (2009). Analysis of the monosaccharide composition of fucoidan by precolumn derivation HPLC. Chinese Journal of Oceanology and Limnology 27, 578-582. Zhao, Z., Liu, M., Tu, P., (2008). Characterization of water soluble polysaccharides from organs of Chinese Jujube (Ziziphus jujuba Mill. cv. Dongzao). European Food Research and Technology 226(5), 985-989. Zhu, F., Yang, S., Fan, W., Wang, A., Hao, H., Yao, S., (2014). Heavy metals in jujubes and their potential health risks to the adult consumers in Xinjiang province, China. Environmental monitoring and assessment 186(10), 6039-6046.
17
Figure Captions Figure 1. Dendrogram of 15 cultivars of jujube fruits based on reducing sugar content. Cultivar lines with the same color are in the same cluster.
Figure 2. Classification of 15 cultivars of jujube fruits based on their reducing sugar
A
CC E
PT
ED
M
A
N
U
SC R
IP T
content. Groups A, B, C, D, E and F are the classified clusters of jujube fruits.
18
Figure 1
19
A ED
PT
CC E A
M
N U SC R
I
I N U SC R I
PT
ED
M
A
II
A
CC E
III
IV
Figure 2
20
I N U SC R
Table 1. Contents of reducing sugars in 15 cultivars of jujube fruits, mg/100 g FW name of Ziziphus jujuba Mill. cultivars
abbrevi
rhamnose
ation
Bin county Jinzao
glucose
galactose
xylose
arabinose
n.d.
232±7 fg
1.32±0.05
n.d.
3.28±0.0
e
BJ
1.68±0.13
A
efg Banzao
mannos
BZ
2.06±0.11
n.d.
304±8 e
PT
Junzao
ED
Hupingzao
DB
CC E
Jidanzao
CangcountyJinsixiao
Lingbaozao
4.88±0.17 b
n.d.
753±17 b
n.d.
de 2.08±0.04
JD
0.603±0 .049 c
1.97±0.13
n.d.
defg JS
1.63±0.11
n.d.
1.58±0.10 fg
0.422±0
5.48±0.08 a
8g
n.d.
4.29±0.14 c
2.35±0.07 d
36.3±1.6 6a
3.50±0.10
642±13 c
n.d.
360±5 d 21
16.8±0.2 9a
2.76±0.06
n.d.
d n.d.
2.84±0.0 5 gh
c 1.06±0.
4.19±0.1 0f
1.02±0.04
740±10 b
2.86±0.0 4 gh
gh 1.32±0.
3.29±0.0
n.d.
1.29±0.03
196±5 gh
05 b PB
9c
efg
n PingshunJunzao
26.7±1.2
1.61±0.03
173±6 h
3.16±0.0 6g
e
efg
NP
n.d.
1.14±0.06
201±7gh
6.94±0.1 5c
fgh
11 a Neihuangbianhesua
n.d.
0.993±0.0
286±7 e
5.96±0.1 6d
72 gh
def
LZ
n.d.
4.08±0.15
268±4 ef
.09 c Lizao
2.59±0.08 d
2.20±0.08
JB
LB
7g
b
HP
zao
A
M
def
Dabailing
efg
8.34±0.2 3b
1.54±0.05
n.d.
4.96±0.1
I PZ
Xiangzao
N U SC R
Pozao
4.29±0.15 c
XZ
2.06±0.10
YL
1005±20 a
0.467±0
1.95±0.11
n.d.
2e 5.05±0.16
n.d.
a
205±5 gh
.06 c
A
def Yuanlingzao
n.d.
ef
5d 1.19±0.07
fg
187±2 gh
32.5±1.2 0b
n.d.
n.d.
M
1.42±0.09 g
2.98±0.0 8 gh
n.d.
85.9±3 i
0.778±0.0 24 h
ED
YZ
3.21±0.0 8g
defg
BaodeYouzao
6.10±0.1
28.3±1.0 1c
2.30±0.0 6h
Data show the mean value ± standard error (n = 3)
PT
Different letters after the data in the same column mean significant differences (p < 0.05) n.d. means the data were not detected， the limit ofdetection (LOD) of mannose is 0.0003 mg/100 g FW; LOD of galactose is 0.025 mg/100 g
A
CC E
FW; LOD of xylose is 0.530 mg/100 g FW
22
I N U SC R
Table 2. Contents of organic acids in 15 cultivars of jujube fruits, mg/100 g FW oxalic acid
tartaric
malic acid
lactic acid
acetic acid
citric acid
fumaric acid
succinic acid
222±1 ef
21.4±0.6d
11.0±0.2 i
121±1 d
121.±1 c
n.d.
258±3 d
15.0±0.4 e
36.8±1.1 e
67.8±0.5 g
1.48±0.03 h
n.d.
acid 19.3±0.7 h
25.6±0.6 h
B
21.0±0.2 h
42.1±0.9e
D
50.3±0.6 c
36.3±0.4 f
177±3 g
n.d.
54.7±0.8 c
100±1 e
1.40±0.02 h
n.d.
H
50.3±1.8 c
36.8±1.6 f
254±3 d
n.d.
15.2±0.3 hi
181±3 a
1.01±0.02 h
n.d.
J
30.4±0.7 ef
41.6±0.5 e
279±3 c
25.0±0.4 b
180±1 a
160±2 b
0.548±0.007 h
80.7±1.0 c
A
B
P
J
30.3±0.4 ef
38.8±0.4 ef
231±2 e
11.8±0.1 f
2.93±0.03 j
51.5±0.8 h
52.3±0.7 f
35.8±0.4 f
J
12.1±0.8 i
41.3±1.1 e
233±3 e
14.7±0.4 e
26.0±0.8 g
88.2±1.1 f
88.2±1.1 d
n.d.
A
CC E
B
PT
B
ED
Z
M
J
L
62.7±1.2 a
56.1±0.7 c
120±2 i
n.d.
2.74±0.01 j
69.1±1.1 g
2.30±0.05 h
n.d.
L
26.9±0.6 fg
17.4±0.5 i
188±2 g
15.5±0.4 e
40.3±0.6 e
42.9±1.1 i
0.853±0.011 h
95.3±1.0 b
N
36.6±1.0 d
60.7±1.1 b
509±4 a
8.41±0.16
31.5±0.5 f
129±1 cd
129±1 b
163±1 a
D
S
B
Z
P
g 23
I 25.4±0.3 g
73.7±1.4 a
219±3 ef
B
N U SC R
P
23.9±0.34b
29.2±0.4 fg
161±2 b
163±1 a
57.7±0.3 d
n.d.
16.8±0.2 h
131±1 c
1.28±0.02 h
7.06±0.15 g
22.5±0.5 cd
111±1 b
29.4±0.6 j
1.30±0.04 h
43.7±0.5 e
c
P
543.7±8.2 b
14.7±0.5 i
162±2 h
X
29.0±0.5 fg
31.8±0.9 g
157±2 h
Y
32.9±0.9 e
46.8±1.0 d
207±3 f
31.5±0.6 a
16.1±0.5 h
34.2±0.4 j
34.2±0.4 g
2.27±0.03 h
Y
32.6±0.5 e
48.5±0.9 d
464±5 b
8.72±0.16
49.3±1.0 d
63.4±1.3 g
64.2±1.3 e
57.9±0.9 d
A
Z
Z
ED
L
M
Z
g
Data show the mean value ± standard error (n = 3)
PT
Different letters after the data in the same column mean significant differences (p < 0.05)
A
CC E
n.d. means the data were not detected; LOD of succinic acid is 0.750 mg/100 g FW; LOD of lactic acid is 0.500 mg/100 g FW
24
I N U SC R
B
Pb
0.245±0.001 abcd
0.385±0.007 e
0.245±0.008 ab
16.5±0.8 ab
2.59±0.14 bcd
n.d.
