Radical Scavenging Ability of Soymilk Fermented with Compound Lactic Acid Bacteria towards DPPH Free Radical

Yongyan GENG, Wenjun LI, Jianming WANG

Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin 300457, China

Nowadays, it is well known that many chronic non-communicable diseases are somehow related to the reactive oxygen species which is produced during oxidation and may cause biological aging[1-2]. A free radical is a highly active atom,molecule or ion with unpaired electrons, which can cause peroxidative damage to cell and cellular membrane and consequently give occasion to disease, cancer and senility[3]. It is the key orientation of scientific research to scavenge the superfluous free radicals on account that they are directly related to human health.A variety of methods to assess radical scavenging ability in vitro have been set up. Different free radical artificial species have been used such as 2, 2-azinobis-3-ethyl benzothiazoline-6-sulfonicacid(ABTS),1,1′-diphenyl-2-picrylhydrazyl(DPPH)and N, N-dimethyl-p-phenylendiamine(DMPD), among which DPPH method was applied more commonly[4]. The scavenging of 1, 1′-diphenyl-2-picrylhydrazyl free radical (DPPH·), which shows a characteristic UV-vis spectrum with a maximum of absorbance close to 517 nm in methanol, was employed in the present paper.The addition of an antioxidant results in a decrease of absorbance proportional to the concentration and antioxidant activity of the compound itself[5].It is easy to perform and highly reproducible.Meanwhile, this method presents the advantage of using a stable and commercially available free radical and has been extensively applied on the study of free radical scavenging.

Fermented soymilk is a novel food which is produced from soybean through lactic acid fermentation. This product has the merits of low fat, high protein and active probiotics. It shows favorable nourishment and healthpromoting effect, and thus arousing widespread interest among researchers[6]. Therefore, this study inves-tigated the effects of fermentation conditions on the ability of fermented soymilk to scavenge radicals, to provide a technical and theoretical support for the research and exploitation of soybean and lactic acid bacteria associated functional food.

Materials and Methods

Materials

Raw materials and soybean were purchased from Desco Super Market.Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus bulgaricus and Streptococcus thermophilus were obtained from Key laboratory of Food Nutrition and Safety(Tianjin University of Science & Technology, Tianjin,China). Ethanol and N-hexane were provided by Tianyi Chemical Reagent Factory(Tianjin,China).1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH·)was obtained from Tianjin, China. 2-(N-morpholino) ethanesulfonic acid(MES)were bought from BBI company(America). Other analytical chemical reagents and doubled water were used throughout the experiment to prepare all solutions.

Experimental instruments

The main instruments used in the present study included UV-spectrophotometer, DHP-781 electro-heating standing-temperature cultivator(from the Medical Instrument Factory in Huangshi,Hubei),JA2003 electronic balance (from Shanghai Jingke Scientific Instrument Co., Ltd), vacuum freeze dryer (from Thermo Electronic Company), TG16-W high speed centrifuge (from Hunan Xiangyi Laboratory Instrument Development Co, Ltd.),LRH-250-A microbiological incubator(from Beijing Instrument Equipment Factory), separating refiner, steam sterilizer and thermostat water bath cauldron.

Preparation of fermented soymilk

Soymilk was prepared from food grade materials with the procedure which could resemble the process of household. Soybeans were obtained from Desco Super Market. Similarsized soybeans were chosen and a portion of 200 g were washed with water and soaked in 600 g of water for 10-12 h. Then the rehydrated beans underwent grinding with a soymilk maker. The soymilk maker cycles were set as follows: soybeans and water were heated to 85 ℃(in approximately 5 min)and once the temperature was reached, the soybeans were ground for 5 s.After this short grinding cycle, the soymilk temperature was brought up near boiling point (1 min)and then four grinding cycles were performed, each cycle lasting for 40 s with 5 s pause between cycles. After grinding, soymilk was heated at just below boiling temperature for 10 min.The hot soymilk was then immediately poured through a strainer to remove the okara, and then passed twice through a cheese cloth. After filtration the soymilk was cooled to refrigeration temperature.

Then Glucono-delta-lactone and calcium sulfate were chosen to add to the soymilk. By adding different proportions of naturalized lactobacillus suspension,the soymilk was fermented at an appropriate temperature. The last process was ripening.

