YAN Zhen, ZHENG Li-jing , NlE Ji-yun Ll Zhi-xia CHENG Yang

1 Laboratory of Quality & Safety Risk Assessment for Fruit (Xingcheng), Ministry of Agriculture/Institute of Pomology, Chinese Academy of Agricultural Sciences, Xingcheng 125100, P.R.China

2 Beijing Agriculture Technology Extension Station, Beijing 100029, P.R.China

YAN Zhen, E-mail: yanzhen01@caas.cn; Correspondence NIE Ji-yun, Tel: +86-429-3598178, E-mail: jiyunnie@163.com

＊These authors contributed equally to this study.

? 2018 CAAS. Publishing services by Elsevier B.V. All rights reserved.

1. lntroduction

Flavor is an important component of fruit quality (Nie and Dong 2014; Zhenget al. 2016). With social and economic developments and the improvement of living standards,fruit flavor is receiving greater attention from fruit breeders,producers, sellers, consumers, and processing industries(Carewet al. 2004; Zhanget al. 2008; Maet al. 2016).Organic acids are the main flavor substances of fruit. Their composition and quantity are closely related to the quality of fruit, and directly affect the flavor of fruit (Mahmoodet al.2012; Shangguanet al. 2015). In apples, flavor mainly depends on acid content, while sugar content has little effect on apple flavor (Guoet al. 2012; Berüter 2004).

China is the world’s largest apple producer and consumer(Wuet al. 2007). Its apple production in 2014 reached 40.923 million tons (NBSC 2015a), accounting for 24.67%of its fruit output and 47.31% of the world’s apple output(86.5 million tons) (NBSC 2015b). Apples have become an important part of the Chinese diet, and they have many components that are beneficial to human health (Zhanget al. 2010). Currently, there are many studies on organic acid in apples. These studies cover the changes in organic acids in leaves of ‘Honey crisp’ apple (Wanget al. 2010),organic acid comparison between different cultivars (Heckeet al. 2006), as well as the effects of fruit bagging (Liuet al.2013), and the effects of location within the tree canopy on organic acids in the fruit peel and flesh (Fenget al. 2014).

However, few apple cultivars are used in these studies,and there is currently no report available on grading standards of evaluation indices for apple sour flavor and the quantitative relationships between these indices (Wuet al. 2007; Maet al. 2015). Thus, the results of these studies can neither fully reflect the composition properties of organic acids in apple, nor provide scientific and quantitative standards for evaluating apple sour flavor (Zhanget al. 2010;Sadaret al. 2016). Given that, the present study selected the mature fruits of 106 apple cultivars and tested the following indices: malic acid (Mal), oxalic acid (Oxa), citric acid (Cit), lactic acid (Lac), succinic acid (Suc), fumaric acid(Fum), pH value, titratable acidity (TiA), acidity value (AcV),and total organic acids (ToA). The present study clarified the level of each index and the relationships between the indices, established the grading standard of evaluation indices for apple sour flavor, and provided a scientific basis for evaluating apple flavor and selecting apple cultivars.

2. Materials and methods

2.1. Sampling and preparation

A total of 106 apple cultivars, as shown in Table 1, were collected from the National Repository of Apple Germplasm Resources (Xingcheng City, Liaoning Province, China). Tenyear-old trees onMalus baccata(L.) Borkh rootstock were planted in sandy loam soil. The orchard where they were planted is flat and is managed at a medium level. Fruits of each cultivar were not bagged during development. For each cultivar, three trees were selected. At the suitable maturity stage for harvest when the seeds began to turn brown (Wanget al. 2003), 30 fruits per cultivar were randomly picked from the middle of the canopy. From these fruits, 10 fruits that were consistent in shape, size, and color were chosen and sampled by quartering, the edible part was chopped and immediately frozen in liquid nitrogen, and then stored at –20°C in a refrigerator. Each sample was repeated three times for testing.

