Flavor Quality Analysis of Ten Actinidia arguta Fruits Based on High-Performance Liquid Chromatography and Headspace Gas Chromatography–Ion Mobility Spectrometry

Actinidia arguta is a fruit crop with high nutritional and economic value. However, its flavor quality depends on various factors, such as variety, environment, and post-harvest handling. We analyzed the composition of total soluble sugars, titratable acids, organic acids, and flavor substances in the fruits of ten A. arguta varieties. The total soluble sugar content ranged from 4.22 g/L to 12.99 g/L, the titratable acid content ranged from 52.55 g/L to 89.9 g/L, and the sugar–acid ratio ranged from 5.39 to 14.17 at the soft ripe stage. High-performance liquid chromatography (HPLC) showed that citric, quinic, and malic acids were the main organic acids in the A. arguta fruits. Headspace gas chromatography–ion mobility spectrometry (HS-GC-IMS) detected 81 volatile compounds in 10 A. arguta varieties, including 24 esters, 17 alcohols, 23 aldehydes, 7 ketones, 5 terpenes, 2 acids, 1 Pyrazine, 1 furan, and 1 benzene. Esters and aldehydes had the highest relative content of total volatile compounds. An orthogonal partial least squares discriminant analysis (OPLS-DA) based on the odor activity value (OAV) revealed that myrcene, benzaldehyde, methyl isobutyrate, α-phellandrene, 3-methyl butanal, valeraldehyde, ethyl butyrate, acetoin, (E)-2-octenal, hexyl propanoate, terpinolene, 1-penten-3-one, and methyl butyrate were the main contributors to the differences in the aroma profiles of the fruits of different A. arguta varieties. Ten A. arguta varieties have different flavors. This study can clarify the differences between varieties and provide a reference for the evaluation of A. arguta fruit flavor, variety improvement and new variety selection.


Introduction
Actinidia arguta is a dioecious deciduous vine belonging to the genus Actinidia in the family Actinidiaceae [1].It originates from China and is widely cultivated in various regions, such as Russia, Japan, Korea, the United States, and New Zealand [2].Its fruit has a tart, sweet, and refreshing taste and is rich in vitamins, amino acids, proteins, minerals, and other nutrients [3,4].It also contains bioactive compounds such as flavonoids, polysaccharides, and volatile oils [5,6].It has various health benefits such as antioxidant, antitumor, and antihyperglycemic effects and is known as the "fruit of health" [7][8][9][10].The Institute of Special Animal and Plant Sciences of the Chinese Academy of Agricultural Sciences has been collecting and breeding A. arguta varieties since the 1960 s and has developed 'Kuilv', 'Fenglv', 'Jialv', 'Wanlv', 'Xinlv', 'Pinglv', 'Lvbao', 'Cuiyu' and 'Tianxinbao' among others to promote the A. arguta industry [11].
Flavor is one of the key indicators of fruit quality, and it consists of acidity, sweetness, and aroma [12].Flavor studies have been extensively studied in many fruit crops, such as grape [13], kiwifruit [12,14], and cherry [15].Organic acids are important components of fruit flavor and functionality, affecting biological activity and microbial growth.Citric, malic, and tartaric acids are the most common in fruits, but the types and contents of organic acids vary widely among species and varieties [16].A. arguta mainly contains citric and quinic acids, followed by malic, oxalic, mangiferin, and succinic acids [17].Aroma compounds also contribute to the sensory quality of fruits [18].A variety of aroma compounds, including esters, aldehydes, alcohols, and terpenes, have been detected in A. arguta so far.The expression of aroma is the result of the interaction of various aroma compounds, but generally, the compounds with higher content or distinctive aroma characteristics can largely determine the special aroma attributes of the fruit [19][20][21][22][23].
Nowadays, the main methods for the determination of organic acids in fruits and fruits include spectrophotometry, gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), ion chromatography (IC), and liquid chromatography-mass spectrometry (LC-MS) [24,25].Among them, HPLC has the advantages of good separation, high sensitivity, good accuracy, and a wide range of applications [25].It has become an important separation and analytical technique in various fields such as chemistry, medicine, industry, agronomy, forensic science, and others [24,26,27].
Various methods have been used to detect volatile compounds in food, such as gas chromatography-olfactometry-mass spectrometry (GC-O-MS), gas chromatography-mass spectrometry (GC-MS), headspace solid-phase microextraction, and gas chromatographyion mobility spectrometry (GC-IMS) [28].Among them, GC-MS is the most widely used method for aroma analysis, but it has some drawbacks such as complex sample preprocessing and potential distortion [29,30].GC-IMS is a novel method based on the differences in the migration speed of gas-phase ions in an electric field, which can achieve high separation and low detection limit, and has been widely applied in food volatile analysis [31][32][33].From Table 1, it can be seen that the application of GC-IMS in A. arguta aroma analysis is still rare and needs further exploration.
Table 1.Studies on organic acids and volatile substances in A. arguta fruit.