Measurement of DPPH scavenging activity

Preparation of solutions MES buffer and soymilk-clotting extraction were needed to measure the DPPH scavenging activity. MES buffer was prepared by adding distilled water to a volumetlric flask which contained 4.26 g MES solid medicines till 100 ml, and then adjusting the PH to 6.0. To produce soymilk-clotting extraction, the middle part of soymilk clotting during the process of lyophilization was taken and put into a glass beaker which contained 6 ml of distilled water prior to 1 h of leaching at 25-30 ℃, in the meantime, the glass breaker was shaken occasionally. Then the sample was centrifuged at the speed 3 000 r/min for 10 min to collect the filtrating supernatant.

DPPH free radical-scavenging assay 4 ml of 0.2 mol/L MES buffer and 1 ml of soymilk-clotting extraction were added to 150 μmol/L solution of DPPH. The mixture was shaken immediately and allowed to stand at room temperature in dark for 0.5 h.The decrease in absorbance at 517 nm was measured. The numerical value was indicated by Ai. For blank control,absorbance at 517 nm(Aj)was read by replacing DPPH solution with anhydrous ethanol. In the same way,the solution of 50%ethanol was added instead of soymilk-clotting extraction for determining the absorbance of negative control (Ac). Each processing was measured three times, and the average of the three measurements was finally taken.

The DPPH free radical scavenging ability was calculated as follows:

DPPH free radical scavenging rate=[Ac-(Ai-Aj)]/Ac×100%

wherein, Acwas the A517of DPPH without soymilk-clotting extraction(negative control); Aiwas the A517of DPPH with soymilk-clotting extraction;and Ajwas the A517of soymilk-clotting extraction with absolute ethanol (blank control).

Experimental design for determining the effects of fermentation conditions on DPPH free radicals scavenging ability

To determine the effects of different starter species on the ability of fermented soymilk to scavenge DPPH free radicals, four starters Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus bulgaricus and Streptococcus thermophiles which were all cultured by soymilk were separately added at an inoculation ratio of 3%to soymilk with a soluble solid content of 8%,cultured at 37 ℃for 6 h before their DPPH free radical scavenging ability was measured.

To measure the effects of different combinations of starter species on the ability of fermented soymilk to scavenge DPPH free radicals,S.thermophiles,L.plantarum and L.helveticus were mixed at different ratios 2∶0.5∶0.5 (No.1),3∶1∶1 (No.2),2∶1.5∶1.5(No.3),2∶1∶1(No.4)and 1∶1∶1∶1(No.5)and added at total inoculation ratio of 3%to soymilk with a soluble solid content of 8%,cultured at 37 ℃for 6 h before their DPPH free radical scavenging ability was measured.

To measure the effects of fermentation temperature on the ability of fermented soymilk to scavenge DPPH free radicals, the mixture of S. thermophiles, L. plantarum and L. helveticus at 2 ∶1.5 ∶1.5 was inoculated at a ratio of 3% to soymilk with a soluble solid content of 8%, cultured respectively at 25, 30, 37, 40 and 42 ℃for 6 h before their DPPH free radical scavenging ability was measured.

To measure the effects of fermentation time on the ability of fermented soymilk to scavenge DPPH free radicals,the mixture of S.thermophiles,L.plantarum and L.helveticus(2∶1.5∶1.5)was inoculated at a ratio of 3% to soymilk with a soluble solid content of 8%,cultured at 37 ℃for 1,2,3,4,5,6 and 7 h respectively before their DPPH free radical scavenging ability was measured.

To determine the effects of soymilk solid content on the ability of fermented soymilk to scavenge DPPH free radicals, soymilk samples with soluble solid contents of 6%, 8% 10%and 12%were selected and fermented at 37 ℃for 6 h by adding 3%of the mixture of S. thermophiles, L. plantarum and L.helveticus(2∶1.5∶1.5),before their DPPH free radical scavenging ability was measured.

To investigate the effects of bacterial inoculation volume on the ability of fermented soymilk to scavenge DPPH free radicals,1%,2%,3%,4%and 5%of bacterial mixture described above was respectively inoculated to soymilk containing 8% soluble solids, which had been dissolved by adding 0.15%GDL and 0.075% calcium sulphate,cultured at 37 ℃for 5 h before their DPPH free radical scavenging ability was measured.

Finally, the DPPH scavenging rate in the soymilk, which was fermented at 37 ℃for 6 h and contained 12% soluble solids, was measured and compared with that of Baiyu lactone soymilk and bean curd to comprehensively evaluate its DPPH scavenging ability.

Results and Analysis

Effects of starter species on DPPH free radical scavenging ability

Fig.1 showed that the ability of scavenging DPPH free radicals for L. bulgaricus,L.plantarum,L.helveticus and S. thermophilus was 22.69%,32.27% , 34.36% and 32.73% , respectively. So, it could be concluded that all the four bacterial species had good ability of scavenging DPPH free radicals.However,among them L. bulgaricus exhibited poorer ability of scavenging DPPH free radicals than others.

