Separation and screening of antioxidant peptides from Scomberomorus niphonius based on nano flow liquid chromatography

doi:10.3724/SP.J.1123.2020.04019

Abstract

Abstract:

As a rich source of high activity antioxidant peptides, Scomberomorus niphonius is a key marine natural product with a high processing value. Due to the high complexity of fish tissues, high recovery extraction and high efficiency screening of the active antioxidant peptide species have become the major challenges for the research and development of preparation and separation techniques. Due to the high specificity of hydrolytic enzymes, when different types of enzymes are used for the hydrolysis of fish tissues, the resultant active peptides may have significant differences in chemical structures, biological functions, and physical activities. In this study, in order to obtain antioxidant peptides with high activity and functionality, defatted visceral powder of Scomberomorus niphonius was used as a raw material for sample preparation. Five hydrolytic enzymes (flavor protease, trypsin, acid protease, neutral protease, and alkaline protease) were selected and investigated for their hydrolyzing efficiencies in visceral solutions of Scomberomorus niphonius, according to their optimum hydrolyzing conditions. The diphenyl bitter hydrazine radical (DPPH·) scavenging rate, hydroxyl radical (·OH) scavenging rate, and degree of hydrolysis (DH) were adopted as indicators for hydrolytic enzyme selection and optimization. The data suggested that, among all the hydrolytic enzymes investigated, trypsin presented the best scavenging capabilities for both DPPH· and ·OH species, with scavenging rates of 88.93%±0.82% for DPPH· and 53.09%±0.73% for ·OH, respectively. Based on the single-factor test results, the DPPH· scavenging rate was further utilized as an indicator and was found to depend on the hydrolytic enzyme quantity, hydrolyzation temperature, and hydrolyzation time. According to the Box-Behnken center composed experimental design, a triple factor and triple level response surface method was adopted for further optimization of antioxidant peptide preparation from Scomberomorus niphonius viscera. The preparation method was systematically optimized, and as a result, a degree of hydrolysis of 23.66%, DPPH· scavenging rate of 93.78%, and ·OH scavenging rate of 62.59% were achieved. High performance nano flow liquid chromatography is a new generation micro scale liquid phase separation technique that has the advantages of low sample requirement, low solvent consumption, and high efficiency. In this study, we applied this method to marine natural product purification. In order to screen the most suitable chromatographic stationary phase for the separation and analysis of active antioxidant peptides from Scomberomorus niphonius viscera, a nano flow reversed-phase C18 column (15 cm×100 μm, 5 μm, 30 nm) and a strong cation exchange column (15 cm×100 μm, 5 μm, 100 nm) were investigated. A nano flow high performance liquid chromatography platform with a 1∶1000 splitting ratio was adopted for this study. Using the enzyme-hydrolyzed solution from Scomberomorus niphonius viscera as the sample, following filtration, the antioxidant peptides were separated, collected, freeze-dried, and finally tested for their antioxidant capability. The results showed that the strong cation exchange phase was more suitable for antioxidant peptide isolation and purification from Scomberomorus niphonius viscera, and an antioxidant peptide component with high activity was successfully screened. During the activity test, the 50% inhibition concentration (IC₅₀) value of the DPPH· scavenging capability of this peptide was 0.672±0.051 mg/mL, which was 13.6 times higher than the activity value before the peptide species was purified. The current study for the first time reports the application of high performance nano flow liquid chromatography in the purification and analysis of antioxidant peptides from a marine natural product source and also demonstrates the effectiveness and future prospect of the use of nano flow ion exchange chromatography for high performance separation and high efficiency screening of antioxidant peptides.

Key words: nano flow liquid chromatography, antioxidant peptides, Scomberomorus niphonius, marine natural product

CLC Number:

O658

XUE Yaru, GUO Rui, ZHANG Bo. Separation and screening of antioxidant peptides from Scomberomorus niphonius based on nano flow liquid chromatography[J]. Chinese Journal of Chromatography, 2020, 38(12): 1431-1439.

Figures/Tables 12

Table 1 Hydrolysis conditions of various proteases

Type of enzyme	Enzyme activity/(u/g)	Temperature/ ℃	pH	Time/ h
Flavor protease	15000	50	7	8
Trypsin	250000^*	37	7	8
Acid protease	800000	50	3	8
Neutral protease	200000	50	7	8
Alkaline protease	200000	50	8	8

Table 2 Factors and levels of response surface analysis

Level	Factors
Level	A(Addition of enzyme/%)	B(Hydrolysis temperature/ ℃)	C(Hydrolysis time/h)
-1	0.2	35	2.0
0	0.4	40	2.5
1	0.6	45	3.0

Fig. 1 Effect of various proteases on degree of hydrolysis, DPPH· and ·OH scavenging ability (n=3)

Table 4 Analysis of variance for regression equation model

Source	Sum of squares	df	Mean square	F value	p-value (Prob>F)	Significance
Model	220.21	9	24.47	77.59	<0.0001	**
A	9.22	1	9.22	29.25	0.001	**
B	8	1	8	25.37	0.0015	**
C	2.11	1	2.11	6.7	0.0361	*
AB	4.35	1	4.35	13.79	0.0075	**
AC	1.04	1	1.04	3.3	0.1122
BC	14.18	1	14.18	44.95	0.0003	**
A²	59.65	1	59.65	189.17	<0.0001
B²	84.94	1	84.94	269.36	<0.0001
C²	19.45	1	19.45	61.66	0.0001
Residual	2.21	7	0.32
Lack of fit	1.27	3	0.42	1.79	0.2879
Pure error	0.94	4	0.24
Cor total	222.41	16

Table 3 Design and results of Box-Behnken test

Test	A	B	C	Y(DPPH· scavenging ability/%)
1	0.2	35	2.5	84.73
2	0.6	35	2.5	85.16
3	0.2	45	2.5	88.91
4	0.6	45	2.5	85.17
5	0.2	40	2	88.94
6	0.6	40	2	87.32
7	0.2	40	3	90.37
8	0.6	40	3	86.71
9	0.4	35	2	83.95
10	0.4	45	2	89.62
11	0.4	35	3	89.36
12	0.4	45	3	87.50
13	0.4	40	2.5	94.04
14	0.4	40	2.5	93.71
15	0.4	40	2.5	94.51
16	0.4	40	2.5	94.03
17	0.4	40	2.5	94.95

Fig. 2 Surface and contour plots of influence for the addition of enzyme and hydrolysis temperature against DPPH· scavenging ability

Fig. 3 Surface and contour plots of influence for the addition of enzyme and hydrolysis time against DPPH· scavenging ability

Fig. 4 Surface and contour plots of influence for hydrolysis temperature and hydrolysis time against DPPH· scavenging ability

Fig. 5 nano-HPLC chromatogram of APS using C18 column Mobile phase: 60% ACN; flow rate: 0.6 mL/min; split ratio: 1∶1000; chromatographic column: C18, 15 cm×100 μm, 5 μm, 30 nm.

Fig. 6 Nano-HPLC chromatogram of APS using SCX stationary phase Mobile phase: 60% ACN; flow rate: 0.6 mL/min; split ratio: 1∶1000; chromatographic column: SCX, 15 cm×100 μm, 5 μm, 100 nm.

Fig. 7 DPPH· scavenging activity of fractions obtained on C18 column The letters indicate significant difference at 0.05 level (data=mean±standard, n=3).

Fig. 8 DPPH· scavenging activity of fractions obtained on SCX column The letters indicate significant difference at 0.05 level (data=mean±standard, n=3).

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