Analysis of Sugar-Acid Content of A. arguta Fruits of Different Varieties
The sugar-acid ratio can be derived by calculating the ratio of total soluble sugar content to titratable acid content, as a comprehensive index, it can reflect the sweet and sour taste of the fruit, which largely affects the flavor performance [12,39].The total soluble solid, titratable acid content and sugar-acid ratio of each variety of A. arguta are shown in Figure 1, with the highest total acid content of 'Fenglv' (12.99 g/L) and the lowest of 'Pinglv' (4.22 g/L); the highest total sugar content of 'Longcheng No.2' (89.9 g/L), and the lowest was 'Cuiyu' (52.55 g/L).The largest sugar-acid ratio among the 10 varieties was 'Pinglv' (14.17), followed by 'Tianxinbao' (12.19) and the lowest was 'Cuiyu' (5.39).

Analysis of Sugar-Acid Content of A. arguta Fruits of Different Varieties
The sugar-acid ratio can be derived by calculating the ratio of total soluble sugar content to titratable acid content, as a comprehensive index, it can reflect the sweet and sour taste of the fruit, which largely affects the flavor performance [12,39].The total soluble solid, titratable acid content and sugar-acid ratio of each variety of A. arguta are shown in Figure 1, with the highest total acid content of 'Fenglv' (12.99 g/L) and the lowest of 'Pinglv' (4.22 g/L); the highest total sugar content of 'Longcheng No.2' (89.9 g/L), and the lowest was 'Cuiyu' (52.55 g/L).The largest sugar-acid ratio among the 10 varieties was 'Pinglv' (14.17), followed by 'Tianxinbao' (12.19) and the lowest was 'Cuiyu' (5.39).

Analysis of Organic Acids in A. arguta Fruit of Different Varieties
The type and content of organic acids affect the acidity, texture, and flavor of A. arguta fruits.Different tree species have different organic acids in their fruits, resulting in different flavors.For example, kiwifruit and summer orange contain malic acid, quinic acid, and citric acid; grape has malic acid and tartaric acid; pear and apple have mainly malic acid [40][41][42][43][44][45].Table 2 shows the organic acid fractions and contents of different A. arguta varieties.Oxalic acid, quinic acid, malic acid, mangiferin, lactic acid, and citric acid were detected in A. arguta fruits, except for lactic acid in 'Cuiyu'.Citric acid, quinic acid, and malic acid were the dominant organic acids in the ten varieties, accounting for more than 90% of the total acid content, which is consistent with previous studies [17,34,46].Based on the organic acid components, the fruits can be categorized into citric acid dominant and quinic acid dominant groups [34].'Kuilv', 'Fenglv', 'Wanlv', 'Xinlv', 'Pinglv', 'Lvbao', 'Cuiyu', and 'Longcheng No.2' were citric acid dominant, while 'Jialv'