BZ
0.209±0.004 cd
0.585±0.055 bc
0.257±0.021 ab
14.5±0.2 def
1.63±0.03 ef
0.130±0.011 a
DB
0.194±0.002 d
0.463±0.021 bcde
0.295±0.034 a
13.8±0.1 f
2.34±0.09 cde
0.054±0.002 c
HP
0.227±0.029 bcd
0.580±0.031 bcd
0.231±0.012 ab
14.1±0.4 ef
2.09±0.10 def
0.091±0.001 b
JB
0.214±0.011 cd
0.648±0.037 b
0.241±0.004 ab
14.5±0.4 def
2.08±0.06 def
n.d.
JD
0.203±0.006 cd
0.384±0.014 e
0.219±0.003 b
14.8±0.3 bcdef
2.52±0.28 bcd
n.d.
JS
0.266±0.028 abc
0.394±0.001 de
0.235±0.014 ab
16.8±0.4 a
2.27±0.35 cdef
n.d.
LB
0.217±0.007 cd
0.411±0.048 cde
0.233±0.004 ab
14.6±0.3 cdef
2.05±0.03 def
0.125±0.006 a
0.251±0.014 abcd
0.530±0.040 bcde
0.250±0.007 ab
14.8±0.1 bcdef
3.11±0.09 ab
0.105±0.009 ab
NP
PT
ED
A
BJ
0.282±0.006 ab
0.400±0.010 cde
0.243±0.013 ab
16.2±0.2 abc
2.85±0.18 abc
0.082±0.012 b
PB
0.284±0.009 ab
0.382±0.006 e
0.249±0.012 ab
15.8±0.3 abcd
2.37±0.06 cde
0.023±0.005 d
PZ
0.204±0.008 cd
0.991±0.094 a
0.236±0.017 ab
14.2±0.2 def
2.11±0.09 def
n.d.
XZ
0.190±0.006 d
0.536±0.012 bcde
0.238±0.005 ab
14.5±0.1 cdef
1.59±0.04 f
n.d.
YL
0.309±0.004 a
0.471±0.031 bcde
0.234±0.002 ab
15.6±0.2 abcde
3.59±0.04 a
n.d.
YZ
0.207±0.001 cd
0.366±0.005 e
0.234±0.012 ab
14.6±0.1 cdef
2.26±0.06 cdef
n.d.
CC E
LZ
A
Al
M
Table 3-1 Heavy metals in 15 cultivars of jujube fruits, mg/100 g DW Ti Cr Ni
Data show the mean value ± standard error (n = 3) Different letters after the data in the same column mean significant differences (p < 0.05), n.d. means the data were not detected; limit of detection (LOD) of Pb is 0.0012 mg/100 g DW. 25
I N U SC R
0.5088±0.017 h
6.92±0.42 cde
BZ
0.833±0.011 b
12.5±0.5 a
DB
1.07±0.02 a
HP
0.517±0.026 gh
JB
0.766±0.014 bcd
JD
0.723±0.008 cde
JS
1.55±0.17 abcd
28.8±1.1 ab
62.3±2.1 b
0.605±0.005 bcde
1.61±0.02 abc
19.3±0.7 ghi
59.8±1.6 bc
7.23±0.11 bcde
0.480±0.039 efg
1.71±0.01 ab
30.2±0.4 a
70.0±0.9 a
6.70±0.09 cde
0.486±0.004 efg
1.34±0.02 bcde
19.7±0.3 gh
51.2±0.4 d
8.38±0.30 bcd
0.536±0.008 def
1.62±0.11 ab
18.0±0.6 hi
61.4±1.4 b
6.23±0.06 de
0.603±0.005 bcde
1.76±0.05 a
25.0±0.9 cde
64.7±1.1 ab
0.479±0.012 h
6.40±0.33 cde
0.438±0.013 fg
1.23±0.02 de
27.8±0.5 abc
61.5±1.3 b
LB
0.688±0.053 de
6.34±0.40 de
0.381±0.012 g
1.12±0.02 e
21.9±0.4 efg
53.4±1.1 cd
LZ
0.826±0.024 bc
7.39±0.63 bcde
0.659±0.007 bcd
1.61±0.10 abc
21.6±0.4 fg
62.5±0.3 b
NP
0.758±0.018 bcd
6.35±0.02 cde
0.674±0.048 bc
1.55±0.08 abcd
23.6±0.6 def
69.7±2.6 a
PB
0.712±0.014 de
6.55±0.31 cde
0.656±0.048 bcd
1.61±0.05 abc
26.2±0.1 bcd
58.6±0.8 bc
PZ
0.621±0.010 efg
9.42±1.11 b
0.472±0.005 efg
1.36±0.03 bcde
28.4±0.5 ab
63.5±1.4 ab
XZ
0.580±0.015 fgh
7.69±0.24 bcd
0.555±0.009 cdef
1.24±0.01 cde
21.4±0.5 fg
54.1±1.4 cd
YL
0.693±0.003 de
8.56±0.23 bc
0.812±0.030 a
1.37±0.08 bcde
16.2±0.7 i
58.9±0.5 bc
YZ
0.643±0.005 ef
5.27±0.28 e
0.601±0.034 bcde
1.62±0.06 ab
26.7±0.5 bcd
63.3±0.4 ab
ED
PT
CC E A
Mg
0.708±0.034 ab
A
BJ
Ca
M
Table 3-2 Contents of minerals in 15 cultivars of jujube fruits, mg/100 mg DW Mn Fe Cu Zn
Data show the mean value ± standard error, (n = 3) Different letters after the data in the same column mean significant differences (p < 0.05), n.d. means the data were not detected
26
I N U SC R
Table 4. Data of ellipses for different groups of jujube cultivars in PCA method a
b
θ
2.64
45.3°
2.86
134°
6.39
134°
2.02
44.6°
46.6
Group B
41.2
Group C
26.9
Group D
34.6
Group E
28.9
1.84
44.7°
Group F
25.0
0.523
134°
M
A
Group A
ED
a is the semi-major axis of ellipse; b is semi-minor axis of ellipse; θ is the angle between the semi-major axis of ellipse and the x-axis of the
A
CC E
PT
coordinate.
27
I N U SC R
Table 5. Percentages of principal components (PCs) for different components in jujube fruits reducing sugars
percentage of PC2, %
cumulative percentage, %
64.6
21.0
85.6
33.2
22.4
55.6
25.6
21.6
47.2
A
organic acids
prcentage of PC1, %
A
CC E
PT
ED
M
minerals
28
S0889-1575(18)30573-8 https://doi.org/10.1016/j.jfca.2018.07.008 YJFCA 3117
To appear in: Received date: Revised date: Accepted date:
12-2-2018 24-6-2018 17-7-2018
Please cite this article as: Wang L, Fu H, Wang W, Wang Y, Zheng F, Ni H, Chen F, Analysis of Reducing Sugars, Organic Acids and Minerals in 15 Cultivars of Jujube (Ziziphus jujuba Mill.) Fruits in China, Journal of Food Composition and Analysis (2018), https://doi.org/10.1016/j.jfca.2018.07.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Analysis of Reducing Sugars, Organic Acids and Minerals in 15 Cultivars of Jujube (Ziziphus jujuba Mill.) Fruits in China Lina Wang a,b, Haiyan Fu b,c, Weizheng Wang a, Yaqi Wang a, Fuping Zheng a,b, Hui Ni b,d, Feng Chen a,b *
a. Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing
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Technology and Business University, Beijing 100048, China
b. Department of Food, Nutrition and Packaging Sciences, Clemson University, Clemson, SC
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29634, USA；
c. School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan 430074, China
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d: College of Food and Biology Engineering, Jimei University, Xiamen, Fujian Province
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361021, China
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*Corresponding author:
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Dr. Feng Chen Professor
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Department of Food, Nutrition and Packaging Sciences Clemson University, Clemson, SC 29634, USA Tel: 864-656-5702
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Email: [email protected]
Highlights:
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Different cultivars of Chinese jujube were collected from Shanxi Province, China
Compositions of reducing sugars and organic acids were determined by HPLC-UV
Composition of minerals was determined by ICP-OES
Principal component analysis and hierarchical cluster analysis were used for classification 1
ABSTRACT
Ziziphus jujuba Mill. has been used as both an edible fruit and Chinese medicine for a long time. In this study, the chemical profiles in terms of reducing sugars, organic acids, and minerals of 15 cultivars of jujube were analyzed. The reducing sugars and organic acids were
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measured by HPLC-UV (high performance liquid chromatography with ultraviolet detection).
