3.1 Screening of pea varieties with sprout characteristics
Seed germination refers to the process in which the embryo breaks through the seed coat after the dry seed absorbs water and under suitable environmental conditions, completes the transition from a dormant state to a physiologically active state, and grows into an autotrophic seedling. A total of 34 pea seeds were germinated, and the growth index after harvest was shown in Fig. 1. The results showed that the total weight of pea sprouts varied from 15.59 g to 32.96 g, among which Bawan 1 was the heaviest and Suwan 4 was the lightest. The average total weight was 23.97g, and 18 varieties were higher-than-average (Fig. 1(a)). Pea sprouts are cotyledon-soil-retaining sprouts, and the edible part of pea sprouts is usually epicotyl, which is also the most valuable part. It was found that the edible portion fresh weight of 34 pea sprouts varied from 3.45 to 13.10 g. The heaviest and lightest varieties were 9618-2 and Suwan 4, respectively, and 16 varieties had a higher-than-average fresh weight of the edible portion of pea sprouts (7.99 g) (Fig. 1(b)). Figure 1 (c) showed that the edible rate of pea sprouts varied from 21.66 to 46.37%, among which the varieties with the highest and the lowest edible rates were Chengduzhushawan and 16WDS007, respectively, and 14 varieties had higher-than-average edible rate (33.31%). The root weight of the 34 pea sprouts varied from 3.77 to 12.18 g. The heaviest variety was Bawan 1 and the lightest was Dingwan 6, twenty-two varieties were below the average root weight (6.48 g) (Fig. 1(d)). The variation range of pea sprouts’ epicotyl length was 3.54 ~ 20.66 cm, among which the longest and shortest cultivars Chengduzhushawan and 16WDS007, respectively, and 14 cultivars were higher than the average epicotyl length (10.91cm) (Fig. 1 (e)). It can be seen from Fig. 1(f) that the variation in stem diameter of pea sprouts ranged from 1.37 to 2.05 mm, with the thickest and thinnest stem varieties being Caoyuan 28 and Chengduzhushawan, respectively, and 15 varieties were wider than the average stem diameter (1.68 mm). The variation range of pea sprouts’ water content was 89.02 ~ 92.53%. The highest and lowest varieties were Dingwan 8 and 16WDS007, respectively, and 17 varieties were higher than the average water content (90.74%) (Fig. 1(g)). Figure 1 (h) showed that the dry matter content of 34 pea sprouts varied from 7.47 to 10.98%. The highest and lowest varieties were 16WDS007 and Dingwan 8, respectively. The dry matter content of 17 varieties was higher than the average (9.26%). Output ratio is one of the critical factors for screening unique varieties of sprouts. It can be seen from Fig. 1 (i) that the variation range of pea sprouts’ output ratio was 0.91–3.03, and the greatest variety was Taiwanxiaobaihua, which was 3.32 times higher than that of Suwan 4, sixteen varieties were greater than the average output ratio (1.82).
3.2 TPC, TFC and AAs of pea sprouts
To screen pea varieties with health-care functions, the TPC, TFC, and AAs of 34 pea sprouts were determined (Fig. 2). Figure 2 (a) showed that the TPC of pea sprouts ranged from 2.33 ~ 4.61mg GAE/g DW, with the highest and lowest content of 16WDS021 and Lvshangu, respectively. Fifteen varieties showed higher TPC than the average level (3.46 mg GAE/g DW). As shown in Fig. 2(b), the TFC varied from 7.14 to 13.54 mg RUT/g DW among 34 pea sprouts, with Zhongwan 6 had the highest and Lvshangu had the lowest TFC, respectively. Specifically, 15 varieties were higher than the average TFC level (9.93mg RUT/g DW). It was found that the ABTS free radical scavenging capacity of pea sprouts ranged from 46.67 to 65.65 µM TE/g DW, and 16 varieties were higher than the average level (53.33 µM TE/g DW) (Fig. 2(c). The FRAP of pea sprouts varied from 14.95 to 31.26 mM Fe2+/g DW, and 19 varieties were higher than the average ferrous reduction capacity (22.10 mM Fe2+/g DW) (Fig. 2 (d)). The varieties with the strongest and weakest antioxidant capacity were 16WDS021 and Wuxudoujian 2, respectively.
In addition, compared with the pea seeds in our previous study (Zhao et al. 2020), TPC, TFC, and AAs of germinated pea sprouts were significantly improved (Fig. 3), which has the potential to be developed into functional foods, which is of great significance for promoting human health.
