Determination of Antioxidant Properties and Antimicrobial Activity of Vinyl Phenolic Compounds Extracted from Saccharomyces Cerevisiae Against Uropathogenic Bacteria

Introduction

The major objective of this work is to determine the vinyl  phenolic compounds with antioxidant properties of this yeast by acetone and methanol extracts of S. cerevisiae and determine the antimicrobial activity.^1,2,3,4 The  HPLC-UV technique was employed for the identification of the vinyl phenolic compounds present in the yeast extract.^5,6,7,8 The Ferric and Ferrous reducing anti-oxidant power assay along with radical scavenging method using 1,1-diphenyl-2-picryl hydrazyl were applied to determine the anti-oxidant properties of the yeast extract^4,9,10,12. The biochemical properties of the yeast extract were determined by different chemical methodologies.^13,14,15 The antimicrobial properties of the yeast extract was determined by the standard antibiotic sensitivity test methodology designed by the Kirby and Bauer.^7,8,16 The reactive oxygen species generally gets produced by the cellular metabolism of aerobic bacteria in which the molecular oxygen plays a vital role.^11,17 They are contributors for the signal transduction of various cellular physiological processes for the human. The diseases such as cardiovascular, Alzheimer, Parkinson, Rheumatoid arthritis, etc. can be caused due to the excess production of reactive oxygen species beyond the threshold  antioxidant capacity limit of our biological system which can lead to oxidative stress.^14,18 The oxidative stress can be minimized by restoring the balance between the oxidant and antioxidant  in the human system for the proper function of the physiological system.¹⁴ The administration of synthetic antioxidant substances can be a remedy but also results in harmful side effects which can affect the human organs due to its residual effect. In the recent studies the harmful effect of the synthetic chemical compounds on humans was once again debated and hence there is a need for the alternate solution.^7,16 The alternative solutions should be able to restore the balance between the oxidant and antioxidant  in the human system for the proper function of the physiological system  and at the same time it should not cause any negative harmful side effects like that of the synthetic compounds.¹⁴ The best solution to overcome is to rediscover the nature blessed natural resources used by the ancestors to diagnose various dangerous diseases.^7,8 The natural products should also be considered only if it is as effective as the synthetic chemical substances⁹. The commonly available brewer’s yeast used in the brewing industries proved to be one of the best natural substance to overcome the synthetic chemical substances and is much more effective.^3,4,9 This  study is intended  to study this yeast’s potential reactive oxygen capacity. The brewer’s yeast employed to our study is S. cerevisiae .This yeast possess vinyl phenol compounds 4‐vinylphenol and 4‐vinylguaiacol in 1:1 ratio which promotes it to be one of the best biopharmaceutical substance.^4,11,13 The enzymatic activity of S. cerevisiae is excellent with substituted cinnamatecarboxy‐lyase which converts coumaric and ferulic acids into vinylphenols by its capability of transforming non‐oxidative decarboxylation of phenolic acid.^18,19,20 This endo- cellular activity of this yeast is a potential source which can be used for the prophylaxis of oxidative stress related disorders¹⁴. The metabolites produced by the yeast exhibits vital pharmaceutical important substances such as analgesic, antipyretic, anti-proliferative and cytotoxic antimicrobial properties.^7,8 The antimicrobial property of the yeast extract was experimented against uropathogenic bacterial infections from isolated specimens collected, purified and identified from the urinary tract infected patients.^7,8,21

Materials and Methods

Reagents

Chemicals like methanol, DPPH, acetone, FeCl₃, Ninhydrin reagent, Fehlings, Benedicts, Salkowski’s reagents were procured from Sigma-Aldrich chemicals (USA). Microbiological selective isolation culture media along with biochemical identification reagents and standard antimicrobial discs were employed. All other chemicals were analytical grade.

Yeast Collection and Identification

A purified and isolated standard strain sample of the yeast S. cerevisiae from one of the ATCC (American Type Collection Centre) specimen collection centre in India and was used for this experiment. The confirmation identification for the pure culture of the yeast was verified by employing various in vitro fungal identification technique.

