In the present study, galactan exopolysaccharide (EPS) from Cistanche KR780676 was evaluated for its potential to alleviate oxidative stress using in vitro assays and in vivo studies, saccharomyces cerevisiae (wild type) and its antioxidant (sod14, sod24, tsa14, cta2, and ctt12)anti-apoptotic (pep4 and fs14) and anti-aging (sod24, tsa1, and ctt12)) isogenic gene deletion mutants. Galactan exhibited strong DPPH and nitric oxide scavenging activity with a lCso value of 450 and 138 ug/mL respectively. ln the yeast mutant model, oxidative stress generated by H,0.was extensively scavenged by galactan in the medium as confirmed using spot assays followed by fluorescent DCF-DA staining and microscopic studies. Galactan treatment resulted in a reduction in the ROS generated in the yeast mutant cells as demonstrated by decreased fluorescence intensity. Furthermore, galactan exhibited protection against oxidative damage through H,O, -induced apoptosis inhibition in the yeast mutant strains (pep4 and fs1) leading to an increased survival rate by neutralizing the oxidative stress. In the chronological life span assay, WT cells treated with galactanEPS showed 8% increase in viability whereas sod2 mutant showed 10-15% increase indicating pronounced anti-aging effects. Galactan from W. confuse KR780676 has immense potential to be used as a natural antioxidant for nutraceutical, pharmaceutical and food technological applications. As per our knowledge, this is the first report on an in-depth assessment of in vivo antioxidant properties of abacterial EPS in a yeast deletion model system.

Click to Get Cistanche Rich In EPS

Oxidation is an essential process to maintain biological processes and also for the production of energy in all living organisms. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) radicals are produced from normal catabolism of oxygen and nitrogen molecules respectively. Severe oxidative stress leads to various degenerative conditions like DNA damage, cellular degeneration, and carcinogenesis. These may result in many health impairments such as aging, cardiovascular diseases, cancer, cirrhosis, atherosclerosis, diabetes and rheumatoid arthritis2-6, Antioxidants are the molecules that scavenge free radicals generated in food or living system and lead to the prevention of oxidative damage-related health conditions-9. Although many synthetic antioxidants are available as strong radical scavengers, some of them have been linked to some undesirable side effects. In view of this, there has been increased interest and demand for natural antioxidants in the use food and pharma industries. Most of the plant and mushroom-based polysaccharides have been reported as significant protective agents against ROS11–21. Various microbial exopolysaccharides (EPS) including from lactic acid bacteria (LAB) have also been reported for their substantial antioxidant properties. These are considered to be safer alternatives to synthetic ones. In recent years, many EPS from LAB has been studied for their antioxidant potential and prevention of oxidative damage22,23. Antioxidant potential and protective role of Weissella EPS have also been reported from a variety of EPS isolated from Weissella many strains like Cistanche EPSWWC, W. confusa OF126, W. cibaria GA44, W. cibaria YB-1, W. confusa W4 and W. cibaria SJ1424–29. In vivo antioxidant properties can be studied using various model systems such as cell lines30, C. elegans31, yeast32, and mice33. Previously, various compounds have been screened for their potential antioxidant properties using Saccharomyces cerevisiae yeast gene deletion mutant model system32,34–37.

This study is focused on the antioxidant potential of galactan EPS produced by the probiotic strain Cistanche KR780676 from an Indian traditional fermented food (Idli batter)38,39. In this paper, the galactan EPS is screened for in vitro antioxidant potential using DPPH, nitric oxide, and hydroxyl radical scavenging assays and in vivo antioxidant, anti-apoptotic and anti-aging properties using yeast gene deletion mutant model system.

Materials and methods

All the chemicals including media supplements were procured from Hi-Media Laboratories Pvt. Ltd., India, and DCF-DA (2,7 Dichlorodihydrofuorescein diacetate) from Sigma. 

Microbial culture. Cistanche KR780676 isolated from an Indian acidic fermented food (Idli batter), reported to produce galactan EPS was used in this present study 38. 

Detection of EPS production. Te EPS production of W. confusa was observed from the colony level. Briefly, the strain was cultured on MRS agar (supplemented with 2% sucrose). Afer 48 h at 30 °C, the appearance of slimy/ mucous colonies was observed. Production of galactan EPS was further verified under scanning electron microscopy (SEM) analysis.