3.3 Correlation analysis among various indexes of pea sprouts
Correlation analysis was carried out on the growth index, TPC, TFC, and AAs of 34 pea sprouts (Fig. 4). According to the correlation heat map, significantly high linear correlation coefficients were reported among TPC, TFC, ABTS, and FRAP of pea sprouts. The results showed that TPC was positively correlated with TFC (r1 = 0.7563, p < 0.0001), ABTS free radical scavenging activity (r2 = 0.6387, p < 0.0001) and FRAP (r3 = 0.5174, p < 0.0001). In addition, TPC had a significant positive correlation with ABTS free radical scavenging activity (r = 0.711, p < 0.0001) and FRAP (r = 0.7697, p < 0.0001). However, ABTS free radical scavenging ability and FRAP also had a significant positive correlation (r = 0.7697, p < 0.0001).
The correlation among the growth indexes of pea sprouts was strong. Results showed that the total weight was significantly positively correlated with the fresh weight of edible parts (r = 0.5778, p༜0.0001) and root weight (r = 0.6940, p༜0.0001); The fresh weight of edible parts was related to epicotyl length (r = 0.8560, p༜0.0001), edible rate (r = 0.8187, p༜0.0001), water content (r = 0.6593, p༜0.0001) and stem diameter (r=-0.3714, p༜0.0001). Root weight was negatively correlated with epicotyl length (r=-0.4577, p < 0.0001), edible rate (r=-0.5728, p < 0.0001) and positively correlated with stem diameter (r = 0.5678, p < 0.0001). Meanwhile, a significant negative correlation was reported between stem diameter and edible rate (r=-0.6435, p < 0.0001). The epicotyl length of pea sprouts had a significant negative correlation with stem diameter (r=-0.6925, p༜0.0001) and dry matter content (r=-0.6194, p༜0.0001), and was significantly positive correlation with the edible rate (r = 0.9443, p༜0.0001) and water content (r = 0.6195, p༜0.0001). The edible rate had a significant positive correlation with water content (r = 0.6420, p < 0.0001), and a significant negative correlation with dry matter content (r=-0.6420, p < 0.0001). The output ratio of pea sprouts was negatively correlated with root weight (r=-0.4262, p༜0.0001), stem diameter (r=-0.6412, p༜0.0001) and dry matter content (r=-0.6415, p༜0.0001), and positively correlated with fresh weight of edible parts (r = 0.6957, p༜0.0001), epicotyl length (r = 0.7703, p༜0.0001) and edible rate (r = 0.8784, p༜0.0001).
In addition, there were also significant correlations between phenolic contents, antioxidant activities, and growth indicators. The pea sprouts with higher stem diameter and dry matter content had richer TPC, TFC, and stronger AAs.
3.4 Principal component analysis (PCA) of the variables of pea sprouts
Based on the significant correlation among the variables of pea sprouts, PCA was conducted to investigate the impact of individual traits. The chi-square test showed that the significance level was 0.000 (less than 0.01), which indicated that each index had a high correlation and was suitable for PCA analysis. It was found that the total variance contribution rate of the three principal component factors reached 84.50%. Among them, the variance contribution rates of principal component 1 (PC1), principal component 2 (PC2), and principal component 3 (PC3) were 54.98%, 17.06%, and 12.46%, respectively, and the eigenvalues were 7.15, 2.22, and 1.62, respectively (Table 2).
Table 2
Contribution percentage and major characters associated with the first three principal components of 34 pea varieties and their eigenvectors
X loadings | PC1 | PC2 | PC3 |
Explained proportion of variation (%) | 54.98 | 17.06 | 12.46 |
Cumulative proportion of variation (%) | 54.98 | 72.04 | 84.5 |
Traits | Eigenvectors |
X1 | -0.127 | 0.84 | 0.115 |
X2 | -0.202 | 0.912 | 0.161 |
X3 | -0.248 | 0.852 | -0.059 |
X4 | -0.459 | 0.75 | 0.088 |
X5 | 0.301 | -0.004 | 0.92 |
X6 | 0.891 | -0.24 | 0.271 |
X7 | -0.3 | 0.176 | 0.872 |
X8 | 0.83 | -0.422 | -0.164 |
X9 | -0.437 | 0.533 | 0.528 |
X10 | 0.884 | -0.299 | -0.284 |
X11 | 0.835 | -0.311 | 0.076 |
X12 | -0.835 | 0.31 | -0.076 |
X13 | 0.884 | -0.013 | -0.242 |
In each extracted principal component, there were some traits make a significant contribution. Among them, PC1 mainly reflected the sprouting characteristics of pea sprouts, in which the fresh weight of edible parts, epicotyl length, edible rate, water content, dry matter content, and output ratio had higher loads, which could be used as important reference indicators; PC2 embodied the nutritional and health care function of sprouts. The traits with the positive and high load in the eigenvector included TPC, TFC, ABTS free radical scavenging power, and FRAP; total weight and root weight played a major role in PC3 (Table 2).
