Accepted Manuscript Changes in Physicochemical Characteristics and Free Amino Acids of Hawthorn (Crataegus pinnatifida) Fruits during Maturation Wei-Qin Li, Qing-Ping Hu, Jian-Guo Xu PII: DOI: Reference:
S0308-8146(14)01863-9 http://dx.doi.org/10.1016/j.foodchem.2014.11.125 FOCH 16810
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22 September 2014 18 November 2014 20 November 2014
Please cite this article as: Li, W-Q., Hu, Q-P., Xu, J-G., Changes in Physicochemical Characteristics and Free Amino Acids of Hawthorn (Crataegus pinnatifida) Fruits during Maturation, Food Chemistry (2014), doi: http://dx.doi.org/ 10.1016/j.foodchem.2014.11.125
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Running Title: Changes in Physicochemical Characteristics of Hawthorn
Changes in Physicochemical Characteristics and Free Amino Acids of Hawthorn (Crataegus pinnatifida) Fruits during Maturation Wei-Qin Li a, Qing-Ping Hu b, and Jian-Guo Xu a*
College of Food Sciences, Shanxi Normal University, Linfen 041004, China
College of Life Sciences, Shanxi Normal University, Linfen 041004, China
Corresponding author at: College of Food Sciences, Shanxi Normal University, Linfen 041004, China. Tel.: +86 357 2051714; Fax: +86 357 2051000. E-mail address: [email protected]
(J.-G. Xu). 1
Abstract In this study, changes in physicochemical characteristics associated with fruit quality and free amino acids were investigated during maturation of hawthorn fruits. Significant differences in these parameters were found during maturation. The color turned progressively from mature green to semi-red, to reach bright red; the shape changed gradually from oval to round or approached round; the size, weight, and edible part (flesh/core ratio) of hawthorns increased while the density of intact fruits did not change. The content of moisture, total soluble sugars, soluble pectin, reduced ascorbic acid, total ascorbic acid, fructose, and sucrose increased while crude protein content decreased significantly. The levels of starch, sucrose, titratable acidity, protopectin, pectin, total free amino acids, and total essential amino acids initially increased and then decreased gradually during maturation. The outcomes of this study provide additional and useful information for fresh consumption and processing as well as utilization of dropped unripe hawthorn fruits. Keywords: physicochemical characteristics; free amino acids; hawthorn; maturation
1. Introduction Hawthorn is one of the most widely consumed horticultural products, either in fresh or processed form. It is also an important component of many processed food products because of its excellent flavour, attractive colour and high content of many macro- and micro-nutrients (Cao, Feng, & Qin, 1995; Özcan, Hacıseferoğulları, Marakoğlu, & Arslan, 2005). In China, the hawthorn species is widely cultivated for its edible fruits (Cui et al., 2006), and have recently attracted increasing attention in the field of nutraceuticals and medicine because their leaves, flowers, and both green (unripe) and red (ripe) berries are widely reported health benefits besides rich nutrient contents (Wang et al., 2011; Kirakosyan et al., 2003), e.g., the reduction of the risk of cardiovascular diseases (Pittler, Schmidt, & Ernst, 2003; Chang, Dao, & Shao, 2005) and offering antioxidant, anti-inflammatory, vasorelaxing, antityrosinase and hypolipidemic effects (Kim, Kang, Kim, & Kim, 2000; Bahorun et al., 2003; Quettier-Deleu et al., 2003; Kao et al., 2005; Chai et al., 2014). However, some pre-harvest and postharvest factors affect the physicochemical properties, nutritional value and active components in fruit and vegetables (Kirakosyan et al., 2003; Lee, & Kader, 2000), and some studies showed that the nutritional compositions, physical and biochemical properties of fruits and vegetable were most affected by maturity and ripening stages (Menz, & Vriesekoop, 2010; Opara, Al-Ani, & Al-Rahbi, 2012; Zheng, Kim, & Chung, 2012; Wang et al., 2012), and hawthorns were no exception (Liu, Kallio, & Yang, 2011). Besides, in the orchards, lots of unripe fruits such as thinning or drop fruits (from natural, drought, pests or physiological diseases) which may have great value 3