Extraction of EPS. EPS extraction process was performed as per the method described in Kavitake et al.38. Fresh inoculum (10%) of W. confusa was added to 2% sucrose-enhanced MRS medium at 30 °C for 48 h under static conditions. The suspension was in this manner centrifuged (12,000×g for 15 min) to isolate the biomass and further treated with trichloro acidic acid to eliminate the protein moieties. The galactan-EPS was precipitated using ice-cold ethanol (threefold the volume), centrifuged (19,200×g for 15 min) and the resultant EPS was dissolved in Milli-Q water. The crude EPS was dialyzed at 12–14 kDa (48 h, 4 °C) and freeze-dried by lyophilization for 48 h.

In vitro antioxidant properties of galactan EPS. DPPH assay for galactan was executed according to the earlier report by Ye et al.9 and percent scavenging activity (%) was calculated by the following equation.

where Ao and As is the absorbance of the control (blank, without EPS) and sample respectively. Nitric oxide (NO) radical scavenging assay for galactan was performed according to Sreejayan et al.40 and calculated as following equation,

where Ao is the absorbance of the control (blank, without EPS) and As is the absorbance in the presence of the EPS. Reducing power activity was measured for galactan EPS as reported by Ye et al.9 . Te absorbance was read at 700 nm and the reductive potential is indicated by a high absorbance capacity of the reaction mixture. Ascorbic acid (Vc) was used as a positive control. The Hydroxyl radical scavenging activity of galactan EPS was evaluated as described by Yang et al.41. Te absorbance was read at 536 nm and the scavenging percentage was calculated as:

where A sample is the absorbance of the sample, A blank is an absorbance in the absence of a sample and H2O2 solution, and A control is an absorbance in the absence of the sample

In vivo antioxidant properties of galactan EPS in yeast mutant strains. 

The Yeast, S. cerevisiae, BY4741 wild type (WT) (MATa his3∆:leu2∆:met15∆:ura3∆), and gene deletion mutant strains were procured from Thermo Fisher Scientific, USA. Yeast strains were grown in yeast peptone dextrose (YPD) medium supplemented with or without 200 µg/mL of Geneticin (G418 sulfate) for the selection of mutants. YPD solid medium was prepared by the addition of 2% Bacto agar to the YPD liquid medium42. 

Effect of EPS on yeast growth. Exponentially growing yeast wild type (WT) culture (approximately 1× 104 cells) was treated with different concentrations (0–400 μg/mL) of EPS in a microwell plate and the final volume was made up to 200 μL with YPD broth. Te culture was incubated for 18 h at 30 °C followed by serial dilution and spreading on YPD agar plates. Te plates were incubated at 30 °C for 2 days and colony forming units (CFUs) were counted and the viability was expressed as percent CFU43.

Measurement of biomarkers of oxidative stress. Exponentially growing yeast WT cells were pre-treated with or without 300 μg/ml of EPS for 2 h. Ten the cells were exposed to 1 mM H2O2 for 1 h at 30 °C in a shaker incubator and processed for the measurement of SOD activity and lipid peroxidation levels as per the method described in earlier reports44–46

The antioxidant property of EPS in S. cerevisiae gene deletion mutants.

Exponentially growing cultures of yeast WT and antioxidant-deficient mutant strains (sod1∆, sod2∆, tsa1∆, cta1∆, ctt1∆, glr1∆ , and yhb1∆) were treated with 300 μg/mL EPS for 2 h followed by exposed to 1 mM H2O2 for 1 h. Serially diluted cells were spread on YPD agar plates, incubated for 2 days at 30 °C, and viability was calculated. For spot assay, cultures were serially diluted in 10-folds, 4 μL of which was spotted on YPD agar plates and incubated at 30 °C for 2 days and photographed43,47. Detection and measurement of ROS. Exponentially growing WT and antioxidant-deficient mutant strains (sod1∆, sod2∆, tsa1∆, cta1∆ , and ctt1∆) were pretreated with or without EPS for 2 h and exposed to 1 mM H2O2 for 1 h at 30 °C. Cell pellets afer centrifugation at 5000 rpm for 5 min were washed twice with PBS buffer, re-suspended in 200 μL of PBS, and incubated with 20 μM DCF-DA in dark for 15–20 min at room temperature. Immediately after the incubation, cells were washed twice with PBS, mounted on the slides and observed under an Olympus Ix71 fluorescence microscope under 40×objective using a blue filter. For quantification of ROS, DCFDA stained cells were resuspended in 200 μL of PBS after washing and the intensity of DCF fluorescence was measured using a spectrofluorometer at the excitation maximum and emission wavelengths of 495/529  nm. Fluorescence units were plotted against each treated and untreated culture and compared48,49.