Minerals were detected by ICP-OES (inductively coupled plasma-optical emission
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spectrometry). Glucose (85.9‒1005 mg/100 g FW), malic acid (120‒509 mg/100 g FW), citric acid (29.4‒181 mg/100 g FW), iron (5.27‒12.5 mg/100 g DW), calcium (16.2‒30.2 mg/100 g DW) and magnesium (51.2‒70.0 mg/100 g DW) were found to be the major components in
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the fruits. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were
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used, in order to classify these cultivars based on the aforementioned chemical profiles.
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According to the PCA, classification based on the content of reducing sugars is more
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categorized into 6 groups.
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reasonable and reliable than other parameters. In this classification, 15 cultivars were
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Keywords: Jujube, Reducing Sugars, Organic Acids, Minerals, Classification, Quantification,
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HPLC-UV, ICP-OES, Food analysis, Food composition
1.
Introduction
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Ziziphus jujuba Mill. is a common fruit, which has been cultivated in Asia for more
than one thousand years. It was reported that there are more than 700 varieties and cultivars of the fruits in China (Chen et al., 2013), which are distributed in different regions, including Henan, Shanxi, Shandong, Shaanxi, Hebei, Gansu provinces, and Xinjiang autonomous region, in the Peoples’ Republic of China (see the supplemental Fig. S1). These jujube fruits 2
have been found to have quite different chemical profiles because of the influence of various environmental conditions, in terms of location, climate, soil, precipitation, etc.. Jujube has a long history of being used as a herbal medicine in ancient China, and is considered as a functional food, due to its rich amounts of nutrients, including sugars, fatty acids, amino acids, minerals, vitamins, polyphenols and other antioxidants (Li et al., 2007). In addition, the
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essential oil extracted from the seeds of jujube was reported to possess anti-inflammatory activity (Al-Reza et al., 2010); the polysaccharides from jujube have hepatoprotective activity
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(Wang et al., 2012) and immunomodulatory activity (Zhang et al., 2013; Zhao et al., 2008); betulinic acid from sour jujube fruits was reported to be able to inhibit breast cancer cells (Sun et al., 2013). In addition, jujube fruits were found to have antioxidant capacity (Xue et
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al., 2009).
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Primary metabolites, such as carbohydrates, proteins, lipids, and amino acids play
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important roles in growth and development of plants, animals and humans (Azmir et al.,
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2013). Sugars such as glucose, fructose and sucrose are the major sugars in fruits, such as
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papaya (Kelebek et al., 2015), berries, peach, apple, watermelon, and cherry, (Ma et al., 2014) etc.. They can be measured by HPLC with detectors like UV-Vis and fluorescence detector
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(FD) after sugar derivatization, for which 1-phenyl-3-methyl-5-pyrazolone (PMP) is a popular chemical reagent (Bai et al., 2015; Dai et al., 2010; Lv et al., 2009; Myron et al., 2015; Zhang
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et al., 2009). This method is used for its high recovery, lack of isomerization, high sensitivity and better accuracy for detection by UV or DAD detectors. In previous studies, fructose,
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glucose and sucrose were found to be the main sugars in jujube fruits; while rhamnose could not be found in most cultivars. In addition, it was found that the sugars in jujube fruits increased as their maturity increased (Guo et al., 2015). However, other reports on reducing sugars in jujube fruits are limited. Organic acids are very important for fruit quality in terms of taste acidity, color, 3
texture, and flavor (de Jesús Ornelas-Paz et al., 2013). In many fruits, such as melon, peach (Flores et al., 2012), pomegranate (Gundogdu and Yilmaz, 2012), strawberry (de Jesús Ornelas-Paz et al., 2013), malic and citric acids are the most common and major acids. It is well known that environmental factors can significantly affect the contents of organic acids in fruits (Arena et al., 2013; Sweetman et al., 2014). Gao et al. (2012b) analyzed organic acids in
could not be detected in the cultivars Zaowangzao and Junchangyihao.
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10 jujube cultivars, and found that malic acid was the major organic acid, while succinic acid
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Minerals often serve as important cofactors of enzymes and/or are involved in biological reactions. Inductively coupled plasma-optical emission spectrometry (ICP-OES) is an efficient instrument to determine mineral content. It can simultaneously analyze the
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micro-minerals (e. g., manganese, zinc, iron, copper, magnesium, selenium, iodine, chromium)
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and macro-minerals (e. g., chloride, sodium, potassium, calcium, phosphorus) in foods
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(Danbaba et al., 2015; Gülfen and Özdemir, 2016). Minerals are also important in commercial
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products such as dried jujube, jujube jelly, etc. (Kao and Chen, 2014). Since the
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overwhelming majority of minerals in plants are from the soil, their contents and relevant fruit quality can be affected by plantation locations. Therefore, minerals could be used for possible
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classification of jujubes.
Ziziphus jujuba Mill. has been widely planted in China. Its plantation area was
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increased from 2.95 million acres to 3.78 million acres from 2004 to 2011. Based on the data from the Chinese Jujube Market Competition and Development (Wang and Hu, 2016), the
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yield of jujube in China approached 7.4 million tons in 2014. Various jujube products, such as fresh jujube, dried jujube, jujube beverage, wine, and vinegar, as well as jujube extracts, have been consumed and/or used as food additives in the world market. Since jujube fruits have high economic value, accurate determination of the jujube composition and their authenticity is very important for customers and the industry. In this study, nutritional compounds of 15 4
cultivars of jujube that were collected from the same farm in Shanxi province of China were analyzed. Reducing sugar-PMP derivatives, organic acids, as well as lactic acid and acetic acid, were analyzed by HPLC/UV; 12 minerals including heavy metals were analyzed by ICP-OES. In general, the objectives of this research were to: (1) obtain the chemical profiles of
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the aforementioned jujube cultivars, and identify their compositional differences which can
help customers to choose the correct fruits for different uses; (2) use principal component
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analysis (PCA) and hierarchical cluster analysis (HCA), based on the aforementioned
nutritional chemical data, to classify the jujube cultivars so as to facilitate the potential
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development of authentic jujube products.
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2. Materials and methods
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2.1 Jujube sample collection
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All the jujube samples were collected from the same farm in Shanxi province, China
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in October, 2015. The specified site of the farm is shown in Supplemental Fig. S2. Intact fruits of similar shape and size were collected, without any visible blemishes. Samples were
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transported to the lab and frozen at ‒-80 °C. All the fruits were peeled to remove the seeds to just keep the pulp for analysis. The analyzed cultivars of Ziziphus jujuba Mill. included cv.
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Banzao (BZ), Dabailing (DB), Cang county Jinsixiaozao (JS), Huping (HP), Lingbao (LB), Yuanling (YL), Jidan (JD), Lizao (LZ), Baode Youzao (YZ), Bin county Jinzao (BJ), Junzao
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(JB), Pingshun Junzao (PB), Xiangzao (XZ), Pozao (PZ), and Neihuangbianhesuan (NP).