of utilization and development are discarded during the ripening of fruits every year, which no doubt cause enormous waste of resources. The reason for this lies in lacking the effective evaluation on physicochemical properties, nutritional value of unripe fruits. As we all known, biological substances can be both synthesized and degraded during the mature process of plant, so we expected that the components from unripe hawthorns may heavily depend upon their growth stage, even the content of some components in unripe hawthorns were higher than those in ripe hawthorns. Therefore, understanding the physicochemical and nutritional properties of hawthorn fruits at different stages of maturity is important in utilization of wastes, optimization of bioprocesses in food manufacture and nutrition planning, and selection of the maturity stage of the hawthorn fruit can provide optimum benefits as a functional food. Liu, Kallio and Yang (2011) studied changes of phenolic compounds in hawthorn (Crataegus grayana) fruits during ripening. However, few works have been conducted on the changes in physicochemical characteristics of hawthorn (Crataegus pinnatifida) fruits during maturation. The objective of this study was to investigate the changes in physicochemical properties, and free ammo acids during the maturation of Dajinxing hawthorn cultivars, a variety typically cultivated for the production of table hawthorns. We aim to increase the understanding of maturation process, which may be useful for optimal harvest timing, and the processing and utilization of hawthorns, especially unripe hawthorns. 2. Materials and methods 2.1. Hawthorn materials
Hawthorn cultivar (Crataegus pinnatifida) Dajinxing, is one of the most popular cultivar with wide adaptability across different hawthorn-growing regions of China and was used in the study. Hawthorn fruits were picked by hand in the production base of hawthorn (Linfen, Shanxi, China) from the 88th to 148th day after full bloom day (namely, from August 11 to October 10, 2013) in seven stages (M1−M7) of maturity, at roughly 10-day intervals. Each sample was picked from three trees at five randomly selected collection points from different sides of each tree. Immediately after harvesting, the partial samples were used for the analyses of physical properties, and the remaining samples were frozen and stored at -40 oC until chemical analyses. 2.2. Reagents Methanol (HPLC grade), acetone (HPLC grade) and acetonitrile (HPLC grade) were from Merck (Germany). D-Fructose, D-Glucose, Sucrose, and L-ascorbic acid were purchased from Sigma Chemical Co. Sigma (USA). All other chemicals and reagent used in the experiments were of analytical grade. 2.3. Physical analyses Physical characteristics of hawthorn fruits such as weight, size, index of shape, external color, and so on were measured in 10 fruits for each maturation stage. Ten fruits were taken from each group, and their linear dimensions-diameter and length were measured. Linear dimensions were measured by a micrometer to an accuracy of 0.01mm. Fruit color was measured through the CIE L*a*b* system using a Minolta chroma-meter CR-330 (Minolta, Ramsey, NJ). During measurement, CIE L*, a* and b* values were
obtained, representing lightness (L*), redness (a*) and greenness (b*). The results reported are the average of at least 10 replications. 2.4. Chemical analyses Moisture was measured by oven drying at 105 °C for 24 h. The crude protein content was determined by AACC approved method 46-10 (N×6.25), and starch was determined according to AACC approved method 76-13. Total soluble sugar (TSS) level was determined by using the phenol-sulfuric acid method, and a glucose standard curve was used to calculate the sugar level. The titratable acidity (TA) of the fruit, determined by titration with 0.1 N NaOH, was used as an indirect measurement of the citric acid concentration. The content of soluble pectin (SP), protopectin (PP) and pectin was determined by carbazole colorimetry method, and galacturonic acid was used for calibration of standard curve. Reduced ascorbic acid (RAA) was measured by titration with 2,6-dichloroindophenol in acidic solution according to AOAC method (2000) while total
spectrophotometric method as reported by Al-Ani, Opara, Al-Bahri and Al-Rahbi (2007). Different concentrations of working standard ascorbic acid calibrators were used to plot a standard curve based on spectrophotometric readings at 520 nm. All measurements were carried out in triplicate. 2.5. HPLC analysis of individual sugars A high-performance liquid chromatographic technique (HPLC) was developed to identify and quantify the sugars. The analysis was carried out by a Schimadzu apparatus equipped with a (LC-10 AT) pump and a (model RID-10Avp) detector. The column was 6