Analysis of Organic Acids in A. arguta Fruit of Different Varieties
The type and content of organic acids affect the acidity, texture, and flavor of A. arguta fruits.Different tree species have different organic acids in their fruits, resulting in different flavors.For example, kiwifruit and summer orange contain malic acid, quinic acid, and citric acid; grape has malic acid and tartaric acid; pear and apple have mainly malic acid [40][41][42][43][44][45].Table 2 shows the organic acid fractions and contents of different A. arguta varieties.Oxalic acid, quinic acid, malic acid, mangiferin, lactic acid, and citric acid were detected in A. arguta fruits, except for lactic acid in 'Cuiyu'.Citric acid, quinic acid, and malic acid were the dominant organic acids in the ten varieties, accounting for more than 90% of the total acid content, which is consistent with previous studies [17,34,46].Based on the organic acid components, the fruits can be categorized into citric acid dominant and quinic acid dominant groups [34].'Kuilv', 'Fenglv', 'Wanlv', 'Xinlv', 'Pinglv', 'Lvbao', 'Cuiyu', and 'Longcheng No.2' were citric acid dominant, while 'Jialv' and 'Tianxinbao' were quinic acid dominant.The differences in organic acid content were reflected in the titratable acidity level.'Fenglv' had the highest titratable acidity level of 12.99 g/L: oxalic acid, quinic acid, malic acid, and citric acid all had the highest levels of 0.4 g/L, 8.48 g/L, 4.4 g/L, and 11.58 g/L, respectively, which were significantly higher than those of the other nine varieties (p < 0.05).The malic acid in fruits inhibits bacterial damage to the pulp, which facilitates fruit preservation [43,47].'Lvbao' had the highest mangiferin content of 0.1 g/L, which was significantly higher than the other nine varieties (p < 0.05).'Tianxinbao' had the highest lactic acid content of 0.83 g/L, which was significantly higher than the other nine varieties (p < 0.05).The heat map analysis can better reflect the characteristics of organic acids in different A. arguta samples.According to the heat map analysis of each variety (Figure 2), ten samples can be classified into three categories.The first category is 'Fenglv'; the second category is 'Kuilv', 'Longcheng No.2', 'Lvbao', 'Xinlv', and 'Wanlv'; the third category is 'Tianxinbao', 'Cuiyu', 'Jialv', and 'Pinglv'.The content of organic acids varied greatly among the samples.The highest total amount of organic acids (oxalic, quinic, malic, lactic, and citric acids were all higher) was found in 'Fenglv'; the total amount of organic acids were higher in the second category; and the total amount of organic acids was lower in the third category.

Two-Dimensional Top-View Spectrogram Analysis
Figure 3a shows the ion mobility spectra of the volatile substances in ten varieties of A. arguta fruits.The horizontal coordinate represents the relative drift time/RIP (no unit), and the vertical coordinate represents the GC retention time (s).The color depth indicates the content of the volatile substances, with darker colors indicating higher concentrations.The results show that GC can separate the volatile components in different va-

Analysis of Volatile Flavor Substances in Different Varieties of A. arguta Fruits 2.3.1. Two-Dimensional Top-View Spectrogram Analysis
Figure 3a shows the ion mobility spectra of the volatile substances in ten varieties of A. arguta fruits.The horizontal coordinate represents the relative drift time/RIP (no unit), and the vertical coordinate represents the GC retention time (s).The color depth indicates the content of the volatile substances, with darker colors indicating higher concentrations.The results show that GC can separate the volatile components in different varieties of A. arguta fruits within 30 min and that there are differences in the volatile flavor substances among them.This suggests that the proportion composition of volatiles is one of the key material bases for the flavor diversity of A. arguta fruits.

Difference Spectrum Analysis
To better compare the differences in the volatile components of different varieties of A. arguta fruits, we used the 'Kuilv' variety as a reference and subtracted its signal peaks from the rest of the spectra to obtain the difference spectra.The blue area indicates that the content of the substance in this sample is lower than that in 'Kuilv', and the red area indicates that the content of the substance in this sample is higher than that in 'Kuilv'.Again, the darker the color, the greater the difference.Figure 3b shows the difference

Difference Spectrum Analysis
To better compare the differences in the volatile components of different varieties of A. arguta fruits, we used the 'Kuilv' variety as a reference and subtracted its signal peaks from the rest of the spectra to obtain the difference spectra.The blue area indicates that the content of the substance in this sample is lower than that in 'Kuilv', and the red area indicates that the content of the substance in this sample is higher than that in 'Kuilv'.Again, the darker the color, the greater the difference.Figure 3b shows the difference spectrum, from which it can be seen that 'Lvbao' contains richer volatile flavor substances, followed by 'Tianxinbao'.The concentration of volatile substances in 'Kuilv' is similar to that in 'Fenglv'.'Jialv', 'Wanlv', 'Xinlv', and 'Pinglv' are more similar to each other.'Cuiyu' has the lowest volatile flavor substance concentration.