2.2 Chemicals Chemical standards, including oxalic acid, malic acid, citric acid, fumaric acid, succinic acid, lactic acid, tartaric acid, acetic acid, rhamnose, mannose, glucose, galactose, 5
xylose, and arabinose, were bought from J. T. Baker Chemical Co. (Fair Lawn, NJ); 1-Phenyl-3-methyl-5-pyrazolone (PMP) (99%) was purchased from Sigma-Aldrich (St Louis, MO). HPLC grade methanol and acetonitrile were bought from Thermo Fisher Scientific (Waltham, PA). 2.3 Analysis of reducing sugars
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One half gram of each individual cultivar sample was placed in a 50-mL test tube with
10 mL of water and shaken well. Then the tube was kept in an 80 °C water bath for 1 hour
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before the samples were centrifuged at 5000 rpm for 15 minutes at room temperature
(Johnson et al., 2013). The supernatant was collected and diluted to 25 mL. One milliliter of the diluted solution was dried by mild nitrogen gas purge. The dried sample was reacted with
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0.5 mol/L of the PMP-methanol reagent and 0.3 mol/L of NaOH in a 70 °C water bath for 60
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minutes. After the sample was cooled to room temperature, 0.3 mL of 0.3 mol/L HCl were
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added. Then, 1 mL of chloroform was added to extract the excess PMP (Lv et al., 2009). The
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aqueous phase was filtered through a 0.22-μm membrane prior to HPLC analysis.
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A Thermo U3000 (Thermo Fisher Scientific, Waltham, MA) series HPLC-UV was used to detect the sugars, for which a Thermo Hypersil Gold C18 column (4.6 × 250 mm, 5
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μm) was used to separate the different sugar-PMP derivatives under isocratic conditions. The mobile phase was composed of 0.1 mol/L phosphate solution: acetonitrile (82:18, v/v) at pH
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7.0. The flow rate was 1 mL/min; the column temperature was controlled at 25 °C; injection volume was 15 μL, and the detected wavelength was 245 nm.
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2.4 Analysis of organic acids This method was based on a previous study (Gao et al., 2012a) with some
modifications. An amount of 1 gram of jujube pulp sample was mixed with 30 mL of deionized water and extracted under ultrasonic treatment for 30 minutes at room temperature. The solution was centrifuged at 5000 rpm for 10 minutes under room temperature to collect 6
the supernatant. The residue was re-extracted three times using the same procedure and under the same conditions. After four extractions, the supernatants were combined, and then evaporated at 65 ºC. The supernatant was filtered through a 0.45-μm nylon filter (Macherey-Nagel Inc., Bethlehem, PA) before HPLC analysis. Concentrations were expressed as mg/100 g fresh weight (FW).
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Organic acids were also analyzed using Thermo U3000 series HPLC-UV with the aforementioned reverse phase Hypersil Gold column and detected at 210 nm. Mobile phase
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was 0.5% NH4H2PO4 (pH 2.6); the flow rate was 0.8 mL/min. The concentration (expressed
in mg per 100 g FW) of the different compounds was calculated based on the external standard method.
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2.5 Analysis of minerals
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This method was based on Hong et al. (2016) with some modifications. The pulps of
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jujube fruits were dried using a conventional oven at 65 °C. Then, 1 g of the dried sample was
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placed in a digestion tube, mixed with 10 mL HNO3 and 2 mL HClO4, which was kept at
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room temperature overnight. Then, the tube was moved into a digestion oven at 280 °C until the solution became clear. After that, distilled water was added to make a final volume of 50
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mL. The mineral contents in the jujube samples were measured by ICP-OES (Perkin Elmer Inc., Waltham, MA). The operating power of the instrument was 1.20 kW, the plasma flow
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rate was 15.0 L/min, the auxiliary flow rate was 1.50 L/min, and the nebulizer pressure was 200 kPa.
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2.6 Statistics
All the samples were analyzed in triplicate. Evaluation of the significant difference
level was by one-way analysis of variance (ANOVA) using JMP Pro 12.2.0 (SAS Institute Inc., Cary, NC). Tukey test was used to evaluate the significance level (p < 0.05). Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were conducted using 7
Matlab (MathWorks, Inc., Natick, MA).
3 Results and discussion 3.1 Analysis of reducing sugars Previous researchers have demonstrated that the environmental conditions are a major
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factor affecting sugar synthesis (Rosa et al., 2009; Ruan et al., 2010; Nguyen-Huu et al., 2015; Paul and Pellny, 2003).. In many previous studies, sucrose, fructose and glucose were
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reported as the major sugars in jujube fruits (Gao et al., 2012a; Gao et al., 2012b; Guo et al., 2015; Li et al., 2007), but information about other reducing sugars is limited. It was first
reported by Honda et al. (1989) that reducing sugars could react with the PMP reagent to form
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sugar-PMP derivatives, which can be easily detected by UV detectors due to their strong UV
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absorbance. Among the jujube cultivars, significantly different concentrations of their
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reducing sugars (see Table 1) were observed. For example, mannose was only detected in the
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cultivars of JB, LB, LZ, NP and XZ. Although there was no significant difference (p < 0.05)
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among the cultivars of JB, LB and XZ based on their mannose content, there were significant differences between the cultivars of LZ and NP and the aforementioned three cultivars (p <
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0.05). Xylose was only detected in cultivars JB, LB, XZ and YZ. The contents of xylose in the cultivars JB and YZ were not significantly different (p < 0.05); but there was a significant
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difference in the contents (p < 0.05) of LB and XZ.
The content of rhamnose was within a
range of 1.42 to 5.48 mg/100 g FW, which was lower than that in a previous report (Gao et al.,
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2012b). The content of glucose was measured in a wide range from 85.9 to 1005 mg/100 g FW, for which the cultivar YZ showed the lowest content while the cultivar PZ contained the highest amount. Arabinose was also measured in jujube; the content varied from 2.30 to 16.8 mg/100 g FW. 3.2 Analysis of organic acids 8
Organic acids contribute to the acidity of fruits and are important to the fruit texture and flavor. Malic, citric, and succinic acids have been found in jujube (Gao et al., 2012a; Gao et al., 2012b). In this study, oxalic, tartaric, malic, lactic, acetic, citric, fumaric and succinic acids were also determined (Table 2). Similar to the contents of reducing sugars, the contents of organic acids had a significant difference in different jujube cultivars (p < 0.05).
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Particularly, lactic and succinic acids were not detected in some cultivars, such as DB, HP and LB. The predominant organic acid was malic acid, of which the amount (120 to 509 mg/100 g
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FW) was the highest among the organic acids in all the tested cultivars. Ziziphus jujuba Mill.
cv. JB was determined to contain 279 mg of malic acid/100 g FW, which was similar to another cultivated JB (294 mg/100 g FW), which is grown in Yulin county in China (Gao et
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al., 2012b); Strawberry was reported to contain around 200 mg/100 g FW of malic acid (de
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Jesús Ornelas-Paz et al., 2013), which was similar to most cultivars of jujube fruits; grapes
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(1095 mg/100 g FW) and peaches (2183 mg/100 g FW) were reported to contain higher malic
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acid content than jujube, while lemons (228 mg/100 g FW) and oranges (131 mg/100 g FW)
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have similar contents (Flores et al., 2012). Ziziphus jujuba Mill. cv. JS contained the lowest amount of oxalic acid (12.1 mg/100 g FW), while the cultivar LB contained the highest
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amount (62.7 mg/100 g FW). The content of tartaric acid varied from 73.7 to 147 mg/100 g FW. Lactic acid could not be detected in the DB, LB, PZ and HP cultivars but showed
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significant difference (p < 0.05) from 8.41 to 31.5 mg/100 g FW in the other cultivars. Acetic acid in the LB and JD cultivars was below 3 mg/100 g FW, but its value was above 10 mg/100
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g in the other cultivars. Its highest content (111 mg/100 g FW) was found in the cultivar XZ. The content of citric acid (29.4 to 181 mg/100 g FW) was higher than other acids in most cultivars. This result is similar to that in a previous report (Gao et al., 2012b). Fumaric acid was detected in all cultivars. Its lowest content was only 0.548 mg/100 g FW in JB, while its highest content was 163 mg/100 g FW in PB. Succinic acid was not detected in some cultivars 9
(i.e., BZ, DB, LB, HP, BJ, JS). In the cultivar YL, its amount was only 2.27 mg/100 g FW, while it was 163 mg/100g FW in the cultivar NP. 3.3 Analysis of minerals Contents of minerals of 15 jujube cultivars are listed in Table 3. The data indicated that calcium, magnesium, and iron were the major minerals in these jujube fruits. According
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to Table 3-1, lead was only detected in Ziziphus jujuba Mill. cv. BZ, DB, HP, LB, LZ, NP, and
PB, of which the content was in a range from 0.023 to 0.130 mg/100g DW. The contents of
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nickel (0.219 to 0.295 mg/100g DW), aluminum (13.8 to 16.8 mg/100 g DW), boron (1.59 to
3.59 mg/100 g DW), titanium (0.194 to 0.309 mg/100 g DW), and chromium (0.366 to 0.991 mg/100 g DW) were not significantly different among most cultivars (p < 0.05). The detected
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ranged from 4.63 to 6.53 mg/100 g (San et al., 2009).