Anti‑apoptotic activity of galactan. 

Spot and CFU assays. Exponentially grown WT and anti-apoptotic-defcient mutant (pep4∆ and fs1∆) cells were pre-treated with EPS and incubated along with respective untreated controls for 2 h. For CFU counts, cultures were treated or untreated with EPS for 2 h and incubated with 0.5 mM H2O2 for 1 h. Each serially diluted culture was spread on YPD agar plates, and incubated at 30 °C for 2 days and cell viability was represented as percent CFU. For spot assay, cultures were allowed for serial dilution followed by spotting on YPD agar plates with or without 1 mM H2O2. Plates were incubated at 30 °C for 2 days and photographed47. 

Detection of an anti‑apoptotic marker of EPS

 using yeast mutant strains. To further confirm the rescue action of EPS on yeast cells from the apoptotic cell death induced by hydrogen peroxide in yeast, WT and anti-apoptotic deficient mutant strains (pep4∆ and fs1∆) were examined for apoptosis markers. Exponentially growing WT, pep4∆ , and fs1∆ cells were treated or untreated with 300 µg/mL EPS for 2 h and then were exposed to 1 mM H2O2 for 1 h. Both treated and untreated cells were stained with acridine orange and ethidium bromide (AO/EB) and observed under a fluorescent microscope for chromatin condensation50,51. For DAPI staining, the treated and untreated cells were fixed with 4% paraformaldehyde and incubated with 1 μg/mL DAPI for 5–10 min in dark at room temperature. Cells were mounted on the slides after washing with PBS and observed under Olympus IX71 fluorescence microscope (UV filter, 40× objective) for nuclear fragmentation52,53.

Anti‑aging effect of EPS by chronological lifespan assay. 

Yeast wild type and antioxidant deficient mutant (sod2∆, tsa1∆ , and ctt1∆) cultures were grown to reach the stationary phase and incubated with or without EPS for chronological lifespan (CLS) assay. The survivability of every strain was calculated at different time intervals from 0 to 30 days. Cell viability was expressed as percent CFU for both treated and untreated cultures54,55. Statistical analysis. All the experiments were carried out in triplicates (±SD) and statistically analyzed using IBM SPSS 20 software in a one-way ANOVA model. Tukey’s HSD comparison test (p<0.05) was used to measure the significance level. 

Results and discussion Lactic acid bacteria (LAB) isolated from fermented foods have fetched enormous attention of food technologists over the last few years because of their proven probiotic properties. Among the bacteria isolated from Indian fermented foods are Lactobacillus spp., Lactococcus spp., Leuconostoc spp. and the less explored Weisella spp. Like the other beneficial LABs, the Cistanche strain KR780676 which was isolated from fermented idli batter in our lab has been reported to act as a potential probiotic candidate39. Earlier reports, this galactan has been characterized as a linear homopolysaccharide and also screened for its physicochemical, functional and emulsifying properties38,56–58. We have also reported that the cells and cell supernatants of W. confusa KR780676 showed strong anti-oxidant activity39. EPS production from W. confusa KR780676 was observed on MRS agar plate enriched with 2% sucrose, incubated for 48 h (Fig. 1A-i), which revealed Weissella slimy colonies and it was further confirmed in SEM image (of the Weissella colony) showing the presence of galactan EPS along with cells (Fig. 1A-ii). Step-by-step EPS production is overviewed with the pictorial representation in Fig. 1B.

In vitro antioxidant properties of galactan EPS. As shown in Fig. 2A, the DPPH scavenging activity is observed to be concentration-dependent, the scavenging activity increased with the galactan concentration. The half-maximal effective concentration (IC50) of ascorbic acid was 8.8 µg/mL, while galactan EPS was 450 µg/mL. 1,1-diphenyl-2-picrylhydrazyl (DPPH) is a stable free radicle that delocalizes the unpaired electrons against the molecule as a whole, thus preventing the molecules from dimerization and resulting in deep violet color59. When DPPH solution is added to different concentrations (50 to 500 µg/mL) of galactan, it gives rise to a reduction of violet color as the concentration increases. Antioxidants when reacting with an electron of DPPH, the deep violet color of DPPH turns to light violet color; and the intensity of the color depends on the antioxidant activity of the substrate60. Galactan showed 60% DPPH scavenging potential which is higher than EPS from the endophytic bacterium Paenibacillus polymyxa EJS-3 (<45.40%)33.