Preparation of the Yeast Extracts

10g of the dried yeast samples were  sequentially extracted with 250 mL of acetone and methanol at 37ºC  for 24 hours^1,2. A rotatory evaporator was employed to dry the concentrated extract under reduced pressure with the temperature of 40°C  and  stored in the sterile test tubes at -20°C. The yield of respective yeast extracts were  estimated as Yield (%) = (dry weight of extract/dry weight of samples) x100

Identification of the Vinyl Phenolic Compound in the Yeast

Based on Huneck and Yoshimura  the identification of the vinyl phenolic compounds present in the yeast S. cerevisiae were performed by HPLC-UV technique analytical technique ²⁰. The stored dried yeast  extracts were dissolved in 500 µL of acetone and analyzed by using Agilant Technologies, 1200 Series HPLC instrument with C18 column 25 cm x 4.6 mm, 10µm with UV spectrophotometric detector.

Determination of  anti-oxidant properties in the yeast

Based on the Oyaizu Ferric and Ferrous Reducing -Antioxidant Power (FRAP) assay methodology the anti-oxidant properties of  yeast extract of  cerevisiae  was determined by the reducing powers of the dried yeast  extracts by using various concentrations of the extract ranging from 50 to 1000 µg/mL  diluted with saline buffer 0.2 M, pH 6.6  &  2.5 ml of  ferric chloride (1%) and ferrous solution (1%) respectively and incubated at 50°C for 30 min. After incubation period  2.5 ml of 10% trichloroacetic acid (TCA) was added to the mixture to stop the reaction. The mixture was centrifugated at 3000g for 10 min  and 2.5 ml distilled water added to 25 ml of the supernatant with 0.5ml  FeCl₃ (0.1%) and then the optical density  was observed at 700 nm with  an American made UNICO spectrophotometer  which determined that the higher absorbance of the reaction mixture which  indicates greater reducing power of the yeast. Suitable Positive and negative controls were used for the test. The test was repeated for the five times and the mean value was recorded for the accuracy of the results.

Based on Kosanic et al radical scavenging methodology using 1,1-diphenyl-2-picryl hydrazyl, the anti-oxidant properties of the yeast extract of S. cerevisiae to scavenge DPPH free radicals was estimated by the reduction of the reaction color between DPPH solution and sample extracts was determined by adding  2 mL of 0.12 mM solution DPPH in methanol to the 1 mL of various concentrations ranging from 50 – 1000 µg/mL of the yeast extract respectively. The mixture was incubated at 37ºC for 30 min and the absorbance of the reaction mixture was measured at 517 nm with an American made UNICO spectrophotometer to determine the  radical scavenging activity of the yeast extract. Suitable positive and negative controls were used for the test. The test was repeated for the five times and the mean value was recorded for the accuracy of the results.

Bio-Chemical Analysis of the Yeast Extract

The biochemical properties of the yeast extract of S. cerevisiae was determined by Mayer’s test, Fehling’s test, Salkowski’s test and Ninhydrin test, Ferric Chloride test, Libermann Burchard’s Test,  Benedict’s Test,   Keller-kilani test and ammonia test ¹³.

Antimicrobial Property of the Yeast Extract

The antimicrobial activity of the yeast extract was performed against the clinically isolated uropathogens from the urinary tract infected persons.

Isolation and Identification of Uropathogenic Specimen

The  clinical urinary sample was collected from the suspected urinary tract infected patients  and inoculated on the selective media Cystine Lactose Electrolyte Deficient Agar with pH indicator bromothymol blue. The inoculated plates were incubated at 37ºC for 24 hours and biochemical reaction test were done for all the isolated uropathogens from the isolated colonies by using IMVIC test and results were observed respectively²¹.

Antibiotic Susceptibility Testing  by Kirby-Bauer Disc Diffusion Method

Based on the standard antibiotic sensitivity methodology developed by Kirby and Bauer the antimicrobial activity of the yeast extract were determined^7,8,16  by incorporating the standard antibiotic discs coated with the yeast extract on the various Mueller Hinton agar plates inoculated with different uropathogenic samples such as E.coli, Klebsiella, Pseudomonas and Proteus isolated from the clinical urine specimens respectively. The inoculated plates impregnated with the antibiotic disc of yeast extract were incubated for 24 hours at 37ºC. The zone formation around the disc indicates the susceptibility of the organism and no zone formation indicates the resistance of the organism towards the antibiotic disc of the yeast extract.