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content of boron in our test cultivars was less than that in the jujubes of Turkey, where values
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Iron is an essential mineral for humans. The iron content in these jujube cultivars was
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from 5.27 to 12.5 mg/100 g DW (see Table 3-2). Most cultivars in this study contained similar
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amounts of iron to those in five cultivars of jujube fruits grown in Jinan in China (Li et al., 2007), which included Yazao, Jianzao and Sanbianhong, as well as Junzao (JB) and
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Jinsixiaozao (JS) that were also analyzed in this study,. However, Ziziphus jujuba Mill. cv. BZ, PZ, YL contained higher content of iron (8.56 to 12.5 mg/100g DW) than those five cultivars
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of jujube (4.68 to 7.90 mg/100 g DW). In this study, the content of iron in the cultivars JS (6.40 mg/100g) and JB (8.38 mg/100g) was higher than that in the same cultivars (4.68 and
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7.90 mg/100 g DW) reported in the previous study (Li et al., 2007). The content of iron in all the cultivars in this study was higher than that (0.67 to 1.43 mg/100 g DW) in the Turkey cultivars (San et al., 2009). The average content of iron (131.9 μg/g) in jujube fruits that were planted in Xinjiang autonomous region of China (Zhu et al., 2014) was higher than the iron content of jujube planted in Shanxi province. This difference could be attributed to the quite 10
different climate and soil conditions in these two regions. Copper is a co-factor of many antioxidants (Gropper and Smith, 2012). In Table 3-2, the content of copper was determined in a range of 0.381 to 0.812 mg/100 g DW. This value was higher than the amount in Li’s report (0.19 to 0.42 mg/100 g), but similar to that in the jujubes planted in Bayikuleng region (4.62 to 8.28 μg/g) in Xinjiang autonomous region (Zhu
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et al., 2014).
Zinc is important for many enzymes, such as carbonic anhydrase, alkaline phosphatase,
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etc. (Gropper and Smith, 2012). Ziziphus jujuba Mill. cv. LB contained the lowest content of
zinc (1.12 mg/ 100 g DW), while Ziziphus jujuba Mill. cv. JD had the highest value (1.76 mg/100 g). The content of zinc in the cultivars in this study was higher than that in Li’s report
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(0.63 mg/100 g compared with 0.35 mg/100 g; (Li et al., 2007)) and San’s report
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(0.53mg/100g to 1.27 mg/100g) (San et al., 2009). However, our result was in agreement with
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the value of zinc in jujubes that were grown in the Xinjiang autonomous region (Zhu et al.,
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2014)
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Calcium is well known to be essential for growth of bones and teeth. Table 3-2 shows that the lowest content of calcium was 16.2 mg/100 g DW found in Ziziphus jujuba Mill. cv.
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YL, while the highest content was 30.2 mg/100 g DW in Ziziphus jujuba Mill. cv. DB. However, its content in all the cultivars was found to be lower than that in both Li’ report
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(45.6 to 118 mg/100g) (Li et al., 2007) and San’s report (79.33 to 121.33 mg/100g) (San et al., 2009).
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Based on the data shown in Table 3-2, magnesium was obviously a primary mineral
because its content was significantly higher than that of all the other minerals. Ziziphus jujuba Mill. cv. HP had the lowest content (51.2 mg/100 g DW), and Ziziphus jujuba Mill. cv. DB contained the highest content (70.0 mg/100 g DW). These values were higher than those of jujube (15.8 to 20.8 mg/100 g) found in Turkey (San et al., 2009). 11
The content of manganese of jujube in this study varied from 0.479 to 1.07 mg/100 g DW. All values were higher than those in the jujube fruits from Turkey (0.10 mg/100 g to 0.20 mg/100 g), and similar to the jujubes in the Xinjiang autonomous region in China . Overall, the contents of minerals in the different cultivars were significantly different (p < 0.05). However, regarding the same mineral, most of the cultivars did not have
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significant differences, which is attributed to the main factor that all of the cultivars were grown and collected from the same farm, so the environmental factors, such as climate and
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soil, were excluded from the major factors that can significantly affect the minerals’ absorbance. In this context, the aforementioned differences among the cultivars are considered to be caused, to a large degree, by the genotype of jujube, though it needs more
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investigations.
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3.4 Hierarchical cluster analysis and principal component analysis
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Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were
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used to categorize the cultivars into subgroups in an effort to explore the existing differences
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among the groups. The former statistical method indicates the similarity among the different cultivars, while the latter method indicates the significant differences among the cultivars. For
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the HCA method, cultivars in the same group are more similar than the cultivars in the other group. In this study, the classifications of HCA were based on the distance equal to 39. In
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contrast, all the ellipses of the constant distance of the PCA method were calculated with a 95% confidence interval. For example, Fig. 1 profiles the dendrogram of all the cultivars based on
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the contents of reducing sugars. According to the mean values of the components, the studied jujube cultivars were categorized into six groups. Ziziphus jujuba Mill. cv. BZ, PZ, DB, NP were in the same group; HP, JD, JS, BJ, PB were classified into another group; YZ and LB were clustered together into the third group, and Ziziphus jujuba Mill. cv. XZ and JB were clustered together into the fourth group. Moreover, the remaining two cultivars YL and LZ 12
were separated into two independent different groups. Fig. 2 shows the score plot of PCA for reducing sugars. After reducing the variable dimensions, the first two PCs (i.e., PC 1 and PC 2) could explain 85.6% of total data variability. As shown in Fig. 2, group A which included DB and PZ was in quadrant I of the coordinate; group D including YL, HP, JS, PB and JD, as well as group E (BZ) and group F (DB), were located in quadrant II; group C including YZ, XZ,
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JB and LB was in quadrant III; and group B (NP and LZ) was observed in quadrant IV. Table 4 lists the ellipse data obtained from the PCA method, and Fig. 2 profiles the corresponding
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ellipse plots for the different groups. If there is no intersection between two ellipses (groups), it means those two groups of clusters have significant differences. In this case, group A, B, C and D had significant differences between each other, and they were all different from group
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E and F. However, since the group E and F had an intersection between them, they could not
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be distinguished from each other. That means there was not a significant difference between
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groups E and F.
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Based on the results of PCA and HCA of the reducing sugars, organic acids, and
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minerals, different jujube cultivars were categorized into different clusters. However, compared with the PCA of organic acids and minerals, only the PCA of reducing sugars might
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be reliable, since it could explain more than 80% of the data variance (See Table 5). In other words, the classified subgroups from the PCA and HCA based on the concentrations of
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organic acids and minerals might not be accurate and convincing enough because of insufficient variability between the samples. Nevertheless, the classification according to the
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reducing sugars was more reliable in comparison to other components.
4. Conclusions In this study, reducing sugars, organic acids, and minerals of 15 cultivars of jujube fruits were analyzed. As a result, the contents of reducing sugars and organic acids were found 13
to be significantly different (p<0.05) among the cultivars. According to PCA, only the reducing sugars could be used as a reliable parameter to classify the cultivars because its first two principal components could explain more than 80% of data variance. In contrast, other jujube nutritional components like the organic acids and minerals were not convincingly reliable for the jujube classification by PCA. Nevertheless, the obtained data of the
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components in the jujube fruits have provided insights into the nutritional value of jujube. In addition, it is a useful attempt to classify different jujube cultivars by PCA and HCA based on
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the aforementioned data, in an effort to improve processing quality, and avoid adulteration of the final products. Finally, these systematical analyses of jujube fruits can increase the
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utilization of the jujube as a functional food.