Nitric oxide is produced when sodium nitroprusside is decomposed in an aqueous solution at a pH of 7.2. Nitrate and nitrite are produced under aerobic conditions when nitric oxide binds with oxygen61. As shown in Fig. 2B, galactan has the potential to reduce nitric oxide generation from sodium nitroprusside as the half-maximal efective concentration (IC50) is 138 µg/mL, while standard ascorbic acid was 11 µg/mL. As shown in Fig. 2C, the reducing power activity of EPS is positively correlated with its concentration in increasing order. Standard (ascorbic acid) showed higher reducing capacity than galactan EPS which was in agreement with Ye et al.9 . Te reducing power activity of galactan indicates the potential antioxidant activity. When potassium ferricyanide [K3Fe (CN)6] is added in galactan EPS, the antioxidants present in it reduces into potassium ferricyanide [K4Fe(CN)6]

Figure 2. In vitro antioxidant properties of galactan. (A) DPPH radical scavenging activity, (B) Nitric oxide assay, (C) Reducing power assay and (D) Hydroxyl radical scavenging activity of galactan EPS.

The Hydroxyl radical scavenging activity of galactan is shown in Fig. 2D. Galactan showed 41.93% as the highest activity, whereas 58.10% by the standard at the same concentration (1 mg/mL). Results trend is in agreement with previous reports for EPS from Lactobacillus plantarum C88 (85.21% for 4 mg/mL concentration of EPS)62 and Paenibacillus polymyxa EJS-3 (68.55% for 1 mg/mL concentration of EPS)63. Compare to standards, galactan showed 72.16% efficiency whereas EPS from Lactobacillus plantarum C8862 and Paenibacillus polymyxa EJS-363 showed 95.19 and 68.55% efficiency respectively.

In vivo antioxidant properties of galactan EPS in a yeast model. Earlier reports have shown different health-promoting biological properties such as anti-proliferative, anti-ulcer, cholesterol-lowering, antioxidant, anti-inflammatory and immunomodulatory activities of EPS derived from LAB64. In this view, it becomes important to evaluate the in-detail antioxidant effect by the EPS that could support to their prebiotic and/or probiotic potential, mainly to maintain gut homeostasis by alleviating oxidative stress. The cellular anti-oxidant machinery plays a critical role in alleviating the unavoidable ROS generated through crucial cellular metabolic pathways. The imbalance between the cell’s innate antioxidant defense and the ROS generated can be very detrimental to the cells. Severe oxidative stress causes potential damage to cellular vital components, proteins, nucleic acids, and lipid molecules which in turn affects many essential signaling pathways, and induces apoptosis. The dietary intake of antioxidants has been shown to lower the frequency of cellular damage markers such as ROS levels, ROS-mediated DNA damage, apoptosis and cellular transformation which further results in the lowered incidence of the age-associated disorders65 Microbial EPS like xanthan and levans have demonstrated anti-oxidant activity in in vitro assays (DPPH and hydroxyl radical assays)5 and against human gastric cancer cells BGC-823, respectively66. Previously, In this study, we evaluated the antioxidant, antiapoptotic, and antiaging effects exerted by galactan EPS isolated from W. confusa KR780676 using yeast Saccharomyces cerevisiae BY4741 as a model organism.

Effect of EPS on yeast growth. Wild-type yeast cells treated with different concentrations of EPS did not show any growth defects confirming that the galactan did not induce any cytotoxicity or growth defects at any of the concentrations ranging from 0 to 400 μg/mL (Fig. 3A). Cells treated with 300 μg/mL or higher concentration of galactan showed significantly higher growth indicating that galactan had yeast growth stimulatory activity.

A galactan concentration of 300 μg/mL is used for subsequent experiments with yeast strains. Galactan reduces SOD enzyme activity. Following the strong in vitro antioxidant activity displayed by galactan EPS, further subjected to check its effect on SOD activity in the yeast WT cells. Our results (Fig. 3B) show that the oxidative stress induced by H2O2 treatment caused a sharp increase in the SOD activity of H2O2-treated WT cells compared to untreated cells. In contrast, galactan pre-treatment of WT cells followed by exposure to H2O2 resulted in an a~twofold reduction in the SOD activity compared to that of the cells treated by H2O2 alone indicating galactan helps the yeast cells in managing the superoxide radical-induced oxidative stress47,67.

