Effects Of Fermented Red Beetroot With Water Kefir Grains On Oxygen Carrying Capacity Of Erythrocytes And MCF-7,HepG2 Cells

Red beetroot contains many bioactive substances,such as betalains,phenols,flavonoids and saponins,etc.,which have anti-oxidation,anti-inflammation,anti-cancer and other functions.The antioxidant capacity of red beetroot juice was further improved during natural and Lactobacillus inoculation fermentation.Water kefir grains are a kind of multi-species starter culture,lactic acid bacteria,acetic acid bacteria and yeast are the main microorganisms in it.Kefir is a kind of probiotic drink fermented by water kefir grains,with the function of anti-oxidation,anti-cancer ect.However,no report on red beetroot as substance fermentation with water kefir grains mentioned.In this paper,the process of red beetroot juice fermented by water kefir grains and isolated strains,the effects of fermented red beetroot juice on oxygen carrying and releasing ability of erythrocytes and on breast cancer cell line MCF-7 and liver cancer cell line HepG2 were evaluated.The main results were as follows:（1）The conditions for the fermentation of red beetroot juice with water kefir grains were determined as 4%inoculation of water kefir grains,28℃and 8%sugar addition,and the red beetroot juice was fermented under these conditions for 48 h.Samples were taken each 6 h and the fermentation process was analysed.The results showed that the titratable acid content of red beetroot juice increased and the pH,reducing sugar and soluble solids content decreased significantly（p<0.05）.The wet weight of water kefir grains increased during 48 h fermentation,with a growth rate of 45%.Viable counts of acetic acid bacteria and yeasts reached to maximum at 18 h of fermentation,7.41 lg cfu/mL and 7.45 lg cfu/mL,respectively.At the end of the fermentation,the viable counts of lactic acid bacteria reached to 7.19 lg cfu/mL,the content of total phenol increased by 19.80%,the flavonoid and betaxanthins content decreased to 2.06 mg/mL and 1.35 mg/100mL.The content of betacyanins increased by4.9%during 24 h of fermentation.A total of 14 kinds of betalains were identified by LC-MS,including a precursor of betalains,8 kinds of betacyanins and 5 kinds of betaxanthins,and betanidin,miraxanthin V and neobetanin were newly produced at 6 h of fermentation.The correlation analysis revealed that the enhanced activity of fermented red beet juice by water kefir grains in scavenging DPPH·,ABTS⁺·,·OH and·O₂^-was mainly positively correlated with 17-decarboxy-betanidin,betanidin,neobetanin and 6’-O-feruloyl-betanin.（2）Bacterial and fungal diversity in red beetroot juice and water kefir grains which fermented red beetroot juice for 24 h were analyzed using 16S and ITS.Lactobacillus,Acetobacillus and Saccharomyces cerevisiae isolated from fermented red beetroot juice,and inoculate them to red beetroot juice in a ratio of 1︰1︰1.The results showed that pH decreased to 3.54,soluble solids content decreased significantly（p<0.05）from 6.3%to 3%,a significant increase in total titratable acid（p<0.05）,and a decrease in reducing sugar content to 28.44 mg/L.The viable counts of lactic acid bacteria,acetic acid bacteria and yeasts all reached more than 7 lg cfu/mL at the end of fermentation.The content of betacyanin was maintained above 6 mg/100mL during fermentation,with a retention rate of 96.99%,while the content of betaxanthin decreased from 2.59 mg/100mL to 1.51 mg/100mL,with a retention rate of 58.30%.Moreover,the color difference of the fermented red beetroot juice increased significantly（p<0.05）,and the color became brighter and purplish-red.The red beetroot juice fermented for 42 h had the highest DPPH·scavenging capacity,with a 79.86%increase compared to the initial.The fermented red beetroot juice at 24 h showed the greatest scavenging capacity for ABTS⁺·with a 13.55%increase.The greatest scavenging of·OH was achieved by the red beetroot juice at 18 h of fermentation,with an increase of 5.05%.And the greatest scavenging of·O₂^-was achieved by the red beetroot juice at 42 h of fermentation,with an increase of 12.28%.The antioxidant capacity was significantly（p<0.05）enhanced during fermentation and was mainly positively correlated with cycle-dopa,6’-O-feruloyl-betanin,2-decarboxy-2,3-dehydro-neobetain,betanidin and 17-decarboxy-betanidin.12 betalains were identified by LC-MS,of which cyclo-DOPA,four betacyanin such as 17-decarboxy-betanidin,betanidin,17-decarboxy-neobetanin,neobetanin and one betaxanthin such as miraxanthin V were newly produced during fermentation.（3）The inhibition of red beetroot juice fermented by water kefir grains and isolated strains on the oxidative hemolysis of model erythrocytes induced by H₂O₂was 82.08%and83.38%,respectively,and 64.87%and 70.44%for model erythrocytes induced by AAPH,respectively.And the inhibition was positively correlated with the betalains generated newly during fermentation.The fermented red beetroot juice were able to inhibit H₂O₂and AAPH induced lipid peroxidation of erythrocyte membranes,and fermented red beetroot juice reduced the content of MDA by 48.05%and 46.52%,respectively.The content of MDA were reduced by 32.19%and 49.92%in H₂O₂induced model erythrocytes,pretreated with red beetroot juice fermented by water kefir grains and isolated strains,respectively.In the model erythrocytes induced by AAPH,red beetroot juice fermented by water kefir grains and isolated strains reduced MDA levels by 48.05%and 46.52%,respectively.In addition to inhibiting lipid peroxidation,fermented red beetroot juice was able to enhance the antioxidant capacity of the erythrocytes themselves by mitigating the degree of SOD activation and increasing the GSH content.The ATP content of model erythrocytes pretreated by red beetroot juice fermented with water kefir grains and isolated strains in combination were significantly restored（p<0.05）,where red beetroot juice fermented with water kefir grains increased the ATP content of model erythrocytes induced by H₂O₂and AAPH by 75.27%and 22.51%,respectively,and in the erythrocytes model given red beetroot juice fermented with isolated strains,the ATP content increased by 101.08%and 39.11%,respectively.The content of 2,3-DPG in model erythrocytes pretreated by red beetroot juice fermented with water kefir grains increased by 23.88%、19.94%.After administration of red beetroot juice fermented by isolated strains,the content of2,3-DPG in model erythrocytes induced by H₂O₂and AAPH increased by 6.39%and 60.96%,respectively.Fermented red beetroot juice effectively protects erythrocytes and improves their ability to carry and release oxygen.（4）The growth inhibition of MCF-7 by red beetroot juice fermented 24 h with water kefir grains reached a maximum,with the inhibition rate rising from 47.96%to 78.17%,mainly associated with the increase of 17-decarboxy-betanidin,betanidin and neobetanin.The growth inhibition rate of HepG2 was 77.72%,which was mainly related to the increase of 17-decarboxy-betanidin.The maximum growth inhibition of red beetroot juice fermented with isolated strains on MCF-7 and HepG2 were 85.71%and 81.43%,respectively,which was positively correlated with cyclo-dopa and miraxanthin V.The assays of apoptosis,intracellular reactive oxygen content and cell cycle revealed that the inhibitory of fermented red beetroot juice on MCF-7 was mainly achieved by promoting late apoptosis,increasing the amount of intracellular reactive oxygen species production and blocking the cycle of most cells in the G1phase,and the inhibitory effect on HepG2 was mainly achieved by promoting early apoptosis,increasing generation of reactive oxygen species and blocking the cell cycle in the G1 phase.