Qualitative Analysis of Volatile Compounds in Different Varieties of A. arguta Fruits
We calculated the retention index of each volatile compound in A. arguta fruits using C4~C9 ortho-ketone as the external standard reference, based on the GC retention time and IMS migration time of the volatile compounds.We characterized the volatile compounds in A. arguta fruits by using the built-in NIST 2020 database and the IMS database of HS-GC-IMS.Table S1 shows that the volatile components characterized in 10 different varieties of A. arguta contained a total of 81 monomer and dimer substances, including 24 esters, 17 alcohols, 23 aldehydes, 7 ketones, 5 terpenes, 2 acids, 1 Pyrazine, 1 furan, and 1 benzene.These substances collectively constitute the characteristic flavor of A. arguta fruits.

Content Analysis of Volatile Compounds in A. arguta Fruits
The aroma and flavor of A. arguta fruits depend on the content and proportion of aroma components, especially the variation of the component composition [13], which differs significantly among different varieties.Figure 5 shows that aldehydes were the main contributing components in 'Kuilv', 'Fenglv', 'Jialv', 'Wanlv', 'Xinlv', 'Pinglv', 'Cuiyu', 'Tianxinbao' and 'Longcheng No.2'; and esters were the main contributing components in 'Lvbao'.The ten varieties of A. arguta had the same types of volatile compounds detected in the fruit, but the contents varied greatly.The highest content of volatile compounds was detected in 'Lvbao' (21,733.59µg/kg), followed by 'Tianxinbao' (18,322

Content Analysis of Volatile Compounds in A. arguta Fruits
The aroma and flavor of A. arguta fruits depend on the content and proportion of aroma components, especially the variation of the component composition [13], which differs significantly among different varieties.Figure 5 shows that aldehydes were the main contributing components in 'Kuilv', 'Fenglv', 'Jialv', 'Wanlv', 'Xinlv', 'Pinglv', 'Cuiyu', 'Tianxinbao' and 'Longcheng No.2'; and esters were the main contributing components in 'Lvbao'.The ten varieties of A. arguta had the same types of volatile compounds detected in the fruit, but the contents varied greatly.The highest content of volatile compounds was detected in 'Lvbao' (21,733.59μg/kg), followed by 'Tianxinbao' (18,322.

Esters
Esters play an important role in the formation of the aroma profile of A. arguta [22,23,38].Table S2 shows that esters are the most abundant types of volatile compounds detected in each variety, and some important esters, such as ethyl acetate, butyl acetate, methyl isobutyrate, and isopentyl acetate, can give A. arguta fruits a strong fruity and floral aroma.The highest content of esters was found in 'Lvbao' (11,145

Alcohols
The content of alcohols in each variety varied from 1607.69 µg/kg to 3639.13 µg/kg, with the highest content found in 'Tianxinbao' (3639.13

Ketones
The content of ketones ranged from 1281.44 µg/kg to 2499.92 µg/kg, accounting for 7.54-18.23% of the total volatile components, with the highest content in 'Cuiyu' and the lowest in 'Kuilv'.The ketones detected in the fruits of the 10 A. arguta varieties were mainly 1-penten-3-one, 2-pentanone, and acetone, which had a green, pungent, buttery flavor [29,30].

PCA Analysis of A. arguta Fruit Aroma Substances
To analyze the differences between different A. arguta fruit samples more intuitively, we performed PCA analysis on the volatile compounds identified by HS-GC-IMS.The ten samples were well differentiated by their aroma characteristics and varieties.PC1 accounted for 42.9% of the variance, and PC2 accounted for 14.1%.The ten groups of samples showed a clear separation trend on the 2D graph, with no outliers.The samples of the same species of A. arguta fruit clustered well.The PCA results indicated significant differences in the overall aroma substances of the ten groups of samples.Figure 6 shows that 'Kuilv', 'Fenglv', 'Jialv', 'Wanlv', 'Xinlv' and 'Pinglv' were closer to each other, 'Longcheng No.2' and 'Tianxinbao' were closer to each other, while 'Lvbao' was far from the other nine varieties, suggesting significant differences in the aroma characteristics of different samples.The HS-GC-IMS technique can distinguish different varieties of A. arguta fruits and explore their characteristics.The method is fast and non-destructive.