Results and Discussion

The yeast  extract results were exemplary for all the tests performed. Based on Huneck and Yoshimura  the identification of the vinyl phenolic compounds present in the S. cerevisiae  were performed by the HPLC-UV analytical technique  analysis of  acetone and methanol extracts of  the yeast extract was used to identify their major vinyl phenolic compounds (Fig.1).

Figure 1: Analysis of avinyl phenolic compounds present in the Saccharomyces cerevisiae (acetone and methanol extracts) by HPLC-UV technique. Click here to View figure

By comparing their retention times (tR) yeast extract substances were identified. The main compounds in acetone extract of  yeast were physodalic acid (tR = 3.295 ± 0.012 min), atranorin (tR = 8.642 ± 0.020 min) and chloratranorin (tR = 11.918 ± 0.026 min). The most abundant substance in the acetone yeast extract was physodalic acid where as in methanol yeast extract only small amount of physodalic acid was observed in the chromatogram. It is evidenced in the chromatograms of  ferric and ferrous reducing power assay of oxidant/antioxidant based on Oyaizu technique as shown in Fig. 2.

Figure 2: Chromatograms of ferric and ferrous reducing power assay of oxidant/antioxidant. Click here to View figure

The chromatogram showed  evernic acid, (tR = 3.045 ± 0.029 min), usnic acid (tR = 7.854 ± 0.025 min), atranorin (tR = 8.236 ± 0.028 min) and chloratranorin  (tR = 11.554 ± 0.028 min) are present in the acetone extracts. The most abundant compound in acetone extract is  evernic acid while in methanol extract also evernic acid and usnic acid detected but the methyl  lecanorate (tR = 1.515 ± 0.009 min) is the most abundant compound.

Based on Kosanic´ et al radical scavenging methodology using 1,1-diphenyl-2-picryl hydrazyl, the anti-oxidant properties of the yeast extract of  S. cerevisiae  to scavenge DPPH free radicals was estimated by the reduction of the reaction color between DPPH solution and sample extracts was determined results showed a statistically significant data as the antioxidant activity was elevated from 2.01 ± 0.009%  to 86.14 ± 0.013% in accordance with the increase of the concentration of the extracts from 50 to 1000 µg/mL of yeast extract (Fig. 3).

Figure 3: Radical scavenging methodology using 1, 1-diphenyl-2-picryl hydrazyl, the anti-oxidant properties of the yeast extract of Saccharomyces cerevisiae to scavenge DPPH free radicals. Click here to View figure

The acetone yeast extract with 1000 µg/mL showed largest DPPH radical scavenging activity: 86.14 ± 0.013% . According to  test results the antioxidant activities obtained from the methanol yeast extracts were obtained but lower than that measured for the acetone yeast extract which showed the highest DPPH radical scavenging activity with an IC 50= 240.220 ±15.165 µg/mL.

The biochemical properties  analysis of  the yeast S. cerevisiae  extract showed the presence of alkaloids, reducing sugar, steroids, protein, phenol, glycosides, cardiac glycosides and amino acids as shown in Table 1.

Table 1: Biochemical properties of yeast

Test	Observation	Result	Interpretation
Mayer’s test	Formation of a creamy substance	Positive	Presence of alkaloids.
Fehling’s test	Brick red color formation	Positive	Presence of reducing sugar
Salkowski’s test	Reddish brown color formation	Positive	Presence of steroids
Ninhydrin test	Violet color formation	Positive	Presence of protein
Ferric chloride test	Blue black coloration	Positive	Presence of Phenol
Libermann Burchard’s test	Violet to bluish green color formation	Positive	Presence of Glycosides
Benedict’s test	Formation of orange red precipitate	Positive	Presence of reducing sugar
Keller-kilani test	Brown ring at the interface	Positive	Presence of Cardiac glycosides
Ammonia test	Yellowish color formation	Positive	Presence of Amino Acids

Further, collected specimens from the urinary tract infected persons were isolated by culturing on a selective media Cystine Lactose Electrolyte Deficient Agar with pH indicator Bromothymol blue showed Yellow lactose fermenting colonies for the Klebseilla and E.coli whereas blue non lactose fermenting colonies for the Proteus and Pseudomonas. The  isolated organisms were confirmed using biochemical reactions of IMVIC and the result observed were tabulated (Table 2) and confirmed the identification of the pathogens.