2.2. Reagents

Sodium hydroxide (GR, guaranteed reagent) was obtained from Tianjin Shengao Chemical Reagent Co., Ltd. (China).Phenolphthalein indicator (AR, analytical reagent) was purchased from Tianjin Damao Chemical Reagent Factory(China). Both were used for the determination of titratable acid content. 50% sodium hydroxide solution (AR) was produced by Merck KGaA (Germany). Heptafuorobutyric acid (≥99.5%) for ion chromatography was produced by Fluka (USA). Tetrabutylammonium hydroxide for ion chromatography (0.995 g cm–3) was produced by Sigma Aldrich (USA). These three reagents were used to prepare the mobile phase. Oxalic acid standard (99.5%)and lactic acid standard (90.5%) were purchased from Dr.Ehrenstorfer GmbH (Germany). Fumaric acid standard(99.5%) was obtained from Aladdin Chemistry Co., Ltd.(USA). Citric acid standard (98%), succinic acid standard(99%), and malic acid standard (99%) were purchased from Chem Service (USA).

2.3. Determination of titratable acid and pH

The titratable acid content was determined using indicator titration and the pH value was tested using a FE20K-type pH Meter (Mettler-Toledo, Switzerland) according to Nie (2009).

2.4. Determination of organic acid component

For each treatment, a 200-g sample was collected and pulverized into a powder using a 6870-type Freezer Mill(SPEX Sample Prep, USA). A 5-g sample was accurately weighed and added to 25 mL of deionized water. The sample was then placed in a constant temperature water bath (HWS28, Shanghai Yiheng Instruments Co., Ltd.,China) set to 80°C and extracted for 30 min. The sample was cooled and diluted to 50 mL, after which it was filtered using absorbent cotton. A total of 1 mL of the filtrate was diluted to 10 mL with deionized water. The sample was cleaned up with an RP column (Dionex, USA) followed by filtration using a 0.22-μm pore size filter (Fine, Japan).

The filtrate was tested for content of the six organic acids using an ion chromatograph (ICS-5000, Dionex, USA)with a suppressor (AMMS-ICE 300, Dionex, USA). The eluent was 0.4 mmol L?1heptafluorobutyric acid and the regeneration solution was 5 mmol L?1tetrabutylammonium hydroxide. The flow rate was 1.0 mL min?1. The column(9 mm×250 mm, ICE-AS6, Dionex, USA) temperature was

set at 30°C. The injection volume was 25 μL. Quantification was conducted using an external standard method.

Table 1 A list of apple cultivars studied

2.5. Calculation of acidity

The acidity value of each sample was calculated according to eq. (1):

Where,AcVis the acidity value;Ci(mg g?1) is the content of Cit, Mal, Lac, Fum, Suc, or Oxa; andAiis the acidity intensity of Cit, Mal, Lac, Fum, Suc, or Oxa, with the values of 100, 76, 107, 55, 88, and 50, respectively (Ding 1997; Xia 2008). The intensity of oxalic acid was calculated based on the human saliva flow rate with an oxalic acid concentration of 0.1 mol L?1(Ding 1997).

2.6. Statistical analysis

Minitab 16 software (Minitab Statistical Software,https://it.minitab.com/en-us/support/answers/answer.aspx?log=0&id=2485) was used to draw boxplot graphs.DPS?software (ver. 13.5) (Tang 2010) was used for the normality test of indices; if the Jarque-Bera test statistical probability value was ＞0.05, then the index was normally distributed.@Risk 5.5 (Risk Analysis Software, http://www.palisade.com/about/about.asp) for Excel was used to fit the normal distribution (iterations=10 000) of the indices, and the 10th, 30th, 70th, and 90th percentiles of the normal distribution curve were taken as the grading node values of the index. Other statistical analyses were performed using Excel 2010 Software.