OAV Analysis of Aroma Components of Different Varieties of A. arguta Fruit
The intensity of A. arguta fruit aroma depends on the concentration and threshold value of the aroma components.Only when the concentration is higher than the threshold value (OAV ≥ 1), the component is considered an active aroma component.We calculated that 25 volatile aroma substances with OAVs > 1 were detected in ten A. arguta fruits (Table 3).Among them, nine were aldehydes: (E)-2-hexenal, (E)-2-octenal, (Z)-4-heptenal, 1-hexanal, 1-nonanal, valeraldehyde, valeraldehyde, 3-Methyl butanal and benzaldehyde; nine were esters: ethyl butyrate, butyl isovalerate, butyl acetate, hexyl acetate, hexyl propanoate, isopentyl acetate, isobutyl acetate and methyl butyrate; four were terpenes: α-phellandrene, α-pinene, myrcene and terpinene; two were ketones: 1-penten-3-one and acetoin; and one was alcohol: 1-octen-3-ol.Although the OAV values of the key volatile compounds varied among the 10 A. arguta varieties, aldehydes generally had higher OAV values than other types of volatile compounds.Because of their lower aroma threshold, they contributed more to the aroma of A. arguta fruit even at lower concentrations.The OAVs of 1-octen-3-ol in all varieties ranged from 23.95 to 69.29, which significantly contributed to the A. arguta fruit aroma.

OAV Analysis of Aroma Components of Different Varieties of A. arguta Fruit
The intensity of A. arguta fruit aroma depends on the concentration and threshold value of the aroma components.Only when the concentration is higher than the threshold value (OAV ≥ 1), the component is considered an active aroma component.We calculated that 25 volatile aroma substances with OAVs > 1 were detected in ten A. arguta fruits (Table 3).Among them, nine were aldehydes: (E)-2-hexenal, (E)-2-octenal, (Z)-4heptenal, 1-hexanal, 1-nonanal, valeraldehyde, valeraldehyde, 3-Methyl butanal and benzaldehyde; nine were esters: ethyl butyrate, butyl isovalerate, butyl acetate, hexyl acetate, hexyl propanoate, isopentyl acetate, isobutyl acetate and methyl butyrate; four were terpenes: α-phellandrene, α-pinene, myrcene and terpinene; two were ketones: 1penten-3-one and acetoin; and one was alcohol: 1-octen-3-ol.Although the OAV values of the key volatile compounds varied among the 10 A. arguta varieties, aldehydes generally had higher OAV values than other types of volatile compounds.Because of their lower aroma threshold, they contributed more to the aroma of A. arguta fruit even at lower concentrations.The OAVs of 1-octen-3-ol in all varieties ranged from 23.95 to 69.29, which significantly contributed to the A. arguta fruit aroma.We used hierarchical analysis to cluster the volatile aroma substances with OAVs > 1 in ten A. arguta fruits and clarify the affinity of volatile flavor profiles among different samples.The heat map analysis (Figure 7a) showed that 'Lvbao' was clustered into one class alone, 'Cuiyu' and 'Longcheng No.2' clustered into another class, and 'Kuilv', 'Fenglv', 'Jialv', 'Wanlv', 'Xinlv', 'Pinglv', and 'Tianxinbao' clustered into a third group.The red color indicated that the volatile compound was highly expressed in the sample, and the blue color indicated that the volatile compound was less expressed in the sample.The content of volatile aroma substances with OAVs > 1 varied greatly among the samples.Among them, butyl acetate, isoamyl acetate, isobutyl acetate, (Z)-4-heptenal, and hexyl acetate were highly expressed in 'Lvbao'; α-phellandrene and myrcene were highly expressed in 'Wanlv'; hexyl acetate and (E)-2-octenal were highly expressed in 'Longcheng No.2'; and acetoin was highly expressed in 'Cuiyu'.
2.6.3.OPLS-DA Analysis of Volatile Compounds with OAVs > 1 OPLS-DA is a supervised discriminant analysis statistical method that can model the relationship between substance expression and samples to achieve the prediction of sample categories [50].We performed partial least squares discriminant analysis on the volatile fractions of different A. arguta fruits with OAV values greater than 1 and selected the differential volatiles with VIP > 1 and p < 0.05 as the characteristic volatiles.We screened 13 characteristic volatile substances that contributed significantly to the classification model: four aldehydes (benzaldehyde, 3-methyl butanal, valeraldehyde, (E)-2-octenal), four esters (methyl isobutyrate, ethyl butyrate, hexyl acetate, methyl butanoate), three terpenes (myrcene, α-phellandrene, terpinolene), and two ketones (acetoin, 1-penten-3-one).These substances may be important for distinguishing different varieties of A. arguta (Figure 8a).Myrcene, which had the highest VIP value, was one of the most important markers for the differences in the aroma characteristics of A. arguta fruits from different varieties.Figure 8b shows the structure of the key volatile compounds.
Molecules 2023, 28, x FOR PEER REVIEW 13 of 18 sample categories [50].We performed partial least squares discriminant analysis on the volatile fractions of different A. arguta fruits with OAV values greater than 1 and selected the differential volatiles with VIP > 1 and p < 0.05 as the characteristic volatiles.We screened 13 characteristic volatile substances that contributed significantly to the classification model: four aldehydes (benzaldehyde, 3-methyl butanal, valeraldehyde, (E)-2octenal), four esters (methyl isobutyrate, ethyl butyrate, hexyl acetate, methyl butanoate), three terpenes (myrcene, α-phellandrene, terpinolene), and two ketones (acetoin, 1-penten-3-one).These substances may be important for distinguishing different varieties of A. arguta (Figure 8a).Myrcene, which had the highest VIP value, was one of the most important markers for the differences in the aroma characteristics of A. arguta fruits from different varieties.Figure 8b shows the structure of the key volatile compounds.

