Enzyme extraction method and antioxidant activity of polysaccharides from red clover

doi:10.11686/cyxb2023260

Abstract

Abstract:

Red clover （Trifolium pratense） is a resource-rich perennial leguminous forage species that is cultivated worldwide. Its polysaccharide components have good hypoglycemic and antioxidant properties， and have potential applications in the development of new， high-value products. The polysaccharides can be extracted using enzymes， and milder extraction conditions are conducive to retaining their beneficial properties. In this study， the effects of enzymatic hydrolysis time， enzyme dosage， enzyme ratio， and enzymatic hydrolysis temperature on the extraction of red clover polysaccharides were investigated. The extraction conditions were optimized and screened by orthogonal experiments. The results showed that the best enzyme extraction method was as follows： enzymatic hydrolysis time of 90 min， enzyme dosage of 1.0%， enzymatic hydrolysis temperature of 60℃， and enzyme ratio papain∶cellulase∶pectinase of 7∶2∶2. The crude polysaccharide content in red clover was 8.85%. We compared the enzyme extraction method with two other methods； ultrasonic extraction and hot water extraction. Red clover polysaccharides were extracted using all three methods， and their physical and chemical properties and antioxidant activity were compared. All three polysaccharide extracts showed antioxidant activity， with stronger abilities to scavenge O₂^·- free radicals than that of the positive control， vitamin C. The molecular weight ranges of polysaccharide samples prepared using the different extraction methods were significantly different， and the enzyme extract and hot water extract were more concentrated than the ultrasonic extract. The three polysaccharide samples were composed of eight types of monosaccharides， among which glucose， galactose， and arabinose had relatively high molar ratios， accounting for approximately 3/4 of the monosaccharide components. The proportions of monosaccharides varied widely among the samples， and the molar percentage of glucose was highest in the enzyme-extracted polysaccharides （1.6 times that in hot water-extracted polysaccharides and 1.4 times that in ultrasonically extracted polysaccharides）.

Key words: red clover, polysaccharides, enzyme extraction, process optimization, antioxidant activity

Zhao-ben QI, Xiao-yan REN, Yi-tong LI, Jin-yun MA, Quan LIU. Enzyme extraction method and antioxidant activity of polysaccharides from red clover[J]. Acta Prataculturae Sinica, 2024, 33(6): 105-115.

Figures/Tables 10

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水平 Levels	A	B	C	D
水平 Levels	酶解时间 Enzymatic hydrolysis time （min）	酶用量 Enzyme dosage （%）	酶解温度 Enzymatic hydrolysis temperature （℃）	酶比例 Enzyme ratio
1	80	0.7	50	3∶1∶1
2	90	0.9	60	7∶2∶2
3	100	1.0	65	4∶1∶1

水平 Levels	A	B	C	D
水平 Levels	酶解时间 Enzymatic hydrolysis time （min）	酶用量 Enzyme dosage （%）	酶解温度 Enzymatic hydrolysis temperature （℃）	酶比例 Enzyme ratio
1	80	0.7	50	3∶1∶1
2	90	0.9	60	7∶2∶2
3	100	1.0	65	4∶1∶1

序号 Serial number	A	B	C	D	多糖含量 Polysaccharides content （%）
序号 Serial number	酶解时间 Enzymatic hydrolysis time （min）	酶用量 Enzyme dosage （%）	酶解温度 Enzymatic hydrolysis temperature （℃）	酶比例 Enzyme ratio	多糖含量 Polysaccharides content （%）
1	1	1	1	1	6.79
2	1	2	2	3	8.83
3	1	3	3	2	6.25
4	2	1	2	2	8.33
5	2	2	3	1	5.84
6	2	3	1	3	8.42
7	3	1	3	3	3.96
8	3	2	1	2	7.24
9	3	3	2	1	8.22
K₁	21.87	19.08	22.45	20.85	—
K₂	22.59	21.91	25.38	21.82	—
K₃	19.42	22.89	16.05	21.21	—
R	3.17	3.81	9.33	0.97	—

序号 Serial number	A	B	C	D	多糖含量 Polysaccharides content （%）
序号 Serial number	酶解时间 Enzymatic hydrolysis time （min）	酶用量 Enzyme dosage （%）	酶解温度 Enzymatic hydrolysis temperature （℃）	酶比例 Enzyme ratio	多糖含量 Polysaccharides content （%）
1	1	1	1	1	6.79
2	1	2	2	3	8.83
3	1	3	3	2	6.25
4	2	1	2	2	8.33
5	2	2	3	1	5.84
6	2	3	1	3	8.42
7	3	1	3	3	3.96
8	3	2	1	2	7.24
9	3	3	2	1	8.22
K₁	21.87	19.08	22.45	20.85	—
K₂	22.59	21.91	25.38	21.82	—
K₃	19.42	22.89	16.05	21.21	—
R	3.17	3.81	9.33	0.97	—

因素 Factors	离差平方和 Sum of squares of deviations	自由度 Degree of freedom	均方 Mean square	F值 F value	显著性 Significance
A	5.553	2	2.777	29.500	P<0.01
B	7.819	2	3.910	41.540	P<0.01
C	45.545	2	22.773	241.940	P<0.01
D	0.474	2	0.237	2.520	P>0.05