3. Results

3.1. lndices of apple sour flavor

The ranges of the 10 tested indices are in Fig. 1. The mean values of total organic acids, titratable acid, acidity value,and pH were 6.10 mg g?1, 0.47%, 8.10 mg g?1, and 3.60,respectively. Among the six organic acids, malic acid had the highest content with an average of 5.80 mg g?1, followed by oxalic acid with an average of 0.162 mg g?1. The other four organic acids (citric acid, lactic acid, succinic acid, and fumaric acid) were very low in content, with averages of 0.083, 0.035, 0.015, and 0.009 mg g?1, respectively. Among the cultivars studied, 9.4% were over 9 mg g?1and 10.4%were below 3 mg g?1in total organic acids; 6.6% were over 9 mg g?1and 15.1% were below 3 mg g?1in malic acid content; 8.5% were above 12 mg g?1and 10.4% were under 4 mg g?1in acidity value; 12.3% were above 4 and 11.3%were under 3.3 in pH value; 5.7% exceeded 0.8% and 9.4%were less than 0.2% in titratable acidity.

The studied indices showed different degrees of dispersion. Cit and Fum had the highest variation (with coefficients of variation up to 50.7 and 65.8%, respectively),followed by Oxa, Suc, Mal, Lac, AcV, ToA, and TiA with coefficients of variation between 31 and 44%. The variation of pH value was the smallest with a coefficient of variation of only 8.2%. The content order of the six organic acids was not the same in each cultivar: 85.8% cultivars were in the order of Mal＞Oxa＞Cit＞Lac＞Suc＞Fum; 6.6% cultivars were in the order of Mal＞Oxa＞Cit＞Lac＞Fum＞Suc; 2.8% cultivars followed the order of Mal＞Oxa＞Cit＞Suc＞Lac＞Fum; and 1.9%cultivars followed the order of Mal＞Oxa＞Lac＞Cit＞Suc＞Fum.Among the organic acids tested, Mal was predominant with average content accounting for 94.5% of ToA, followed by Oxa with average content accounting for 3.1% of ToA (Fig. 2).Cit was the third most common with average content accounting for 1.4% of ToA. Lac, Suc, and Fum accounted for 0.65, 0.28, and 0.17% of ToA, respectively.

Fig. 1 Ranges of 10 sour flavor indices in fruits of apple cultivars tested. Mal, malic acid; Oxa, oxalic acid; Cit, citric acid; Lac,lactic acid; Suc, succinic acid; Fum, fumaric acid; ToA, total organic acids; AcV, acidity value; TiA, titratable acidity. Mal,Oxa, Cit, Lac, Suc, Fum, ToA, and AcV are in mg g–1; TiA is in percentage.

Fig. 2 Content distribution of six acids in fruits of apple cultivars tested. Mal, malic acid; Oxa, oxalic acid; Cit, citric acid; Lac,lactic acid; Suc, succinic acid; Fum, fumaric acid.

3.2. Relationships between indices of apple sour flavor

The correlation analysis of the 10 studied indices showed that 51.1% of the correlation coefficients were significant at α=0.01 (Table 2). There was a significant negative correlation between pH value and six indices (Mal, Cit, Suc,ToA, TiA, and AcV). Mal and five indices (Cit, Suc, ToA, TiA,and AcV) were significantly positively correlated. Similar trends were seen between Oxa and Lac; between Lac and Fum; between TiA and AcV; between Cit and four indices(Suc, ToA, TiA, and AcV); between Suc and three indices(ToA, TiA, and AcV); and between ToA and two indices(TiA and AcV). The correlation coefficients were close to or exceeded 0.85 (some were close to 1) for: Mal and four indices (ToA, TiA, AcV and pH value); ToA and three indices(TiA, AcV, and pH value); TiA and two indices (AcV and pH value); and AcV and the pH value.

Regression analysis indicated that there were significant positive linear relationships between Mal and ToA (Fig. 3-A),Mal and AcV with average fitting errors of 1.0 and 1.3%,respectively (Fig. 3-B). There was also a significant linear relationship (Fig. 3-C) between ToA and AcV with an average fitting error of 0.4%. There were significant logarithmic relationships (Fig. 3-D, E, F, and G) between pH value and one of the four indices (TiA, Mal, ToA, and AcV) with average fitting errors of 2.1, 3.0, 3.1, and 3.1%, respectively.For all of these relationships, the equations had high fitting accuracy and can be used to accurately predict related indices. Significant linear relationships were also found between TiA and each of the three indices (Mal, ToA, and AcV) (Fig. 3-H, I, and J) with average fitting errors of 6.6,6.3, and 6.2%, respectively, indicating that these equations can be used to roughly estimate related indices.