Plant Materials
Ten A. arguta varieties were harvested during in September 2022 from the Actinidia arguta Resource Nursery of the Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Zuojia Town, Jilin City, Jilin Province.A. arguta fruits are shown in Figure S1.Sampling was performed by randomly selecting well-grown, medium-sized fruit trees in the resource nursery, choosing A. arguta with the same degree of exposure to light, the same size, and similar hardness and that was free of pests and diseases.The fruits were analyzed at the eating-ripe stage.Sample information is presented in Table S3.

Overview of the Sampling Sit
Actinidia arguta Resource Nursery was on a gentle slope in the mountains, using   S1.Sampling was performed by randomly selecting well-grown, medium-sized fruit trees in the resource nursery, choosing A. arguta with the same degree of exposure to light, the same size, and similar hardness and that was free of pests and diseases.The fruits were analyzed at the eating-ripe stage.Sample information is presented in Table S3.

Overview of the Sampling Sit
Actinidia arguta Resource Nursery was on a gentle slope in the mountains, using dark brown forest soil.After the soil thawed in spring, we dug a 40 cm diameter and 30 cm deep planting hole in the center of a cultivation ditch, where we planted ten different varieties of A. arguta (a perennial vine fruit plant).The seedlings' roots spread evenly in the hole.We adopted a T-shaped frame cultivation method, with a row spacing of 3.5 m × 2.0 m and a male-to-female plant ratio of 8:1.We applied fertilizer 2-3 times and removed weeds 3-4 times annually.The sampling sites are shown in Table S4.

Determination of Sugar and Acid Content
The total soluble sugar content of A. arguta juice was determined using anthrone and sulfuric acid colorimetry; the titratable acid content of A. arguta juice was determined by the NaOH neutralization titration method [4,13].Sugar-acid ratio = total soluble sugar content/titratable acid content [12].It is a comprehensive index that reflects the balance of sweetness and sourness in the fruit, which largely influences its flavor quality [39].

Detection of Organic Acids Content
The organic acid content of A. arguta was determined by high-performance liquid chromatography (HPLC) [51].
Standard preparation: The following organic acids were weighed: 0.01030 g of oxalic acid, 0.0108 g of quinic acid, 0.0103 g of malic acid, 0.0100 g of shikimic acid, and 0.0103 g of citric acid.Also, 0.1 mL of lactic acid was measured.The concentration of each acid was adjusted to 10 mL with the mobile phase, resulting in the following concentrations: 1.03 g/L for oxalic, malic, and citric acids; 1.08 g/L for quinic acid; 1.00 g/L for shikimic acid; and 10.61 g/L for lactic acid.Gradient dilution was performed to obtain the regression equations and correlation coefficients of the peak areas (x) versus the mass concentrations (Y) of the organic acids.The standard curves of the organic acids were plotted and are shown in Table S5.
Sample pretreatment: The sample was diluted by a factor of two with the mobile phase, filtered through a 0.22 µm microporous filter membrane, and reserved for injection.
Chromatographic conditions: A C18-XT column (4.6 mm × 250 mm, 5 µL) was used at a column temperature of 25 • C and a detection wavelength of 210 nm.The mobile phase was aqueous phosphoric acid (pH 2.3) with a flow rate of 0.3 mL/min and an injection volume of 10 µL.