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Declaration of interest: None. This research did not receive any specific grant from funding
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agencies in the public, commercial, or not-for-profit sectors.
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References
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Al-Reza, S.M., Yoon, J.I., Kim, H.J., Kim, J.S., Kang, S.C., (2010). Anti-inflammatory activity of seed essential oil from Zizyphus jujuba. Food and Chemical Toxicology 48(2), 639-643. Arena, M.E., Zuleta, A., Dyner, L., Constenla, D., Ceci, L., Curvetto, N., (2013). Berberis buxifolia fruit growth and ripening: evolution in carbohydrate and organic acid contents. Scientia Horticulturae 158, 52-58. Azmir, J., Zaidul, I., Rahman, M., Sharif, K., Mohamed, A., Sahena, F., Jahurul, M., Ghafoor, K., Norulaini, N., Omar, A., (2013). Techniques for extraction of bioactive compounds from plant materials: a review. Journal of Food Engineering 117(4), 426-436. Bai, W., Fang, X., Zhao, W., Huang, S., Zhang, H., Qian, M., (2015). Determination of oligosaccharides and monosaccharides in Hakka rice wine by precolumn derivation high-performance liquid chromatography. journal of food and drug analysis 23(4), 645-651. Chen, J., Li, Z., Maiwulanjiang, M., Zhang, W.L., Zhan, J.Y., Lam, C.T., Zhu, K.Y., Yao, P., Choi, R.C., Lau, D.T., (2013). Chemical and biological assessment of Ziziphus jujuba fruits from China: different geographical sources and developmental stages. J Agric Food Chem 61(30), 7315-7324. Dai, J., Wu, Y., Chen, S.W., Zhu, S., Yin, H.P., Wang, M., Tang, J., (2010). Sugar compositional determination of polysaccharides from Dunaliella salina by modified RP-HPLC method of precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone. Carbohydrate Polymers 82(3), 629-635. Danbaba, N., Nkama, I., Badau, M.H., (2015). Application of response surface methodology (RSM) and central composite design (CCD) to optimize minerals composition of rice-cowpea composite blends during extrusion cooking. International Journal of Food Science and Nutrition Engineering 5(1), 40-52. de Jesús Ornelas-Paz, J., Yahia, E.M., Ramírez-Bustamante, N., Pérez-Martínez, J.D., del Pilar Escalante-Minakata, M., Ibarra-Junquera, V., Acosta-Muñiz, C., Guerrero-Prieto, V., Ochoa-Reyes, E., (2013). Physical attributes and chemical composition of organic strawberry fruit (Fragaria x ananassa Duch, Cv. Albion) at six stages of ripening. Food Chem 138(1), 372-381. Flores, P., Hellín, P., Fenoll, J., (2012). Determination of organic acids in fruits and vegetables by liquid chromatography with tandem-mass spectrometry. Food Chem 132(2), 1049-1054. Gülfen, M., Özdemir, A., (2016). Analysis of dietary minerals in selected seeds and nuts by using ICP-OES and assessment based on the recommended daily intakes. Nutrition & Food Science 46(2), 282-292. Gao, Q.-H., Wu, C.S., Wang, M., Xu, B.N., Du, L.J., (2012a). Effect of drying of jujubes (Ziziphus jujuba Mill.) on the contents of sugars, organic acids, α-tocopherol, β-carotene, and phenolic compounds. J Agric Food Chem 60(38), 9642-9648. Gao, Q.H., Wu, C.S., Yu, J.G., Wang, M., Ma, Y.J., Li, C.L., (2012b). Textural characteristic, antioxidant activity, sugar, organic acid, and phenolic profiles of 10 promising jujube (Ziziphus jujuba Mill.) Selections. J Food Sci 77(11), C1218-C1225. Gropper, S., Smith, J., (2012). Advanced nutrition and human metabolism. Cengage Learning. Gundogdu, M., Yilmaz, H., (2012). Organic acid, phenolic profile and antioxidant capacities of pomegranate (Punica granatum L.) cultivars and selected genotypes. Scientia Horticulturae 143, 38-42. Guo, S., Duan, J.A., Qian, D., Tang, Y., Wu, D., Su, S., Wang, H., Zhao, Y., (2015). Content 15
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variations of triterpenic acid, nucleoside, nucleobase, and sugar in jujube (Ziziphus jujuba) fruit during ripening. Food Chem 167, 468-474. Honda, S., Akao, E., Suzuki, S., Okuda, M., Kakehi, K., Nakamura, J., (1989). High-performance liquid chromatography of reducing carbohydrates as strongly ultraviolet-absorbing and electrochemically sensitive 1-phenyl-3-methyl5-pyrazolone derivatives. Analytical biochemistry 180(2), 351-357. Hong, J., Wang, L., Sun, Y., Zhao, L., Niu, G., Tan, W., Rico, C.M., Peralta-Videa J.R., Gardea-Torresdey, J. L. (2016). Foliar applied nanoscale and microscale CeO 2 and CuO alter cucumber (Cucumis sativus) fruit quality. Science of The Total Environment, 563, 904-911. Johnson, C.R., Combs Jr, G.F., Thavarajah, P., (2013). Lentil (Lens culinaris L.): A prebiotic-rich whole food legume. Food Research International 51(1), 107-113. Kao, T.H., Chen, B.H., (2014). Functional components in Zizyphus with emphasis on polysaccharides, Polysaccharides. Springer, pp. 1-28. Kelebek, H., Selli, S., Gubbuk, H., Gunes, E., (2015). Comparative evaluation of volatiles, phenolics, sugars, organic acids and antioxidant properties of Sel-42 and Tainung papaya varieties. Food chemistry 173, 912-919. Li, J.W., Fan, L.P., Ding, S.D., Ding, X.L., (2007). Nutritional composition of five cultivars of chinese jujube. Food Chem 103(2), 454-460. Lv, Y., Yang, X., Zhao, Y., Ruan, Y., Yang, Y., Wang, Z., (2009). Separation and quantification of component monosaccharides of the tea polysaccharides from Gynostemma pentaphyllum by HPLC with indirect UV detection. Food Chem 112(3), 742-746. Ma, C., Sun, Z., Chen, C., Zhang, L., Zhu, S., (2014). Simultaneous separation and determination of fructose, sorbitol, glucose and sucrose in fruits by HPLC–ELSD. Food Chemistry 145, 784-788. Myron, P., Siddiquee, S., Azad, S., Yong, Y., (2015). Tributylamine Facilitated Separations of Fucosylated Chondroitin Sulfate (Fucs) by High Performance Liquid Chromatography (HPLC) into its Component Using 1-Phenyl-3-Methyl-5-Pyrazolone (PMP) Derivatization. Journal of Chromatography & Separation Techniques 2015. Nguyen-Huu, T.D., Gupta, C., Ma, B., Ott, W., Josić, K., Bennett, M.R., (2015). Timing and variability of galactose metabolic gene activation depend on the rate of environmental change. PLoS computational biology 11(7), e1004399. Paul, M.J., Pellny, T.K., (2003). Carbon metabolite feedback regulation of leaf photosynthesis and development. J Exp Bot 54(382), 539-547. Rosa, M., Prado, C., Podazza, G., Interdonato, R., González, J.A., Hilal, M., Prado, F.E., (2009). Soluble sugars: Metabolism, sensing and abiotic stress: A complex network in the life of plants. Plant signaling & behavior 4(5), 388-393. Ruan, Y.-L., Jin, Y., Yang, Y.-J., Li, G.-J., Boyer, J.S., (2010). Sugar input, metabolism, and signaling mediated by invertase: roles in development, yield potential, and response to drought and heat. Molecular Plant 3(6), 942-955. San, B., Yildirim, A.N., Polat, M., Yildirim, F., (2009). Mineral composition of leaves and fruits of some promising Jujube (Zizyphus jujuba miller) genotypes. Asian J Chem 21, 2898-2902. Sun, Y.F., Song, C.K., Viernstein, H., Unger, F., Liang, Z.S., (2013). Apoptosis of human breast cancer cells induced by microencapsulated betulinic acid from sour jujube fruits through the mitochondria transduction pathway. Food Chem 138(2–3), 1998-2007. Sweetman, C., Sadras, V., Hancock, R., Soole, K., Ford, C., (2014). Metabolic effects of elevated temperature on organic acid degradation in ripening Vitis vinifera fruit. J Exp Bot 65(20), 5975-5988. Wang, D., Zhao, Y., Jiao, Y., Yu, L., Yang, S., Yang, X., (2012). Antioxidative and 16
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hepatoprotective effects of the polysaccharides from Zizyphus jujube cv. Shaanbeitanzao. Carbohydrate Polymers 88(4), 1453-1459. Wang, L., Hu, R., (2016). Technical and cost efficiency of jujube growers in Henan Province, China. CUSTOS E AGRONEGOCIO ON LINE 12(2), 279-297. Xue, Z., Feng, W., Cao, J., Cao, D., Jiang, W., (2009). Antioxidant activity and total phenolic contents in peel and pulp of Chinese jujube (Ziziphus jujuba Mill) fruits. Journal of Food Biochemistry 33(5), 613-629. Zhang, J., Chen, J., Wang, D., Hu, Y., Zhang, C., Qin, T., Liu, C., Sheng, X., Nguyen, T.L., (2013). Immune-enhancing activity comparison of sulfated ophiopogonpolysaccharide and sulfated jujube polysaccharide. International Journal of Biological Macromolecules 52, 212-217. Zhang, J., Zhang, Q., Wang, J., Shi, X., Zhang, Z., (2009). Analysis of the monosaccharide composition of fucoidan by precolumn derivation HPLC. Chinese Journal of Oceanology and Limnology 27, 578-582. Zhao, Z., Liu, M., Tu, P., (2008). Characterization of water soluble polysaccharides from organs of Chinese Jujube (Ziziphus jujuba Mill. cv. Dongzao). European Food Research and Technology 226(5), 985-989. Zhu, F., Yang, S., Fan, W., Wang, A., Hao, H., Yao, S., (2014). Heavy metals in jujubes and their potential health risks to the adult consumers in Xinjiang province, China. Environmental monitoring and assessment 186(10), 6039-6046.