Oxidative stress induced by peroxide treatment activates the superoxide enzyme and a change in the level of SOD activity is a direct measure of the level of cellular oxidative stress. In this study, the NBT reduction method was used, where, NBT is an indicator of superoxide radical production. Inhibition of NBT reduction is a direct measure of SOD. Since SOD competes with NBT for superoxide radicals generated by exposing riboflavin to visible light in the presence of oxygen and methionine, which is an electron donor. Superoxide reduces NBT to a blue color product, formazan which can be colorimetrically measured at 560 nm.

Previous reports indicate that LAB EPS has exerted an antioxidant effect in a concentration-dependent manner in in vitro assays, and in colon cancer cell lines and in vivo models. Te ability of EPS to scavenge the ROS might be attributed to their diverse chemical groups68. Other of EPS (500 µg/ml) from Bacillus amyl liquefacient have been shown to greatly influence SOD activity and protect the HepG2 cells from oxidative stress induced by H2O2 68. Similarly, the EPS from L. plantarum showed a concentration-dependent effect on SOD activity in Caco2 cells against H2O2-induced oxidative stress69. Our results, in concordant with these reports show that galactan EPS pre-treatment scavenges the superoxide radicals induced by H2O2 treatment in yeast WT cells.

EPS decreases cellular lipid peroxidation. Malondialdehyde (MDA) is the most studied cellular lipid peroxidation biomarker that indicates the level of oxidative stress. MDA is a chemically stable, highly reactive dialdehyde and can readily bind to proteins, nucleic acids, and lipoproteins. It is highly mutagenic and can greatly afect the biochemical properties of these biomolecules which is deleterious to various signaling pathways70. An increase in MDA levels is an indicator of ROS-induced tissue damage and its increased levels are detected in several human pathologies46. In this study, to examine how EPS pre-treatment influences the lipid peroxidation induced by H2O2 in yeast WT cells, MDA levels were estimated. Results showed an approximately 1.5- fold reduction in the MDA level in EPS pre-treated cells, compared to those exposed to H2O2 alone as shown in Fig. 3C. 

Previously, EPS from L. plantarum C88 has been shown to reduce MDA levels induced by H2O2 treatment, in a dose-dependent manner (50–200 ug/ml) in Caco2 cells, indicating that the peroxide-induced membrane injury of the intestinal cells can be alleviated by the supplementation of EPS69 (zhang 2013). Further, 500 ug/mL of EPS from B. amyloliquefaciens significantly reduced H2O2-induced MDA in HepG2 cells71. Our results on H2O2-induced MDA levels in yeast cells and its significant reduction following pre-treatment of the cells with galactan EPS indicate that the treatment with galactan EPS reduces cellular ROS-induced lipid peroxidation and in turn, may protect the cells from tissue damage caused by peroxide radicals and aid in maintaining cellular integrity 

experiments. *represents P<0.0001 a signifcant increase/decrease in EPS+ H2O2 treated samples compared to those treated with H2O2 alone.

Galactan protects yeast antioxidant gene mutants under oxidative stress. In order to evaluate the antioxidant ability of galactan, different antioxidant gene deficient yeast mutants (that lack various oxidative stress response genes) were treated with galactan and then exposed to a sub-lethal dose of H2O2 42. SOD (sod1∆ and sod2∆), catalase (cta1∆ and ctt1∆), thioredoxin peroxidase (tsa1∆), glutathione reductase (glr1∆), and nitric oxide oxidoreductase (yhb1∆) mutants when treated with H2O2. showed low survival (sod1∆ 10.8%, sod2∆ 13.17%, tsa1∆ 13.55%, cta1∆ 15.34%, ctt1∆ 17.06%, glr1∆ 27.37% and yhb1∆ 33.87%) against WT. In contrast, with the galactan pre-treatment, the tolerance against oxidative stress increased in all the antioxidant gene deficient mutants and their viability increased significantly (sod1∆ 78.43%, sod2∆ 59.96%, cta1∆ 87%, ctt1∆ 87%, tsa1∆ 72.73%, glr1∆ 75.26% and yhb1∆ 82.41%) as shown in Fig. 4A. Tese results suggest that galactan can effectively scavenge the free radicals induced by H2O2 in mutants lacking specific oxidative stress response genes and protects the cells against oxidative stress. Similar protection was observed in the spot assay, where galactan rescued the mutants from oxidative stress and increased the viability as shown in Fig. 4B.