(100 × 7.8 mm) fast carbohydrate, Biorad, and the temperature was maintained at 80 °C. The flow rate was 0.3 mL/min. The mobile phase used was water. A standard mixture solution of sugars (glucose, fructose, and sucrose) was analyzed. Sample concentrations were calculated, based on peak areas compared to those of each of the external standards. 2.6. Analysis of free amino acids Free amino acids were extracted according to the method reported previously by Lin, Peng, Yang and Peng (2008) with some modifications. The samples were blended with 20 mL of 75% ethanol and shaken with a laboratory rotary shaker (JB50–D; Shanghai Shengke Instruments, Shanghai, China) at 250 rpm for 30 min at 50 oC, and then the homogenates were centrifuged at 10 000 g for 15 min at 4 oC in a centrifuge (Eppendorf 5417R, Germany). After centrifugation, the ethanol supernatants were removed and extraction was repeated three times at the same conditions. Then the supernatants were pooled, vacuum–evaporated to dryness at 40 oC, and reconstituted with 0.2 M sodium citrate loading buffer solution (pH 2.2) to a final volume of 10 mL. The concentration of free amino acids was determined following the method described by Xu, Hu, Duan and Tian (2010) with some modifications. Briefly, the free amino acid extracts were filtered through a 0.45 µm of nylon syringe filter (Filtrex Technology, Singapore) prior to analysis and analyzed by a Biochrom 30 series Amino Acid
Na–cation–exchange column (8 µm, 4.6mm × 200 mm). The injection volume was 20 µL, the duration of a single run was 50 min. Amino acids were postcolumn derivatized with ninhydrin reagent and detected by absorbance at 570 nm. The amino acids extracts and 7
amino acid standard solution were analyzed under the same conditions, and all of the above experiments were replicated three times. Identification of amino acids was performed by comparisons to the retention time and UV spectra of authentic standards from Sigma. To determine the tryptophan, sample was hydrolyzed with 5 M NaOH including 5% SnCl2 for 20 h at 110±0.5 °C. 2.7. Statistical analysis All experiments were conducted three times independently and the data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and Duncan’s multiple range tests were carried out to determine significant differences (p < 0.05) between the means by Statistical Product and Service Solutions statistics (SPSS version 13.0). Correlation coefficient and regression analyzes were determined by Data Processing System (DPS, version 3.01) and EXCEL program. 3. Results and discussion The physicochemical changes of Dajinxing hawthorn (e.g., fruit weight, diameter, moisture content, acidity, soluble sugar content, and so on) were investigated during fruit growth. The physicochemical changing profile in fruits during maturation is very important for choosing the harvesting date because harvest maturity significantly affects the quality and shelf-life of fruits (Kulkarni, & Aradhya, 2005). Figure 1 is a collection of digital photographs, which shows the changes in color of the hawthorn fruits from green red. These photographs show easily observable characteristics, which can be related to the physical and chemical composition data. [Figure 1 about here] 8
3.1. Changes in shape and color of hawthorn during maturation During fruit maturation, the changes in shape and color are shown in Table 1. The range of length and diameter of the hawthorn were from 21.7 to 26.1 mm and from 22.5 to 28.8 mm during maturation, and they increased continuously by 20% and 28% from M1 to M7 stages, respectively. The increase of diameter was higher than that of length, so fruit' shape index (length/diameter) of hawthorn also gradually decreased from 0.96 to 0.89, indicating that the shape of hawthorn fruit changed gradually from oval to round or approached round during maturation, and that the growth of hawthorn fruit encompassed a phase of rapid development (mainly due to cell enlargement) from M2 stage. During fruit development, the color of the hawthorn surface changed progressively from mature green to semi red, to reach bright red. The distinctive red color of the fruit was gradually developed up to M5 stages, with minor variations thereafter (Figure 1). As shown in Table 1, CIE L*, a*, and b* values were significantly different among maturation stages. As an objective evaluation of hawthorn fruit, CIE L* values appear to be important and sensitive to color evaluation. The L* value, which can be an indicator of lightness of color, decreased significantly from 54.65 to 33.52 from M1 to M7 stages, however, no difference in L* value was found among stages M5, M6 and M7. Conversely, the CIE a* value that indicates the redness (positive a*) and greenness (negative a*) increased significantly from -11.55 to 17.25 from M1 to M7 stages, while no difference in a* value was found among stages M5, M6 and M7. The CIE b* value indicates the yellowness, and it first increased from 17.22 to 21.54 at M3 stage, and then decreased to 12.42 during
