HIT R&D Institute

Research on Antioxidation

Research on the anti-oxidant effect and antibacterial activity of blueberry anthocyanins:

The oxidation resistance of the blueberry anthocyanin is evaluated by testing the ability of blueberry anthocyanin in eliminating superoxide anion free radicals, hydroxyl radicals (·OH), DPPH free radicals (DPPH·). Its antibacterial activity is assessed according to its inhibiting effect on E.coli. The result shows that the higher the blueberry anthocyanin concentration is, the stronger ability the blueberry anthocyanin has in eliminating superoxide anion free radicals, OH free radicals and DPPH free radicals. Additionally, its oxidation resistance is stronger than ascorbic acid of the same concentration; the blueberry anthocyanin can inhibit the growth of E.coli in an effective way. Hence, the blueberry anthocyanin has very good anti-oxidant activity and certain antibacterial activity.

Anthocyanin, also called anthocyanidin, is a kind of natural water-soluble pigment. As a flavonoids compound, it exists in the form of glucoside and its structural unit is α-phenyl benzopyrane positive ion. Blueberry, also called huckleberry, belongs to perennially deciduous or evergreen shrub of vaccinium of Ericaceae. There are a wide variety of blueberries and they are rich in anthocyanin. In the test, we use the wild fresh blueberry which is rich in anthocyanin. At present, plenty of researches have been done on anthocyanin of the blueberry at home and broad and there are conventional extraction procedures. Based on the effective extraction of anthocyanin from blueberry, the research aims to explore its anti-oxidant and antibacterial effectiveness to accelerate the market development of blueberry anthocyanin.

1 Materials and Methods

1.1 Instruments and Equipment

Wild blueberries from the Lapland Region of Finland, UV spectrophotometer, electric-heated thermostatic water bath, electric heating air-blowing drier, drying cabinet clean bench

1.2 Blueberry Anthocyanin Agent

The blueberry anthocyanin agent is prepared by our laboratory. The production process is as follows: soak and extract smashed fresh blueberry in ethanol, get anthocyanin extraction solution after filtration and centrufugation; absorb by polyamide resin, elute the solution by 60% ethanol, get anthocyanin purified liquid after rotary evaporation, get blueberry anthocyanin lyophilized powder after vacuum drying at a low temperature. Prepare 100mg/ml blueberry anthocyanin agent with ultrapure water at last.

1.3 Experimental Method

1.3.1 Measurement of Anti-oxidant Activity of the Blueberry Anthocyanin

1.3.1.1 Measurement of the Free Radical Scavenging Capacity of Superoxide Anion

Put 4.5ml 50m MpH 8.2 Tris-HCl buffer solution in 25°C water for 20 minutes, add 1ml 100mg/ml blueberry anthocyanin agent sample and 0.5ml 25m M pyrogallic acid solution. Blend equally then put in 25°C water for 5 minutes, add 1ml 8M HCI, stop the reaction. Take Tris-HCl Buffer as a reference, double distilled water as the blank reference and ascorbic acid solution of the same concentration as the reference. Repeat each step for three times, test the absorbance (A) at 425 nm. Take the average value to get the scavenging rate.

1.3.1.2 Measurement of the Scavenging Capacity of the hydroxyl radicals (·OH)

Take 1ml 9m M FeSO4, 1ml 9m M salicylic acid ethanol solution, 1 ml blueberry anthocyanin sample, add 1ml 8.8m M H2O2. Put the solution in 37°C water for 30 minutes. Take distilled water as a reference, distilled H2Oas the blank reference, and ascorbic acid solution of the same concentration as the reference. Repeat each step for three times, test the absorbance (A) at 510 nm. Take the average value to get the scavenging rate.

1.3.1.3 Measurement of the Scavenging Capacity of the DPPH Free Radical (DPPH·)

Prepare 0.04mg/ml DPPH solution with anhydrous ethanol, store it away from direct sunlight. Mix 2ml DPPH solution and 2ml blueberry anthocyanin sample adequately, stand for 30 minutes. Take double distilled water as the blank reference and ascorbic acid solution of the same concentration as the reference. Repeat each step for three times, test the absorbance (A) at 515 nm. Take the average value to get the scavenging rate.