3.5 Clustering analysis
The phenotypic traits of 34 pea sprouts were clustered using the phylogenetic clustering method, and the classification distance ranged from 0 to 25. Figure 5 showed that the tree diagram of the system divided all pea sprouts into four groups. Among them, Suwan 4 was the first group, and its sprouts had poor growth, low phenolic content, and weak antioxidant capacity. The second group included three pea sprouts: ZDW, Qingwandou, and Bawan 1, which root weights were the heaviest among the 34 pea sprouts. The third group consists of 9 pea sprouts with thicker stems and higher TFC: 16WDS018, 16WDS021, Caoyuan 28, Zhongwan 6, 16WDS002, Longwan 3, 16WDS007, 16WDS017, and Chengwan 268-1. The remaining 21 pea sprouts were classified into the fourth group, most of which were varieties with better growth and sprouting characteristics.
3.6 Comprehensive value evaluation of pea sprouts
In order to objectively evaluate the sprout characteristics and functional quality of pea sprouts and screen out the best pea sprouts varieties, the grey relational analysis was used to assess the comprehensive value of 34 pea sprouts (Table S1). The results showed that the cultivar significantly affected the sprout and functional properties of pea sprouts.
The heat map of the influence of each factor on the final result in the grey theory system explained the differences among the varieties of pea sprouts, and more directly reflected the contribution of each index to the final result, which was helpful to explain the difference between the weighted correlation degree of the samples. The heat map for 9618-2 possessed the largest number of green blocks, mainly including TFC, fresh weight of edible part, epicotyl length, edible rate, water content, and output ratio, which indicated that the above indexes of this variety were closer to the ideal variety, that is the main reason why this variety ranked in the forefront. However, Ewan 3 showed more orange and red blocks, with the lowest hierarchy (Fig. 6). The ranking of all pea sprout varieties was shown in Table 3. Generally speaking, 9618-2 (0.819) was the closest pea sprout variety to the ideal sample, followed by Dingwan 4 (0.815), Dingwan 8 (0.805), Baiwandou (0.797), and Taiwanxiaobaihua (0.797).
Table 3
The gray relational grade values of 34 pea sprouts
Code | Variety | WGRD | Code | Variety | WGRD |
1 | 9618-2 | 0.819 | 18 | Dingwan 1 | 0.751 |
2 | Dingwan 4 | 0.815 | 19 | 16WDS017 | 0.751 |
3 | Dingwan 8 | 0.805 | 20 | Chengwan 11 | 0.750 |
4 | Baiwandou | 0.797 | 21 | 16WDS007 | 0.749 |
5 | Taiwanxiaobaihua | 0.797 | 22 | Chengwan 10 | 0.748 |
6 | Chengduzhushawan | 0.795 | 23 | Wuxudoujian 1 | 0.746 |
7 | Dingwan 5 | 0.793 | 24 | Lvshangu | 0.745 |
8 | 16WDS021 | 0.791 | 25 | Chengwan 8 | 0.745 |
9 | Caoyuan 224 | 0.778 | 26 | Longwan 3 | 0.744 |
10 | Caoyuan 28 | 0.775 | 27 | ZDW | 0.740 |
11 | Chengwan 268-1 | 0.772 | 28 | Wushanzihuawan | 0.739 |
12 | Zhongwan 6 | 0.768 | 29 | Qingwandou | 0.739 |
13 | Dingwan 6 | 0.765 | 30 | 16WDS023 | 0.735 |
14 | Xinnongcun | 0.764 | 31 | Bawan 1 | 0.734 |
15 | 16WDS018 | 0.763 | 32 | 16WDS010 | 0.724 |
16 | 16WDS002 | 0.754 | 33 | Suwan 4 | 0.718 |
17 | Wuxudoujian 2 | 0.752 | 34 | Ewan 3 | 0.715 |