maturation of hawthorn fruit. These changes in the CIE L*, a*, and b* values was basically coincident with the observations during the fruit development. [Table 1 about here] 3.2. Changes in weight and edible part of hawthorn during maturation During this growth period, the weight of the Dajinxing hawthorns increased continuously and significantly 1.6-fold from 5.7 to 14.9 g until the M7 stage, but no change was found from stages M6 to M7 (Figure 2A). The changes in fruit volume showed similar increase patterns as the fruit weight change, and the rate of increase in weight and volume was basically the same at each stage (Figure 2A). Consequently, the density of intact hawthorn fruits remained unchanged during maturation (Figure 2A). Özcan, Hacıseferoğulları, Marakoğlu and Arslan (2005) reported the density of ripe hawthorn fruit was 1065.98 kg/m3, which was in accord with our results. The growth of the hawthorns proceeds to the maturation through the cell division and cell enlargement stages, and their pattern shows a typical sigmoidal pattern (Combe, 1976). It is assumed that the cell enlargement stage of the Dajinxing hawthorn begins after M2 stage, in which a steeper weight change was observed than before. [Figure 2 about here] Figure 2 shows the changes in the weight of flesh, core and edible part (flesh/cote ratio) of intact hawthorn during maturation (Figure 2B). Similar to weight and volume of intact fruits, the weight of flesh increased gradually and significantly (p < 0.05) by 1.85-fold from 4.30 to 12.25 g. Interestingly, the weight of hawthorn core did not change significantly during maturation, indicating that the seed has been mature at M1 stage. 10
Correspondingly, edible part of hawthorn increased significantly (p < 0.05) by 1.93-fold from 2.9 to 8.5. It was clear that the flesh contributed significantly to the increase of the intact hawthorn weight during this development period. Based on above analysis, the increase of diameter was higher than that of length which might be one of the features of hawthorn growth. 3.3. Changes in nutritional compositions of hawthorn during maturation The nutritional compositions affecting the quality of hawthorns were investigated during the fruit growth (Table 2). During the fruit growth, starch content first increased significantly (p < 0.05) by 4.56-fold and reached the maximum at stage M5, and then it sharply decreased by 51% (p < 0.05), from 5.68% to 2.75% at M7 stage. The crude protein content had no obvious change from stages M1 to M4, thereafter, there were a significant decrease (p < 0.05) in crude protein and it decreased by 8.9-fold from 5.83% at M4 stage to 0.59% at M7 stage. The content of moisture and total soluble sugars increased continuously by 15.6% and 216% by the end of maturity, respectively. However, they did not change much between stages M6 and M7. The increases in total soluble sugars may be attributed to the hydrolysis of the starch component in the hawthorn with maturity of fruits (Zheng, Kim, & Chung, 2012), which is desirable for hawthorn taste. In addition, the increase in total soluble sugars can bring the hawthorns colors because there was a marked positive correlation between the content of anthocyanin and total sugars of hawthorn fruits (Qi, Li, & Xu, 2005). A similar change in the total sugar content has also been reported in pomegranates (Kulkarni, & Aradhya, 2005) and apples (Zheng, Kim, & Chung, 2012). Similar to starch, the TA values first 11
increased significantly (p < 0.05) by approximately 2.5-fold and reached the maximum at stage M5, and then it decreased by 28.7% (p < 0.05), from 4.11% to 2.93% at M7 stage. Liu et al.(2010) reported that the malic acid, citric acid, quinic acid were the major organic acids and the total acid content of the full mature hawthorn fruits varied from 3.1 to 11.8 g/100 g DM because of differences in the cultivars or species. As is common in most edible fruits (Scheerens, 2006), we report here that TA levels decreased and TSS increased as hawthorns ripened were similar to patterns reported in other fruits (Johnson, Bomser, Scheerens, & Giusti, 2011; Distefano et al., 2009). The content of RAA and TAA increased continuously and significantly (p < 0.05) by approximately 20.9-fold and 8.7-fold by the end of maturity, respectively, which was in accord with their changes in yuzus during maturation (Yoo, Lee, Park, Lee, & Hwang, 2004). However, some variations on this were also found in tomatoes (Opara, Al-Ani, & Al-Rahbi, 2012) and sea buckthorn berries (Kallio, Yang, & Peippo, 2002), which may be concerned in the origins, cultivars, and species of test materials. [Table 2 about here] The levels of the soluble pectin (SP), protopectin (PP) and pectin from hawthorn fruits were measured, which was incoordinately influenced by fruit ripening stages (Table 3). The SP content gradually increased and PP and pectin first increased and then decreased with the processing of maturation. The SP content increased (p > 0.05) approximately 21% during the prophase of maturation (M1-M4). And then a significant increase (p < 0.05) in SP content was detected during the remainder of maturation (M5-M7) and its content increased by 307% as compared to M1 stage. Wang et al. 21 also 12
reported that ripe jujubes had higher pectin than unripe fruits. The content of PP first increased significantly (p < 0.05) by 91% and reached the maximum at stage M5, and then it sharply decreased by 53.5% (p < 0.05) at M7 stage. The pectin comprise the SP, PP, and its content was influenced by the SP and PP. Because the content of PP were much higher than that of SP at each stage, changes of pectin content had a more similar trend to the PP (Table 3), but the variation range of the pectin content was different from that of the PP content. [Table 3 about here] Pectin, a complex polysaccharide, is one of the major components in the primary cell wall and
pentaoligosaccharide from hawthorn was able to up-regulate the gene and protein expressions of peroxisome proliferator-activated receptor α (Li et al., 2013; Zhu et al., 2013). During the early stage of maturation, PP rapidly increased in order to the growth of fruits, and partially PP polymers is more depolymerized to SP and solubilized within cell walls while fruits reached a certain maturity, which resulted in the increase of SP and increased fruit solubility. However, SP can be further decomposed to pectin acid and methanol under pectin methyl esterase, leading to reduced amount of total pectin. 3.4. HPLC analysis of individual sugars of hawthorn during maturation The HPLC quantitative analytical results of the individual sugars extracted from hawthorns are shown in Table 3. The levels of the main sugars varied significantly (p < 0.05) among various stages of maturation. The sucrose first increased from 0.64% to 1.10% (p < 0.05) at M5 stage and then decreased to 0.42% (p < 0.05); while the content 13
of glucose and fructose increased by 4-fold from 0.42% to 2.13% and 5.4-fold from 0.49 % to 3.15% by the end of maturity, respectively. Sucrose was the main component of soluble sugars, followed by fructose and glucose at stages M1 and M2. However, this situation was now changing because fructose and glucose were increasing rapidly along with the ripening of hawthorns. Namely, fructose became the main component of soluble sugars, followed by glucose and sucrose during later period of maturation, which was in agreement with previous report that the fructose content was the highest, followed by glucose in the full mature hawthorn fruits (Liu et al., 2010). Somewhat differently, Liu et al. (2010) reported that the level of fructose and glucose were 17.32, 15.96 g/100 g DM while sucrose was even no found in the full mature Dajinxing hawthorn fruits. These differences in the content of individual sugars from hawthorn fruits may be concerned in the geographical origins and maturity. Also unlike the present results, Qi, Li and Xu (2005) reported that sucrose content in hawthorn fruits increased during later period of maturation, and the glucose content was the highest, followed by fructose and sucrose in the ripe fruits, which may be due to the differences in hawthorn cultivars. 3.5. Changes in free amino acids of hawthorn during maturation The content of free amino acid compositions in all seven growth stages of hawthorn are shown in Table 4. The total free amino acids (TFAA) first increased from 317.4 mg/100g at stage M1 to 727.0 mg/100g at stage M4, and then decreased to 348.5 mg/100g at stage M7. Changes in the content of total essential amino acids (TEAA) and nonessential amino acids (NEAA) were similar to total free amino acids (Table 4). The data also showed that none of the samples contained free tryptophane and methionine, and 14
