Galactan protects yeast cells from apoptotic cell death. 

To check if galactan has anti-apoptotic properties, we treated yeast anti-apoptotic gene-deficient mutants (fs1∆ and pep4∆) with galactan and exposed them to an apoptosis inducer, H2O2. While yeast Fis1 is a mitochondrial fusion protein and prevents apoptosis, yeast Pep4 is a vacuolar aspartyl protease and protects the cells from acetic acid-induced apoptosis80,81. CFU counts showed about 25–30% increase in the survival of both pep4∆ and fs1∆ cells when treated with galactan compared to the cells under apoptotic stress induced by 1 mM H2O2 (Fig. 5A). Spot assay results also showed a concordance with the CFU counts that showed better survival of galactan treated yeast anti-apoptotic mutants under apoptotic stress (Fig. 5B). Our results show that galactan protects yeast cells from apoptotic cell death induced by H2O2.

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Galactan reduces chromatin condensation. In this study, we performed AO/EB staining of the galactan-pretreated yeast cells. Te pep4∆ and fs1∆ cells with H2O2 alone mostly appeared yellow-orange compared to WT, and a notable reduction in the number of yellow-orange color cells was observed in the cells pre-treated with galactan and then, exposed to H2O2. This shows that the number of apoptotic cells significantly reduced in pep4∆ reducing and chromatin condensation. AO/EB staining is a method used to detect apoptosis in both yeast and mammalian cells. Acridine orange is a cell-permeable dye that readily stains nucleic acids in both viable and non-viable cells, whereas ethidium bromide is a DNA intercalating dye that enters only when the cells are disintegrated or apoptotic or dead. This selectivity of AO/EB staining enables the detection of cells in three different phases i.e., viable cells with uniform green color in both nucleus and cytoplasm, early apoptotic cells with bright green colored nucleus due to chromatin condensation which stands out in the cytoplasm and late apoptotic cells which show yellow to orange/bright red color due to the entry of ethidium bromide into the cells50,51.

Galactan reduces nuclear fragmentation. 

Yeast WT and anti-apoptotic deficient mutants (pep4∆ and fs1∆) pre-treated with or without galactan were exposed to H2O2 and subjected to DAPI staining. Yeast anti-apoptotic deficient mutant cells pre-treated with galactan showed lesser nuclear fragmentation and low intensity of DAPI, while those without galactan pre-treatment showed a significantly higher fluorescence and increased nuclear fragmentation (Fig. 5D)52. This indicates that galactan can effectively alleviate oxidative stress-induced nuclear fragmentation leading to cellular disintegration. Previously, it was shown in transgenic mice that Bifidobacterium breve reduces apoptotic features like cell shedding in the intestinal epithelial cells which is mediated by the EPS present on the surface of the Bifdobac term. The report shows a significant dose-dependent reduction in cell shedding and the expression levels of apoptotic markers. Bifidobacterium EPS protected the cells from apoptotic cell death by modulating both intrinsic and extrinsic apoptotic signaling pathways 82. Our results, in agreement with the previous results, indicate that the galactan EPS protects the yeast anti-apoptotic gene-deficient mutants pep4∆ and fs1∆ from the apoptotic stress induced by H2O2.

Galactan extends CLS of S. cerevisiae.

To assess the anti-aging activity of galactan we performed a CLS assay with WT and yeast mutants (sod2∆, tsa1∆ , and ctt1∆)with galactan treatment. SOD2 is a manganese SOD, localized to the mitochondrial matrix. The deletion of SOD2 renders the cells highly sensitive to oxidative stress and a high mutation rate because the mtDNA is more accessible to ROS generated in the mitochondria83 and is one of the genes associated with the chronological life span of yeast. TSA1 is a thioredoxin peroxidase (thiolspecifc antioxidant) which is both cytoplasmic and ribosome associated. TSA1 is involved in conferring the resistance of the cell against the oxidative stress induced by hydrogen peroxide and lack of TSA1 is known to affect the lifespan of yeast cells. Another antioxidant mutant ctt1∆ used in the CLS experiment lacks CTT1, the cytosolic catalase involved in the detoxification of hydrogen peroxide. 