17
Figure Captions Figure 1. Dendrogram of 15 cultivars of jujube fruits based on reducing sugar content. Cultivar lines with the same color are in the same cluster.
Figure 2. Classification of 15 cultivars of jujube fruits based on their reducing sugar
A
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PT
ED
M
A
N
U
SC R
IP T
content. Groups A, B, C, D, E and F are the classified clusters of jujube fruits.
18
Figure 1
19
A ED
PT
CC E A
M
N U SC R
I
I N U SC R I
PT
ED
M
A
II
A
CC E
III
IV
Figure 2
20
I N U SC R
Table 1. Contents of reducing sugars in 15 cultivars of jujube fruits, mg/100 g FW name of Ziziphus jujuba Mill. cultivars
abbrevi
rhamnose
ation
Bin county Jinzao
glucose
galactose
xylose
arabinose
n.d.
232±7 fg
1.32±0.05
n.d.
3.28±0.0
e
BJ
1.68±0.13
A
efg Banzao
mannos
BZ
2.06±0.11
n.d.
304±8 e
PT
Junzao
ED
Hupingzao
DB
CC E
Jidanzao
CangcountyJinsixiao
Lingbaozao
4.88±0.17 b
n.d.
753±17 b
n.d.
de 2.08±0.04
JD
0.603±0 .049 c
1.97±0.13
n.d.
defg JS
1.63±0.11
n.d.
1.58±0.10 fg
0.422±0
5.48±0.08 a
8g
n.d.
4.29±0.14 c
2.35±0.07 d
36.3±1.6 6a
3.50±0.10
642±13 c
n.d.
360±5 d 21
16.8±0.2 9a
2.76±0.06
n.d.
d n.d.
2.84±0.0 5 gh
c 1.06±0.
4.19±0.1 0f
1.02±0.04
740±10 b
2.86±0.0 4 gh
gh 1.32±0.
3.29±0.0
n.d.
1.29±0.03
196±5 gh
05 b PB
9c
efg
n PingshunJunzao
26.7±1.2
1.61±0.03
173±6 h
3.16±0.0 6g
e
efg
NP
n.d.
1.14±0.06
201±7gh
6.94±0.1 5c
fgh
11 a Neihuangbianhesua
n.d.
0.993±0.0
286±7 e
5.96±0.1 6d
72 gh
def
LZ
n.d.
4.08±0.15
268±4 ef
.09 c Lizao
2.59±0.08 d
2.20±0.08
JB
LB
7g
b
HP
zao
A
M
def
Dabailing
efg
8.34±0.2 3b
1.54±0.05
n.d.
4.96±0.1
I PZ
Xiangzao
N U SC R
Pozao
4.29±0.15 c
XZ
2.06±0.10
YL
1005±20 a
0.467±0
1.95±0.11
n.d.
2e 5.05±0.16
n.d.
a
205±5 gh
.06 c
A
def Yuanlingzao
n.d.
ef
5d 1.19±0.07
fg
187±2 gh
32.5±1.2 0b
n.d.
n.d.
M
1.42±0.09 g
2.98±0.0 8 gh
n.d.
85.9±3 i
0.778±0.0 24 h
ED
YZ
3.21±0.0 8g
defg
BaodeYouzao
6.10±0.1
28.3±1.0 1c
2.30±0.0 6h
Data show the mean value ± standard error (n = 3)
PT
Different letters after the data in the same column mean significant differences (p < 0.05) n.d. means the data were not detected， the limit ofdetection (LOD) of mannose is 0.0003 mg/100 g FW; LOD of galactose is 0.025 mg/100 g
A
CC E
FW; LOD of xylose is 0.530 mg/100 g FW
22
I N U SC R
Table 2. Contents of organic acids in 15 cultivars of jujube fruits, mg/100 g FW oxalic acid
tartaric
malic acid
lactic acid
acetic acid
citric acid
fumaric acid
succinic acid
222±1 ef
21.4±0.6d
11.0±0.2 i
121±1 d
121.±1 c
n.d.
258±3 d
15.0±0.4 e
36.8±1.1 e
67.8±0.5 g
1.48±0.03 h
n.d.
acid 19.3±0.7 h
25.6±0.6 h
B
21.0±0.2 h
42.1±0.9e
D
50.3±0.6 c
36.3±0.4 f
177±3 g
n.d.
54.7±0.8 c
100±1 e
1.40±0.02 h
n.d.
H
50.3±1.8 c
36.8±1.6 f
254±3 d
n.d.
15.2±0.3 hi
181±3 a
1.01±0.02 h
n.d.
J
30.4±0.7 ef
41.6±0.5 e
279±3 c
25.0±0.4 b
180±1 a
160±2 b
0.548±0.007 h
80.7±1.0 c
A
B
P
J
30.3±0.4 ef
38.8±0.4 ef
231±2 e
11.8±0.1 f
2.93±0.03 j
51.5±0.8 h
52.3±0.7 f
35.8±0.4 f
J
12.1±0.8 i
41.3±1.1 e
233±3 e
14.7±0.4 e
26.0±0.8 g
88.2±1.1 f
88.2±1.1 d
n.d.
A
CC E
B
PT
B
ED
Z
M
J
L
62.7±1.2 a
56.1±0.7 c
120±2 i
n.d.
2.74±0.01 j
69.1±1.1 g
2.30±0.05 h
n.d.
L
26.9±0.6 fg
17.4±0.5 i
188±2 g
15.5±0.4 e
40.3±0.6 e
42.9±1.1 i
0.853±0.011 h
95.3±1.0 b
N
36.6±1.0 d
60.7±1.1 b
509±4 a
8.41±0.16
31.5±0.5 f
129±1 cd
129±1 b
163±1 a
D
S
B
Z
P
g 23
I 25.4±0.3 g
73.7±1.4 a
219±3 ef
B
N U SC R
P
23.9±0.34b
29.2±0.4 fg
161±2 b
163±1 a
57.7±0.3 d
n.d.
