Background
Probiotics are a group of organism those confer health benefit to consumers [
1]. To be used as probiotic, an organism should possess several attributes such as adhesive ability, acid and H
2O
2 production ability [
2], bile tolerance and significant antibacterial activity and immunomodulatory activity [
3] and must be non-pathogenic [
4,
5]. Microorganisms that are probiotic to humans include yeasts, bacilli,
Escherichia coli, enterococci, and the more commonly used bifidobacteria and lactic acid bacteria, such as lactobacilli, lactococci and streptococci [
6]. Previous reports involving both In vitro and in vivo studies have indicated that
Saccharomyces boulardii is able to prevent intestinal infection caused by
Escherichia coli,
Salmonella typhimurium,
Staphylococcus aureus,
Pseudomonas aeruginosa, Proteus vulgaris,
Yersinia enterocolitica and
Candida albicans [
7]. But probiotics properties of
Saccharomyces cerevisiae haven’t been explored that much.
Saccharomyces cerevisiae is a unicellular yeast and one of the most explored organism in terms of industrial applications and genetic studies [
8]. Several previous studies showed that members of
Saccharomyces genus can possess anti-bacterial and probiotic properties [
9]. Several studies have also been reported with the use of yeasts (
S. boulardii or S. cerevisiae) as a potential bio-therapeutic agent (probiotic) for the treatment of microbes associated diarrhea and colitis [
10]. Anti-bacterial capability of
S. cerevisiae might be due to production of extracellular protease [
11], secretion of inhibitory proteins, stimulation of immunoglobulin A [
12], acquisition and elimination of secreted toxins [
13], killer toxins, sulfur di oxide etc. [
14]. Foods such as milk, fermented foods, fruits, etc. are an important source of probiotic
Saccharomyces cerevisiae [
14,
15].
No such study has been performed in Bangladesh to assess the probiotic potential of indigenous Saccharomyces cerevisiae. This study aims to determine the probiotic properties of a putative probiotic yeast strain, S. cerevisiae IFST 062013.
Discussion
Saccharomyces cerevisiae is one of the most studied microorganisms and for long has been used in different biotechnological applications due to its better fermentation capability. Besides industrial applications, probiotic and health benefit potential of yeast has also been reported in recent times [
47]. Probiotics are defined as the viable microorganisms that exhibit a beneficial effect on health of the host by improving its intestinal microbial balance.
S. cerevisiae and
S. boulardii are clinically proven yeasts being used as a human probiotic and has shown to positively influence host’s health by antimicrobial effect, nutritional effect, inactivation of bacterial toxins, quorum sensing, trophic effects, immuno-modulatory effects, anti-inflammatory effects, cell restitution and maintenance of epithelial barrier integrity [
48].
In this study a potential probiotic yeast strain (
S. cerevisiae IFST 062013) was isolated from fruit and identified and characterized as
Saccharomyces cerevisiae on the basis of morphological and biochemical characteristics and phylogenetic analysis. Many other studies reported probiotic yeast isolated from different samples [
5,
14,
15,
49,
50]. Al Zubaidy and Khidhr [
51] also identified
Saccharomyces cerevisiae var. bouldardii from fruits with probiotic properties (antimicrobial activity, bile salt and gastric acid tolerance). Syal and Vohra [
52] reported probiotic attributes of
Geotrichum klebahnii, a yeast like fungus isolated from cheese.
To be a successful probiotic, any microorganisms must have the capability to be tolerant to stresses that prevail inside human body. The isolate can grow in a wide range of temperature and pH while optimum growth at 37
0C and pH 5.0. It also possesses tolerance to bile salt, high NaCl, simulated gastric juice, intestinal environment, α-amylase, trypsin and lysozyme (Fig.
1). Syal and Vohra [
26] reported yeast isolates that can survive in low pH and high bile salt concentration. It can produce organic acid and showed resistance against tetracycline, ampicillin, gentamycin, penicillin, polymixin B and nalidixic acid. The resistance of the yeast strain to antibiotics make it suitable for use in patients undergoing antibiotic treatment [
52]. Higher resistance to antibiotic provides the yeast strain advantage over bacteria for therapeutic use.
The isolate pose desirable properties to be a potential probiotic. It can assimilate cholesterol (33%), can produce killer toxin, vitamin B12, glutathione, siderophore and strong biofilm. Vitamins play key role in numerous metabolic processes of the body and yeasts have been reported to be able to produce vitamins, especially vitamin B complex, which is a distinctive advantage for yeast to be used as a probiotic over bacteria [
52]. Dubash et al. [
53] reported a number of yeast strains belonging to
Sachharomyces cerevisiae, Candida pintolopesii, Candida tropicalis, Pichia anomala and
Dekkera spp. with killer toxin activity. It showed moderate auto-aggregation ability and cell surface hydrophobicity. Auto-aggregation and cell surface hydrophobility is very important property of a potential probiotic as these properties are involved in adhesion of the microorganisms to intestinal epithelial cells of patients [
54]. To provide health benefits to patients by improving nutrient utilization within the intestine, a probiotic should have the ability to produce related enzymes [
55]. The isolate can produce enzymes such as amylase, protease, lipase, cellulose, but unable to produce galactosidase. The isolate don’t produce gelatinase and DNase indicating its safety to be used for human patients as most of the pathogenic microorganisms produce these enzymes as part of their pathogenesis [
26]. Cholesterol assimilation by yeast with probiotic attributes has also been reported by Chen et al. [
53]. Syal and Vohra [
26] reported yeast isolates that showed high auto-aggregation ability and cell surface hydrophobicity. The isolates were able to produce enzymes such as phytase, β-galactosidase, L-asparaginase, protease and lipase. The isolates can produce vitamin B12 and exopolysaccharide. The isolates can assimilate cholesterol, don’t produce DNase and gelatinase. Sourabh et al. [
15] reported probiotic yeast with surface hydrophobicity and autoaggregation.