Figure 6 shows the effect of galactan on CLS of the yeast mutant strains lacking anti-oxidant genes. While WT cells treated with galactan EPS showed~10% increase in viability, sod2∆, tsa1∆ , and ctt1∆ , showed 15–20% increase in viability (Fig. 6). Mitochondrial ROS leads to senescence and nuclear DNA damage, and SOD2 is involved in scavenging ROS accumulation in mitochondria. TSA1 and CTT1 are involved in the detoxifcation of peroxide accumulated in the cytoplasm which is significantly higher in aging cells. Our CLS assay results indicate that EPS scavenges mitochondrial as well as cytoplasmic ROS buildup, prevents senescence or cell death-like features and extends the CLS in the yeast cells lacking SOD2, TSA1, and CTT154 .

Cellular oxidative stress is closely associated with the aging process and it greatly influences the onset of several age-associated human diseases such as diabetes, cardiovascular diseases, neurodegenerative disorders and cancers. Oxidative stress and inflammation are two interlinked events that play a major role in the pathology of many chronic diseases. Recent reports suggest a close link between the effect of gut microbiota on these age-related disorders. Gut microbiome protects the intestinal epithelial lining from cellular damage induced by oxidative stress-induced inflammatory pathways. Complex gut microbiota interacts with the ROS and the antioxidant defense system, helping in scavenging the free radicals and preventing inflammation. and may also regulate the oxidative state of the central nervous system through the production of neurotransmitters such as GABA, dopamine, and serotonin. The microbiome also has immunomodulatory activity, prevents extensive colonization of pathogenic microbes, and provides immunity against many microbial infections83. Many microbial polysaccharides have been tested in different models and reported to have a high antioxidant potential, anti-inflammatory activity, and cell protective functions evidenced by modulating the respective biomarker levels mediated by these exopolysaccharides82,84. Tese molecules have been found to have neuroprotective effect by preventing amyloid plaques and neuronal death in Alzheimer’s disease experimental models. Also, some of them have been shown to enhance SOD activity, reduce neurotoxic marker levels and inhibit dopaminergic neuron death in Parkinson’s disease experimental models.

The aging process is marked by elevated cellular oxidative leading to an increased accumulation of DNA damage, elevated mutation rate and decrease in cell viability. It is has been previously shown ROS induces apoptotic cell death in chronologically aging yeast cells and the supplementation of cells with the natural compounds can increase the longevity of the aging cells47,85. Our results on the antiaging effect exerted by galactan EPS from W. confusa, on the yeast sod2∆, tsa1∆ and ctt1∆ show that galactan EPS protects the yeast cells from aging-induced ROS and apoptosis, and increases their viability. Te human homologs of these genes are implicated in providing protection against age-related neurodegenerative disorders and cancers. Our results suggest that supplementation of galactan EPS may rescue the cells that lack these anti-oxidant defense genes from age-related cell death and reduce the risk of developing age-related diseases.

Conclusion

Oxidative stress is a cause of many unpropitious processes inside the cells which leads to various diseases and disorders. Galactan from W. confusa KR780676 Weissella confusa KR780676 exhibited pronounced antioxidant properties in both in vitro and in vivo studies. This study clearly showed that galactan protected the yeast antioxidant mutants against extracellular oxidative stress. Galactan also protected the anti-apoptotic deficient mutant cells from oxidative-mediated apoptotic stress as evidenced by the decreased nuclear fragmentation. Galactan decreased ROS levels extending their life expectancy, and shielding the cells from oxidative stress. Overall, the results prove that galactan has the ability to alleviate oxidative stress in the medium by scavenging free radicals. It would be interesting to test the antioxidant and anti-aging potential of galactan in higher eukaryotic cell models such as mammalian cell lines as well as animal models. Galactan is credited with strong prebiotic activity and antioxidant activity can play a significant role in reducing oxidative stress in the gut in addition to maintaining gut homeostasis. With various technological properties credited to galactan such as strong emulsifying properties, it would find immense application in the food and pharma industry as a natural functional ingredient.

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