only the last three growth stages contained free proline, which rapidly increased from 2.5 mg/100g at M5 to 12.2 mg/100g at M7 stage. In addition, isoleucine and tyrosine were found during maturation but disappeared at later period of maturation. Choi et al. (2012) also got similar results while they studied effects of growth stages on the free amino acid in jujube fruit. [Table 4 about here] We calculated the contribution of total free amino acids to the total crude protein determined from Kjeldahl nitrogen (Table 2). The percentage of total free amino acid values showed in the last row of Table 4 increased dramatically from 5.2 at stage M1 to 59.1 at stage M7. In the fully ripe fruit, free amino acids are the major contributor, more than protein, to nitrogen content. This could increase the digestibility of the nitrogen present but also could contribute to reactions such as Maillard browning. 4. Conclusion In conclusion, profiles of changes in physical parameters, chemical characteristics and free amino acids were investigated during maturation of hawthorn fruits. Differences in these parameters were found among different maturation stages. During maturation of hawthorn fruits, the color turned progressively from mature green to semi red, to reach bright red; the shape changed gradually from oval to round or approached round. The size, weight, and edible part (flesh/core ratio) of hawthorn increased while the density of intact fruits did not change. The content of moisture, TSS, SP, RAA, TAA, fructose, and sucrose significantly increased while the crude protein content decreased significantly. Changes in the starch, sucrose, TA, PP, pectin, TFAA, and TEAA during ripening were 15
reported, and their levels initially increased and then decreased gradually during maturation. The outcomes of this study provide additional and useful information for fresh consumption and processing as well as utilization of dropped unripe hawthorn fruits. Acknowledgments This work was financially supported by a project of the Natural Science Foundation of Shanxi Province, China (project no. 2012011031-3). Conflict of interest The authors declare no competing financial interest.
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Figure 1. Digital photographs of eight stages of maturation (M1−M7) of hawthorn fruits. Figure 2. Changes in weight, volume (Figure 2 A) and the edible part (Figure 2 B) of hawthorns during maturation. Data are expressed as the mean values of three independent replicates ± SD. M1-M7 refer to the different maturation stages. Different letters indicate statistically significant differences between the means (p < 0.05) for weight, volume, density, flesh and core.
Table 1. Shape and color of hawthorn fruits at different maturation stages a Fruit shape index Stages
Numbers represent mean values of three independent replicates ± SD. b L/D refer to the
M1-M7 refers to the different maturation stages. Different letters
indicate statistically significant differences between the means (P < 0.05) for each parameter.
Table 2. Content of some nutrients in hawthorn fruits at different maturation Stages a Ascorbic acid (mg/100g)
0.59±0.05d 2.75±0.05d 11.65±0.27a
TA (%) RAA
3.12±0.22b 12.24±1.02d 46.89±5.25d 4.11±0.18a
Numbers represent mean values of three independent replicates ± SD. b M1-M7 refer to
the different maturation stages. Different letters indicate statistically significant differences between the means (P < 0.05) for each nutrient.
Table 3. Content of individual sugars and pectin in hawthorn fruits at different maturation stages a
0.26±0.02d 1.89±0.06d 2.15±0.08e
0.30±0.07d 3.07±0.14b 3.38±0.21bc
0.33±0.03d 3.24±0.10b 3.58±0.13b
Numbers represent mean values of three independent replicates ± SD. b M1-M7 refer to
the different maturation stages. Different letters indicate statistically significant differences between the means (P < 0.05) for each nutrient.
Table 4. Content of free amino acids in hawthorns at different maturation stages a Amino acid content (mg/100g) Amino acids M1b
TFAA/protein (%) a
Numbers represent mean values of three independent replicates ± SD. b M1-M7 refer to
the different maturation stages. c nf, not found.
Abbreviations of amino acids (TEAA,
total essential amino acids; NEAA, nonessential amino acids; TFAA, total free amino acids. 27
Figure 2. 1.2 a
20 weight volume density
12 core flesh flesh/core
e 4 a
fruit weight (g)
d d 8
Density (g/cm )
Weight or volume (g or cm )
The research highlights of the manuscript are as following: 1) Investigated the physical changes during maturation of hawthorn 2) Studied the chemical changes during maturation of hawthorn 4) Evaluated changes in the individual sugars during maturation of hawthorn 5) Analyzed changes in the free amino acids during maturation of hawthorn