Cells are evolved with enzymatic antioxidant defense systems to withstand the endogenous oxidative stress caused by ROS generated through various physiological reactions, which would otherwise have deleterious effects on the well-being of the cell. Previous reports and our results in this study suggest that EPS are able to scavenge ROS and improve cell viability. Our results with yeast oxidative stress response mutant strains suggest that galactan EPS treatment scavenges both cytosolic and mitochondrial superoxide radicals induced by H2O2 treatment, as evidenced by the increased viability of yeast sod1∆ and sod2∆ mutant strains respectively. SODs are primary enzymatic antioxidant defenses in the cells against endogenous and exogenous oxidative stress and mutations in these genes have been implicated in cancer and degenerative disorders. Likewise, the antioxidant rescue of cta1∆ and ctt1∆ (catalase mutants) by EPS treatment indicates that treatment with galactan EPS provides protection against peroxisomal and cytosolic oxidative stress induced by H2O2. Catalase is responsible for the cellular detoxification of hydrogen peroxide and its deficiency is associated with the onset of age-related disorders. TSA1, thioredoxin peroxidase is both ribosomal associated and free cytoplasmic protein that relieves the cells from hydrogen peroxide stress. Reports suggest that TSA1 also has a role in oxidative DNA damage repair. Our results suggest that EPS treatment rescues yeast tsa1∆ cells from peroxide-induced stress. Mammalian peroxiredoxins have a positive effect on cell growth, metabolism, and immune functions. Their deficiency results in elevated cellular oxidative stress that affects crucial signaling pathways and is implicated in neurodegenerative disorders, malignancies, and inflammatory diseases 72. Glutathione antioxidant mechanism is another first-line enzymatic antioxidant defense system present in the cells to overcome oxidative stress. GLR1 is the yeast homolog of mammalian glutathione reductase, localizes to both cytosol and mitochondria73. Glr reduces oxidized glutathione (GSSH) to reduced glutathione (GSH) and plays a role in maintaining GSH levels in the cells which in turn detoxifies superoxide and hydroxide radicals, thus protecting the cells from oxidative stress74. Our results show a high sensitivity of yeast glr1∆ to H2O2 which was alleviated by pre-treatment with galactan EPS suggesting that galactan EPS might protect the cells that are deficient of GLR1 from oxidative stress. Interestingly, glutathione reductase deficiency has been implicated in aging and age-related metabolic, degenerative and cardiovascular disorders75,76. As shown in Fig. 4A, yhb1∆ was also protected by galactan EPS against H2O2 stress which suggests that galactan EPS protects the cells that are deficient of YHB1 from oxidative stress. Yeast YHB1 is a favorite hemoglobin that has been reported to protect the cells from nitric oxide stress77 and oxidative stress78. Evidence suggests that the human homolog of YHB1 appears to rescue cells from alpha-synuclein toxicity which is a biomarker of Parkinson’s disease79. These previous fndings and our results suggest that EPS may promote cell survival against the development of degenerative disorders.

To assess the level of ROS in the yeast mutants with the presence and absence of galactan under H2O2 stress, yeast cells were observed under a fluorescence microscope and the intracellular oxidation level was calculated with a spectrofuorometer47,48. Yeast mutants treated with H2O2 alone showed more green fuorescent cells compared to those pre-treated with galactan. (Fig. 4C). DCF fluorescence intensity increased by approximately 60%, whereas with galactan pre-treatment, it reduced to approximately 30% in H2O2-treated yeast mutants compared to WT and respective control (Fig. 4D). Both microscopic and spectrophotometric results indicate an increased level of ROS in the cells that lack specific antioxidant genes compared to respective controls. Cultures pre-treated with galactan and then exposed to H2O2 showed a diminished fluorescence compared to those without galactan pre-treatment which suggests that galactan reduced the ROS induction H2O2 exposure in yeast mutant cells and promotes cell survival. In all the above experiments, when the cells were washed before the addition of H2O2 showed no significant rescue (supplementary data) indicating that the ROS scavenging by galactan is due to direct scavenging in the medium rather than in the intracellular action.