Detection of Volatile Compounds
The volatile substances of A. arguta fruit were determined by headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS), referring to the previously published literature [28].Sample treatment: Take 3 g of A. arguta fruit homogenate in a 20 mL headspace vial, and add 20 µL of 10 ppm internal standard (4-methyl-2-pentanol).The internal standard concentration was 198 ppb, the signal peak volume was 478.01, and the intensity of each signal was approximately 0.414 ppb.Substances were qualitatively analyzed in terms of C4-C9 ketones to calculate retention indices, using the software's builtin NIST and IMS databases.The analytical conditions and gas chromatographic conditions are shown in Tables S6 and S7.The quantitative calculation of volatile compounds is as follows: C i is the calculated mass concentration of any volatile component, in µg/kg, C is is the mass concentration of the internal standard (4-Methyl-2-pentanol), in µg/kg, and A i /A is is the volume ratio of any signal peak to the signal peak of the internal standard.

Odor Activity Value (OAV) Calculation
The contribution of the overall aroma of A. arguta fruit fruit was evaluated using the odor activity value (OAV).The OAV was calculated using the formula [13,28,52].

OAV =
Cx OTx OAV is the aroma activity value; Cx is the concentration of volatile compound x (µg/kg); and OTx is the aroma threshold of volatile compound x in water (µg/kg), which is mainly referred to in the Compendium of Compound Aroma Thresholds (Second Edition) and the aroma thresholds of compounds reported in the literature [48,49].The OAV was calculated by dividing the concentration of volatile compounds by the odor threshold.Volatile compounds with OAV > 1 were considered aroma-active and played an important role in developing the aromatic properties of A. arguta fruit.

Data Processing and Statistical Analyses
Each measurement was performed in triplicate; the data are presented as mean ± standard deviation and differences between groups were deemed significant at p < 0.05.The data were analyzed using Excel 2016 and SPSS 27.0 for ANOVA and significance testing.Figure 1 was created using Origin pro2021.OPLS-DA and VIP value analysis were conducted using Simca14.1.Heat map analysis, PCA, and correlation analysis were performed using the omicshare tool.(https://www.omicshare.com/tools,accessed 8 August 2023).

Conclusions
Actinidia arguta is a fruit crop with high nutritional and economic value, but its flavor quality is affected by various factors.We analyzed the flavor components and their variation among different varieties of Actinidia arguta, and identified the key compounds that contribute to the unique aroma of this fruit: we measured the total soluble sugars, titratable acids, organic acids and volatile compounds in the fruits of ten Actinidia arguta varieties harvested in 2022 in Zuojia Town, Jilin City, China.We found that each variety had a distinctive flavor.'Lvbao' and 'Tianxinbao' had the highest sugar to acid ratio and the highest total volatile compounds, indicating their superior fruit flavor.High-performance liquid chromatography (HPLC) was used to determine the content of organic acids.The results showed that citric acid, quinic acid and malic acid were the major organic acids, with 'Fenglv' having the highest total organic acid.
Headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) was used to determine the volatile compounds in different varieties of Actinidia arguta fruits, and a total of 81 volatile compounds were identified, including 24 esters, 17 alcohols, 23 aldehydes,

Molecules 2023 , 18 Figure 2 .
Figure 2. Heat map analysis of organic acids in fruits of different A. arguta varieties.

Figure 2 .
Figure 2. Heat map analysis of organic acids in fruits of different A. arguta varieties.

Figure 5 .
Figure 5. Histogram of volatile aromatic compounds in fruits of different A. arguta varieties.

Figure 5 .Figure 5 .
Figure 5. Histogram of volatile aromatic compounds in fruits of different A. arguta varieties.

3 .
Materials and Methods 3.1.Materials and Reagents 3.1.1.Plant Materials Ten A. arguta varieties were harvested during in September 2022 from the Actinidia arguta Resource Nursery of the Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Zuojia Town, Jilin City, Jilin Province.A. arguta fruits are shown in Figure

Table 2 .
Comparison of organic acids in fruit samples of different A. arguta varieties.

Table 3 .
OAV analysis of the main aroma compounds of in fruits of different A. arguta varieties.