Effects of combinations of starter species on DPPH free radical scavenging ability

As shown in Fig.2, the combinations of bacteria species showed different ability of scavenging DPPH free radicals. The DPPH free radical scavenging ability for S. thermophiles, L. plantarum and L. helveticus mixed at 2∶0.5∶0.5 (No.1),3∶1∶1(No.2),2∶1.5∶1.5 (No.3), 2∶1∶1(No.4)and 1∶1∶1∶1(No.5)was 55.3%,57.8%,65.7%, 63.4% and 57.2%,respectively. It was obviously that complex bacteria had higher DPPH scavenging ability than each single bacterium. It proved that fermentation using mixed lactic acid bacteria had positive effect on improving DPPH free radical scavenging rate. Meanwhile,the scavenging rate of the combined use of S. thermophiles, L. plantarum and L. helveticus at 2 ∶1.5 ∶1.5(No.3) was larger than at other ratios.The reason may be that the synergistic effect of these lactic acid bacteria in combination No.3 increased the strains and metabolites those possessed the ability to scavenge DPPH free radicals, while there were less effective components in other combinations.

Effects of fermentation temperature on DPPH free radical scavenging ability

As shown in Fig.3, the DPPH scavenging rate was increased at first with the increasing of fermentation temperature, reached the maximum 63.4% at fermentation temperature of 37 ℃, and then began to decrease when the fermentation temperature was increased from 37 to 42 ℃. The reason may be that the number of lactic acid bacteria was decreased at higher fermentation temperature.

Effects of fermentation time on DPPH free radical scavenging ability

According to Fig.4, the clearance rate of DPPH was increased at first with the extension of fermentation time and reached the maximum level 68.3% when fermentation time was 6 h,and then decreased.The number of active bacteria kept growing during the logarithmic growth phase of compound lactic acid bacteria at early fermentation period,and they began to produce lactic acid with sugar after logarithmic phase.In the meantime,the number of living bacteria reached the maximum level after 6 h of fermentation, and then stopped growing because of the increased acidity.

Effects of soymilk solid content on DPPH free radical scavenging ability

As shown in Fig.5, the clearancerate of DPPH was increased with the increasing of soluble solid content.The ability of soybean curd to scavenge free radicals of DPPH was positively related to the effective components of bacteria. Acid was produced by lactic acid bacteria during their proliferation from soybean milk proteins,so lactic acid bacteria kept increasing when there was enough substrate.Therefore,the removal ability of DPPH free radicals in the soymilk with a soluble solid content of 12% reached the maximum level,77.2%.

Effects of bacterial inoculation volume on DPPH free radical scavenging ability

As shown in Fig.6,the scavenging rate of DPPH free radicals in soymilk was gradually increased with the increasing of inoculated bacterial volume.Thus,with bacteria content growing, the corresponding living bacterial monomers and their ability of scavenging free radicals were increased.

Evaluation on the ability of soymilk fermented with compound lactic acid bacteria to scavenge DPPH free radicals

The results revealed that DPPH scavenging rate in the soymilk, which was fermented at 37 ℃for 6 h and contained 12%soluble solids,reached 84.3% , while that of Baiyu lactone soymilk and bean curd were only 32.1%and 23.2%.

Conclusions

In summary,the clearance rate of soybean milk fermented was far higher than that of other similar products.This was due to the addition of lactic acid bacteria that improved the antioxidant capacity of the product.

As previously seen, fermented soymilk showed high ability to scavenge DPPH free radicals especially for L.helveticus fermented soymilk whose ability was much higher than other bacteria. Besides, the soymilk which was fermented at 37 ℃for 6 h and contained 12% soluble solids showed the highest ability to scavenge DPPH free radicals(84.3%).Thus,lactic acid bacteria own the ability of scavenging DPPH radicals and the more lactic acid bacteria, the stronger activity of lactic acid bacteria, the higher DPPH clearance. Meanwhile, the clearance rate of soymilk fermented is far higher than that of other similar products,indicating that the process of fermentation of lactic acid bacteria can significantly improve the ability of radical scavenging of soymilk.

Acknowledgements

First and foremost, I would like to show my deepest gratitude to my teacher, Dr. Wang, who has provided me with valuable guidance in every stage of the writing of this thesis.Without her enlightening instruction,impressive kindness and patience, I could not have completed my thesis. I shall extend my thanks to Miss. Chen for all her kindness and help. I would also like to thank all my teachers who have helped me to develop the fundamental and essential academic competence.

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