16.8±0.2 h
131±1 c
1.28±0.02 h
7.06±0.15 g
22.5±0.5 cd
111±1 b
29.4±0.6 j
1.30±0.04 h
43.7±0.5 e
c
P
543.7±8.2 b
14.7±0.5 i
162±2 h
X
29.0±0.5 fg
31.8±0.9 g
157±2 h
Y
32.9±0.9 e
46.8±1.0 d
207±3 f
31.5±0.6 a
16.1±0.5 h
34.2±0.4 j
34.2±0.4 g
2.27±0.03 h
Y
32.6±0.5 e
48.5±0.9 d
464±5 b
8.72±0.16
49.3±1.0 d
63.4±1.3 g
64.2±1.3 e
57.9±0.9 d
A
Z
Z
ED
L
M
Z
g
Data show the mean value ± standard error (n = 3)
PT
Different letters after the data in the same column mean significant differences (p < 0.05)
A
CC E
n.d. means the data were not detected; LOD of succinic acid is 0.750 mg/100 g FW; LOD of lactic acid is 0.500 mg/100 g FW
24
I N U SC R
B
Pb
0.245±0.001 abcd
0.385±0.007 e
0.245±0.008 ab
16.5±0.8 ab
2.59±0.14 bcd
n.d.
BZ
0.209±0.004 cd
0.585±0.055 bc
0.257±0.021 ab
14.5±0.2 def
1.63±0.03 ef
0.130±0.011 a
DB
0.194±0.002 d
0.463±0.021 bcde
0.295±0.034 a
13.8±0.1 f
2.34±0.09 cde
0.054±0.002 c
HP
0.227±0.029 bcd
0.580±0.031 bcd
0.231±0.012 ab
14.1±0.4 ef
2.09±0.10 def
0.091±0.001 b
JB
0.214±0.011 cd
0.648±0.037 b
0.241±0.004 ab
14.5±0.4 def
2.08±0.06 def
n.d.
JD
0.203±0.006 cd
0.384±0.014 e
0.219±0.003 b
14.8±0.3 bcdef
2.52±0.28 bcd
n.d.
JS
0.266±0.028 abc
0.394±0.001 de
0.235±0.014 ab
16.8±0.4 a
2.27±0.35 cdef
n.d.
LB
0.217±0.007 cd
0.411±0.048 cde
0.233±0.004 ab
14.6±0.3 cdef
2.05±0.03 def
0.125±0.006 a
0.251±0.014 abcd
0.530±0.040 bcde
0.250±0.007 ab
14.8±0.1 bcdef
3.11±0.09 ab
0.105±0.009 ab
NP
PT
ED
A
BJ
0.282±0.006 ab
0.400±0.010 cde
0.243±0.013 ab
16.2±0.2 abc
2.85±0.18 abc
0.082±0.012 b
PB
0.284±0.009 ab
0.382±0.006 e
0.249±0.012 ab
15.8±0.3 abcd
2.37±0.06 cde
0.023±0.005 d
PZ
0.204±0.008 cd
0.991±0.094 a
0.236±0.017 ab
14.2±0.2 def
2.11±0.09 def
n.d.
XZ
0.190±0.006 d
0.536±0.012 bcde
0.238±0.005 ab
14.5±0.1 cdef
1.59±0.04 f
n.d.
YL
0.309±0.004 a
0.471±0.031 bcde
0.234±0.002 ab
15.6±0.2 abcde
3.59±0.04 a
n.d.
YZ
0.207±0.001 cd
0.366±0.005 e
0.234±0.012 ab
14.6±0.1 cdef
2.26±0.06 cdef
n.d.
CC E
LZ
A
Al
M
Table 3-1 Heavy metals in 15 cultivars of jujube fruits, mg/100 g DW Ti Cr Ni
Data show the mean value ± standard error (n = 3) Different letters after the data in the same column mean significant differences (p < 0.05), n.d. means the data were not detected; limit of detection (LOD) of Pb is 0.0012 mg/100 g DW. 25
I N U SC R
0.5088±0.017 h
6.92±0.42 cde
BZ
0.833±0.011 b
12.5±0.5 a
DB
1.07±0.02 a
HP
0.517±0.026 gh
JB
0.766±0.014 bcd
JD
0.723±0.008 cde
JS
1.55±0.17 abcd
28.8±1.1 ab
62.3±2.1 b
0.605±0.005 bcde
1.61±0.02 abc
19.3±0.7 ghi
59.8±1.6 bc
7.23±0.11 bcde
0.480±0.039 efg
1.71±0.01 ab
30.2±0.4 a
70.0±0.9 a
6.70±0.09 cde
0.486±0.004 efg
1.34±0.02 bcde
19.7±0.3 gh
51.2±0.4 d
8.38±0.30 bcd
0.536±0.008 def
1.62±0.11 ab
18.0±0.6 hi
61.4±1.4 b
6.23±0.06 de
0.603±0.005 bcde
1.76±0.05 a
25.0±0.9 cde
64.7±1.1 ab
0.479±0.012 h
6.40±0.33 cde
0.438±0.013 fg
1.23±0.02 de
27.8±0.5 abc
61.5±1.3 b
LB
0.688±0.053 de
6.34±0.40 de
0.381±0.012 g
1.12±0.02 e
21.9±0.4 efg
53.4±1.1 cd
LZ
0.826±0.024 bc
7.39±0.63 bcde
0.659±0.007 bcd
1.61±0.10 abc
21.6±0.4 fg
62.5±0.3 b
NP
0.758±0.018 bcd
6.35±0.02 cde
0.674±0.048 bc
1.55±0.08 abcd
23.6±0.6 def
69.7±2.6 a
PB
0.712±0.014 de
6.55±0.31 cde
0.656±0.048 bcd
1.61±0.05 abc
26.2±0.1 bcd
58.6±0.8 bc
PZ
0.621±0.010 efg
9.42±1.11 b
0.472±0.005 efg
1.36±0.03 bcde
28.4±0.5 ab
63.5±1.4 ab
XZ
0.580±0.015 fgh
7.69±0.24 bcd
0.555±0.009 cdef
1.24±0.01 cde
21.4±0.5 fg
54.1±1.4 cd
YL
0.693±0.003 de
8.56±0.23 bc
0.812±0.030 a
1.37±0.08 bcde
16.2±0.7 i
58.9±0.5 bc
YZ
0.643±0.005 ef
5.27±0.28 e
0.601±0.034 bcde
1.62±0.06 ab
26.7±0.5 bcd
63.3±0.4 ab
ED
PT
CC E A
Mg
0.708±0.034 ab
A
BJ
Ca
M
Table 3-2 Contents of minerals in 15 cultivars of jujube fruits, mg/100 mg DW Mn Fe Cu Zn
Data show the mean value ± standard error, (n = 3) Different letters after the data in the same column mean significant differences (p < 0.05), n.d. means the data were not detected
26
I N U SC R
Table 4. Data of ellipses for different groups of jujube cultivars in PCA method a
b
θ
2.64
45.3°
2.86
134°
6.39
134°
2.02
44.6°
46.6
Group B
41.2
Group C
26.9
Group D
34.6
Group E
28.9
1.84
44.7°
Group F
25.0
0.523
134°
M
A
Group A
ED
a is the semi-major axis of ellipse; b is semi-minor axis of ellipse; θ is the angle between the semi-major axis of ellipse and the x-axis of the
A
CC E
PT
coordinate.
27
I N U SC R
Table 5. Percentages of principal components (PCs) for different components in jujube fruits reducing sugars
percentage of PC2, %
cumulative percentage, %
64.6
21.0
85.6
33.2
22.4
55.6
25.6
21.6
47.2
A
organic acids
prcentage of PC1, %
A
CC E
PT
ED
M
minerals
28