One of the most desirable properties of probiotic yeasts is the anti-bacterial activity of yeasts against human pathogens. The isolate showed moderate anti-microbial activity against bacteria and fungi in comparison with standard antibiotic (Doxycycline for bacteria and fluconazole for fungi). Cell lysate showed better antimicrobial activity than whole cell and culture supernatant. Again, the isolate showed better anti-bacterial activity against gram negative bacteria than gram positive. Culture supernatant showed least anti-microbial activity indicating that the anti-microbial compounds are not extracellular, rather cell bound. Rajkowska et al. [
56] reported probiotic yeast strains (belonging to
S. cerevisiae and
S. boulardii) which showed antagonistic activity against human pathogens such as
Listeria monocytogenes, Salmonella typhimurium, Pseudomonas aeruginosa, Escherichia coli and
Enterococcus faecalis. Roostita et al. [
14] reported yeast strains with antimicrobial activity against
Pseudomonas aerugenes,
Staphylococcus aureus and
Escherichia coli. Syal and Vohra [
26] isolated yeast with antimicrobial activity against
E. coli, Salmonella sp., Staphylococcus aureus, Vibrio cholerae and
Pseudomonas sp. Further studies on antimicrobial activity of the yeast isolate against other species of pathogenic bacteria and fungi are needed.
The isolate showed strong antioxidant activity, reducing power, nitric oxide and hydroxyl radical scavenging activity, significant brine shrimp cytotoxicity and acute toxicity (Fig.
2) and metal ion chelating activity (Table
4). Foligne et al. [
42] reported yeast possessing significant anti-inflammatory activity in mice. Antioxidant activity of yeast has also been reported by Chen et al. [
54]. Hassan [
29] reported two yeast isolate, whose cell autolysates showed antioxidant and immunostimulating activity such as reducing power, DPPH radical scavenging, nitric oxide scavenging, hydroxyl radical scavenging and metal ion chelating activities. Sourabh et al. [
15] reported probiotic yeast with antioxidant properties, DPPH free radical scavenging activity and siderophore production ability. The isolate also showed strong metal chelating activity, an essential property for antioxidant activity. Hassan [
29] has reported probiotic
Saccharomyces cerevisiae with strong metal ion chelating activity.
Safety assessment is an important criterion to select any potential probiotic for therapeutic applications. To assess the safety of
S. cerevisiae IFST 062013, oral toxicity testing in mice was conducted. After 14 days of post-ingestions period, there were no significant differences in behavior or activity of the mice and no diarrheal death. No
S. cerevisiae was detected in blood samples which indicate that the isolate don’t pose the ability to infiltrate areas outside the intestine. AST level provides a general estimation about any cellular injury occurred as its level increases in case of disease & cellular injury. On the other hand, ALT more specifically indicates liver cell damage & higher serum cholesterol. Increased ALP has been linked with increased osteoblastic activity & lack of bile flow & higher serum cholesterol [
41]. Blood sample analysis also showed that AST, ALP and ALT content is almost similar in both treated and control group mice. But cholesterol content in treated group mice were lower than control group mice further ensuring the isolate’s ability to assimilate cholesterol. These observations indicate that the isolate do not induce any gross acute oral toxicity on general health, growth and development of mice. There were no significant differences in numbers of enterobacteria and
S. cerevisiae in the feces of treated group and control group mice throughout the 14 day observation period, which indicate that the isolate can persist in the intestines. Growth rate of the treated group mice was almost similar to that of the control mice. There were no significant difference between spleen weight index and liver weight ratio of the treated group and control group mice. These results indicate that the isolate cannot induce any systemic infections in mice and is non-invasive.
To test the effect of
S. cerevisiae IFST062013 on the cellular immune response, we examined splenocyte proliferation. On day 10, the spleen lymphocyte proliferation capacity was significantly increased in the
S. cerevisiae-treated groups when compared with the ConA control group (
P < 0.04). The SI values of the higher dose groups (5x10
9 CFU/mouse) reached their maximum values and were higher than for the moderate dose groups (5x10
8 CFU/mouse) (
P < 0.01). On day 20, the results showed a similar trend. These results indicate that the probiotic
S. cerevisiae strain could stimulate a T-lymphocyte specific proliferative response and could potentiate humoral immunity and cell-mediated immunity and consequently have potential antitumor activity. Cytokines play an important role in the development of immune response, we evaluated the effect of the strain on the production of pro-inflammatory cytokines IFN-α and IFN-γ, and the anti-inflammatory cytokine IL-10. IFN-γinduces cell-mediated and inflammatory immune responses. Our results showed that the probiotic strain simultaneously induced pro- and anti-inflammatory mediators and consequently helped to maintain a balance between Th1 and Th2 type cytokines, which is important for host immunity. The probiotic strain modulates gene expression of cytokines in dose dependent-manner (Fig.
4).