Table 2: Biochemical assessment of the isolated uropathogens

ORGANISM	INDOLE	METHYL RED	VOGES PROSKAUER	CITRATE	UREASE	OXIDASE
E.coli	Pink RingPositive	Red ColorPositive	Yellow ColorNegative	Green ColorNegative	Yellow ColorNegative	No purple Color Negative
Klebsiella	Yellow Ring Negative	Yellow ColorNegative	Red ColorPositive	Blue ColorPositive	Pink ColorPositive	No purple Color Negative
Pseudomonas	Yellow RingNegative	Yellow ColorNegative	Yellow ColorNegative	Blue ColorPositive	Yellow ColorNegative	Purple ColorPositive
Proteus	Yellow RingNegative	Red ColorPositive	Yellow ColorNegative	Blue ColorPositive	Pink ColorPositive	No purple color Negative

Then using the Kirby-Bauer disc diffusion method  the antimicrobial susceptibility of the prepared yeast extract antibiotic disc were tested against the isolated uropathogens and the results were exemplary. E.coli shown the best susceptibility towards the yeast extract while the Pseudomonas shown the least susceptibility. The Klebsiella and Proteus shown a moderate susceptibility with the yeast extract antibiotic disc. The antimicrobial susceptibility was excellent for all the isolated uropathogens when compared with the standard antibiotics (Table 3).

Table 3: Antimicrobial susceptibility comparison of yeast extract with standard antibiotics for urinary tract infections

Antimbiotics	E.coli	Klebsiella	Pseudomonas	Proteus
Yeast  Extract	Sensitive25mm	Sensitive23mm	Sensitive19mm	Sensitive 21mm
Trimethoprim	Sensitive21mm	Intermediate 15mm	Resistant2mm	Intermediate14mm
Sulfamethoxazole	Sensitive18mm	Sensitive24mm	Resistant4mm	Sensitive 19mm
Fosfomycin	Sensitive21mm	Sensitive22mm	Sensitive21mm	Sensitive18mm
Nitrofurantoin	Sensitive26mm	Sensitive21mm	Sensitive20mm	Sensitive 18mm
Azithromycin	Sensitive24mm	Sensitive19mm	Sensitive22mm	Sensitive22mm
Ceftriaxone	Sensitive19mm	Sensitive24mm	Sensitive20mm	Sensitive 24mm
Cephalexin	Sensitive20mm	Sensitive21mm	Sensitive18mm	Sensitive21mm
Levofloxacin	Intermediate 16mm	Resistant8mm	Resistant2mm	Resistant 6mm
Ciprofloxacin	Sensitive22mm	Sensitive19mm	Intermediate14mm	Intermediate 13mm

Conclusion

The antioxidant properties along with the vinyl phenolic compounds of the yeast Saccharomyces cerevisiae shows that this solated yeast extract can be used as a potential anti synthetic substance for the treatment of many diseases not only just as an antibiotic but can also help to balance between the oxidant and antioxidant level in the body for the proper function of the physiological system in maintaining the proper body functions as it contains many essential biochemical nutrients required for our body  to produce reactive oxygen species to eradicate many diseases. This study is a small attempt towards a larger future to serve the mankind with natural remedies.

Conflict of Interests

No conflict of interest.

Funding

None

Acknowledgement

We would like to Acknowledge our family members for their constant support and encouragement.

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Abbreviations used 

ATCC (American Type Collection Centre), DPPH (1,1-diphenyl-2-picryl hydrazyl ), FRAP (Ferric and Ferrous Reducing -Antioxidant Power)

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