3.3. Grading standards of sour flavor indices for apples

Based on correlation analysis, five indices (Mal, ToA, AcV,TiA, and pH value) were chosen as the sour flavor evaluation indices for apples. For these indices, the correlation coefficients were all close to or above 0.85. According to the results of normality testing using DPS?Software, Mal,ToA, and AcV were all normally distributed with probability values of the Jarque-Bera statistic over 0.05, TiA was close to normally distributed, whereas pH value was not normally distributed (Table 3). The normal distribution curves of Mal,ToA, AcV, and TiA in fruits of 106 varieties were fitted using@Risk 5.5 for Excel (Fig. 4) .

On the basis of the theoretical probability of a normal distribution, grade medium accounts for 40%, grade low and grade high both account for 20%, both grade lower and grade higher account for 10% (Liu 1996). Thus, each of the four indices (Mal, ToA, AcV, and TiA) were divided into five grades (lower, low, medium, high, and higher) by the 10th,30th, 70th, and 90th percentiles of their normal distribution curve (Table 3). As mentioned above, pH value was not normally distributed. Mal was normally distributed, and there was logarithmic relationship between Mal (x) and pH value (y), namelyy=?0.619ln(x)+4.64. The corresponding grading node values of pH value were obtained using this equation and the 10th, 30th, 70th, and 90th percentiles of the normal distribution curve of Mal (Table 3).

As shown in Table 3, the four indices of Mal, ToA, AcV,and TiA had the same trend that the higher the index value and the grade, the stronger the sour flavor of apple. The index of pH value showed the reverse trend that the lower the index value and the grade, the stronger the sour flavor of apple.

4. Discussion

Differences in the level and proportion of organic acidscharacterize the distinctive flavor of each type of fruit (Gilet al. 2000; Sadkaet al. 2000; Róthet al. 2007; Chenet al.2009). Apples contain a variety of organic acids including oxalic acid (Oxa), fumaric acid (Fum), succinic acid (Suc),tartaric acid, quinic acid, shikimic acid, citric acid (Cit), malic acid (Mal), and acetic acid (Wuet al. 2007; Lianget al. 2011;Rodríguezet al. 2017). Among these acids, the content of malic acid in apples is significantly higher than other organic acids with a value usually close to or more than 90% of ToA(Jinget al. 2016). In this paper, average malic acid content was 94.5% of ToA in 106 apple cultivars. Therefore, apples can be considered a typical fruit that is dominated by malic acid compared to other organic acids (Berüteret al. 2004;Maet al. 2015). In apples, the content of other organic acids is often very low and almost negligible compared to malic acid (Liuet al. 2016). However, not all organic acids found in apples are consistently being monitored. In particular, fumaric acid, shikimic acid, and maleic acid are seldom reported (Wuet al. 2007; Wanget al. 2010). These differences might be due to different conditions of growth and cultivation and different biological time (Petkovseket al.2007; Sadaraet al. 2016).

Table 2 Correlation coefficients between 10 fruit indices of tested apple cultivars1)

Fig. 3 Relationship between five sour flavor indices of different tested apple cultivars (A–J). ToA, total organic acids; Mal, malic acid; AcV, acidity value;TiA, titratable acidity.

Table 3 Grading of five acidity evaluation indices of tested apple cultivars

Fig. 4 Simulated normal distribution curves of four fruit sour flavor indices of different tested apple cultivars. TiA, titratable acidity;ToA, total organic acids; Mal, malic acid; AcV, acidity value.