1.3.2 Measurement of Bacteriostatic Activity of Blueberry Anthocyanin

1.3.2.1 Sketch of E.coli Growth Curve

Prepare 1.25mg/ml blueberry anthocyanin bacteria solution with total plate count of 103 to 105 per ml. Take the solution for shaking culture at 37°C. Sample to test A532 per hour, draw the growth curve of E.coli.

1.3.2.2 Measurement of Blueberry Anthocyanin’s Sensitivity to E.coli

Add 0, 12.5, 25, 50μl blueberry anthocyanin samples to 1.5ml EP tubes respectively, add 5μl E.coli and LB medium to 1ml. Take the sample for shaking culture at 37°C for 1.5 hours. Add 100μl bacteria liquid to LB agar premixed powder, incubate at steady 37°C for 12 hours. Use multiple proportion dilution to test minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Repeat each step for three times.

2 Results and Discussions

2.1 Anti-oxidant Activity of Blueberry Anthocyanin

There are many ways to measure the anti-oxidant activity of blueberry anthocyanin. In this research, we determine the anti-oxidant effectiveness of blueberry anthocyanin by measuring the scavenging activity of the superoxide anion free radical, hydroxyl radicals, DPPH free radical.

2.1.1 Scavenging Effect on the Superoxide Anion Free Radical


Figure 1 Comparison between Blueberry Anthocyanin and Ascorbic Acid upon Superoxide Anion Free Radical Scavenging Abilities

The blueberry anthocyanin has a strong ability to scavenge superoxide anion free radicals and the ability becomes stronger with the increase of concentration in the agent. When the concertation is 0.3mg/ml, the scavenging rate reaches 80%.

2.1.2 Scavenging Effect on the Hydroxyl Radicals


Figure 2 Comparison between Blueberry Anthocyanin and Ascorbic Acid upon OH Scavenging Abilities

The blueberry anthocyanin has a stronger ability over ascorbic acid in scavenging hydroxyl radicals.

2.1.3 Scavenging Effect on the DPPH Free Radical


Figure 3 Comparison of DPPH Scavenging Abilities between Blueberry Anthocyanin and Ascorbic Acid

The blueberry anthocyanin has an outstanding ability to scavenge DPPH free radicals and its scavenging rate is higher than that of ascorbic acid when the concentration is higher than 0.005mg/ml in the agent. When the drug concentration is lower than 0.005mg/ml, however, the ascorbic acid has a better performance.

2.2 Blueberry Anthocyanin’s Effect on the E.coli Growth Curve


Figure 4 Blueberry Anthocyanin’s Effect on the E.coli Growth Curve

The normal logarithmic phase of E.coli is 3 to 8 hours. In LB containing 1.25mg/ml blueberry anthocyanin, the E.coli growth curve shows a noticeable change: after the blueberry anthocyanin agent has worked for 4 hours, the bacteria decrease in quantity. After 6 hours, the bacteria begin to enter the decline phase rather than achieve the summit of growth as normal. The result indicates blueberry anthocyanin mainly inhibits the bacteria split of E.coli in the logarithmic phase.

2.3 Blueberry Anthocyanin’s Effect on Suppressing the Growth of E.coli


Figure 5 Blueberry Anthocyanin’s Effect on Suppressing the Growth of E.coli

The blueberry anthocyanin has a notable effect on inhibiting the growth of E.coli and such effect becomes stronger with the increase of blueberry anthocyanin concentration in the agent. When the blueberry anthocyanin sample has a concentration of 5mg/ml, no E.coli bacterial colony is determined and 100% E.coli. is inhibited from growing. By applying multiple proportion dilution, we get MIC and MBC of E.coli under the influence of blueberry anthocyanin, and they are 0.734 (mg/ml) and 1.36 (mg/ml) respectively.

3 Conclusions

The ability of blueberry anthocyanin to scavenge superoxide anion free radicals, hydroxyl radicals and DPPH free radicals is much greater than that of the ascorbic acid with the same concentration. Such ability becomes stronger with the increase of concentration and it has a good effect to inhibit the growth of E.coli. Accordingly, it can be developed and applied in food industry as a natural anti-oxidant.


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