Regression analysis is a method to study the change in relationship between the dependent and independent variables, and its final result is generally an empirical regression equation (Tang2006). This method has been effective in food quality studies including: characteristic analysis of cherry fruit (Khub 2014); correlations between orange quality indices (Rosaet al. 2016); and differences in organic acids between wine varieties (Geanaet al. 2016).In the present study, a large sample of apples (106 cultivars)and regression analysis were used to discover significant linear or logarithmic relationships between five apple sour flavor indices, namely Mal, ToA, AcV, TiA, and pH value.The average fitting error of each equation was less than 5%,and these equations can therefore be used to accurately predict related indices. Significant correlations between indices are the basis for linear regression or nonlinear regression. The present study showed that there were significant (at α=0.01) correlations between five apple sour flavor evaluation indices (Mal, ToA, TiA, AcV, and pH value),and the correlation coefficients were all close to or more than 0.85, with some close to 1. This is consistent with the finding of a previous report (Fenget al. 2013).

Most of the variables of biological phenomena follow a normal distribution, and thus the normal distribution is the most common distribution in fruit science experiments(Huazhong Agricultural College 1979; Palacioset al. 2015).This study showed that Mal, ToA, and AcV of apples were normally distributed; TiA was close to normally distributed;and pH value had a skewed distribution. Compared to traditional experience grading, probability grading has the advantage of objective, unified standards and comparable results, and therefore, has more guidance value. However,a large sample is needed to represent the normal population(Avilaet al. 2015; Malegoriaet al. 2017). In this study, a large sample of 106 apple cultivars were used, covering early-, medium-, and late-maturing groups.

We have ever studied the flavor evaluation indices for fresh apple juice based on 159 cultivars (Nieet al. 2012),and the results showed that there were significant positive correlation between the sour flavor and the TiA of fresh apple juice, that is the higher the TiA, the stronger the sour flavor, andvice versa. In that paper, the sensory evaluation of the sour flavor of fresh apple juice was carried out by nine experts using oral taste. It is known that fresh apple juice was produced from apple without modification, and its sour flavor is the same as that of raw apple. In this article,as there were significant positive linear relationships or significant logarithmic relationships between TiA and the other four indices (Mal, ToA, AcV, and pH value) of apple(see Fig. 3), the latter four indices (Mal, ToA, AcV, and pH value) were also recommended as the suitable indices for the sensory evaluation of apple sour flavor.

At present, the evaluation indices of sour flavor for apple are generally expressed in the absolute value of titratable acid content (Wanget al. 2005), and grading standards of apple sour flavor evaluation indices have not been reported.In the present study, the grading standards were established.Using the theoretical probability of a normal distribution (Liu 1996), the four evaluation indices of apple sour flavor (Mal,ToA, AcV, and TiA) were divided into five grades (lower, low,medium, high, and higher). These four indices all followed,or nearly followed, a normal distribution. Although pH value did not follow a normal distribution, its grading node values were obtained using the logarithmic relationship between pH value and Mal. Therefore, the grading standards of the above mentioned five apple sour flavor evaluation indices were established and can be used for future apple sour flavor evaluation (Table 3). All of these indices are stable,quantitative, and can be detected accurately.

5. Conclusion

Apple is a typical fruit that is dominated by malic acid content among all organic acids. The average content of malic acid in apples in the present study accounted for 94.5% of ToA, and the content order of organic acids in most apples was Mal＞Oxa＞Cit＞Lac＞Suc＞Fum. There were significant linear or logarithmic relationships between apple sour flavor indices (Mal, ToA, AcV, TiA, and pH value),and most of the equations had high- fitting precision and can be used to accurately predict related indices. Mal,ToA, and AcV were normally distributed; TiA was close to a normal distribution; but pH value had a skewed distribution.The grading standards of Mal, TiA, ToA, and AcV were established based on their normal distribution curves, while the grading standards of the pH value were established using its logarithmic relationship with Mal. For the index of pH value, higher index values and grades meant weaker sour flavor of apple. Conversely, for the other four indices,higher index values and grades meant stronger sour flavor of apple. The grading standards of all five indices can be used to evaluate the sour flavor of apple.

Acknowledgements

This work was financially supported by the earmarked fund for the China Agriculture Research System (CARS-27), the National Program for Quality and Safety Risk Assessment of Agricultural Products of China (GJFP2017003) and the Scientific and Technological Innovation Project of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP) .

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