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Int J Pept Res Ther DOI 10.1007/s10989-017-9632-2

Int J Pept Res Ther DOI 10.1007/s10989-017-9632-2
Int J Pept Res Ther DOI 10.1007/s10989-017-9632-2

Isolation, Purification and Characterization of Antimicrobial Peptides Produced from Saccharomyces boulardii

Alaa Kareem Naimah 1 · Alaa Jabbar Abd Al‑Manhel 1 · Manar Jabbar Al‑Shawi 1

Accepted: 26 September 2017 © Springer Science+Business Media, LLC 2017

Abstract Saccharomyces boulardii was used for anti- microbial peptides production. Separation process of pro- duced antimicrobial peptides was conducted using ultrafil- tration technique through dialysis membranes with porous 10 (MWCO) kDa. The inhibition activity was determined against four bacterial isolates. As a result, higher inhibition zone against Bacillus cereus were 26, 29 and 33 mm after adding 50, 75 and 100 µL of concentrated peptide, respec- tively. After that, peptide passed through the Sephadex G-50 column to achieve purified peptide using gel filtration. The high activity of purified peptide was confirmed based on the second peak reaching to 37 mm of bacterial inhibition zone while other peaks did not show any inhibition against tested bacteria. Some of the important characteristics of purified bioactive peptide were applied. Antimicrobial peptides stability was studied and found to be stable at pH range from 5 to 7 values studied in addition to its inhibi- tion activity reached to 100%. Regarding thermal stability, it was observed that the peptide was fully activity at a both 60–80 °C for 30 min. Moreover, molecular weight of a pep- tide was identified using electrophoresis technique with SDS measured at 5792 Dalton.

Keywords

peptides · Ultrafiltration

Saccharomyces boulardii · Antimicrobial

* Alaa Kareem Naimah

alaakareem2002@hotmail.com

1 Department of Food Science, Agriculture College, Basrah University, Basra, Iraq

Science, Agriculture College, Basrah University, Basra, Iraq Introduction Saccharomyces genus contains several yeasts

Introduction

Saccharomyces genus contains several yeasts including Sac- charomyces cerevisiae (used in bread, pastries wine, and beer production), S. bayanus (used in wine making) and S. boulardii (used as a probiotic yeast in food making as such yogurt and medicine) (Demain et al. 1998; Niamah 2017). The dosage of S. boulardii has given rise to positive health benefits by during regulation of gut microbial bal- ance and prevention of many digestive disorders including Clostridium difficile toxin, traveler’s diarrhea, Crohn’s dis- ease, Helicobacter pylori infection and diarrhea associated with antibiotics (Kelesidis and Pothoulakis 2012). S. boulardii was shown to produce many inhibition fac- tors such as polyamines (Zaouche et al. 2000), protease ser- ine enzyme (Hedstrom 2002), Alkaline phosphomono ester- ases (Buts et al. 2006) and short-chain fatty acids (Murzyn et al. 2010). S. boulardii has inhibition activity against many microorganisms such as Staphylococcus aureus, Escherichia coli, Klebsiella oxytoca, Yersinia enterocolitica, Clostridium perfringens, Clostridium difficile, Salmonella sp., Shigella sp., Candida albicans and Entamoeba hystolitica (Berni Canani et al. 2011). The metabolic extract of S. boulardii has inhibition activity against 26 isolates of bacteria; the highest inhibition zone was 37 mm of Enterobacter spp. while low inhibition zone was 18 mm of E.coli (Niamah et al. 2017). The isolation and purification techniques of bioactive peptides are very import through the bioactive peptides production. Isolation process of peptides from yeast is dif- ficult because of matrix growth media (mixture of glucose, organic acids, ions, and protein). For this reason, ultrafil- tration process are usually used and sodium dodecyl sul- fate–polyacrylamide gel electrophoresis (SDS-PAGE) can be used to confirm purification and molecular weight of active peptide (Sah 2016).

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Int J Pept Res Ther

Bioactive peptides structures range from a 2 to 40 amino acids. The peptide complex has a long chain of amino acids. These bioactive peptides can be synthesized once described for a large-scale manufacture. Synthesis of chemical, synthesis of enzymatic and recombinant DNA technology are three main mechanisms for bioactive peptide synthesis decide mainly by the size of peptide (Yazawa and Numata 2014; Sah et al. 2016). The bioactive peptides have antibacterial, antifungal and antioxidant properties (Jin et al. 2009; Lee et al. 2012; He et al. 2013). The net positive charge of bioactive peptides help an initial reaction with the negative charge of phospho- lipids in bacterial outer membrane by electrostatic bound. The bioactive peptide, after crossing during the membrane of the bacterial cell and binding with cytoplasmic mem- brane (negative charge). Inside the bacterial cell, this pep- tides effect on DNA or RNA (Bahar and Ren 2013). The study aimed to use the ultrafiltration process for produce antimicrobial peptides with small molecular weight from S. boulardii and studying the chemical and microbial proper- ties of these peptides.

Materials and Methods

Microbial Isolates

Saccharomyces boulardii ATCC MYA-796TM was sup- plied from the Swanson laboratories/ Australia. Bacillus cereus, E. coli, Pseudomonas aeruginosa and Staphylococ- cus aureus were obtained from Department of Food Science/ College of Agriculture/University of Basrah/Iraq.

Antimicrobial Peptides Production

0.5 ml of old yeast (18 h) transferred to 100 ml of yeast extract peptone glucose (YEPG) media (Himedia-India) and incubated at 30 °C for 24 h. After incubation, centrifuga- tion at was carried out at 8000 rpm for 15 min and the cell free supernatant of yeast was filtered (0.45 μl of Millipore filter from Millipore company, UK) (Ali et al. 2012). The metabolic extract of yeast passed through ultrafiltration mem- branes (Millipore and Amicon, USA) are porous MWCO 10 kDa. After ultrafiltration process, the metabolic extract of yeast was collected and lyophilized. Lyophilized of meta- bolic extract of yeast was tested against bacteria (Bacillus cereus, E. coli, Pseudomonas aeruginosa and Staphylococcus aureus) by well diffusion agar method after adding 50, 75 and 100 µl of concentrated peptide to solid media (Niamah 2014).

Purification of Antimicrobial Peptides

Sephadex G-50 gel used in the purification of antimicrobial peptides from Saccharomyces boulardii. 30 g of Sephadex

G-50 (Pharmacia, Sweden) was heated in 0.1 M citrate–phos- phate buffer of pH 5.0 for 1 h at 90 °C. The prepared Sephadex G-50 was packed in glass column of 92 cm height and 2.5 cm diameter. Lyophilized metabolic extract of S. boulardii was applied on the Sephadex column. The fractionation of meta- bolic extract was made using 0.1 M citrate–phosphate buffer pH 5.0 and flow speed at 60 ml/h. 225 fractions were collected (3 ml/tube) and recorded absorption of fractions by Spectro- photometer at 205 nm. The fractions were concentrated by Freeze-drier and tested their inhibition activity against bacte- rial test (Agyei and Danquah 2011; Magaña et al. 2015).

Antimicrobial Peptides Properties

Molecular Weight of Peptide

The molecular weight of antimicrobial peptides from Sac- charomyces boulardii was estimated by 12% of polyacryla- mide gel electrophoresis with Sodium dodecyl sulfate (SDS- PAGE) with three standard proteins (Glucagon 3800 Da, Insulin 5800 Da and Lysozyme 14,400 Da were obtained from Sigma Company). 3 mg/ml of peptide and standard proteins were dissolved in sample buffer and transferred onto slab vertical chamber (10 cm high, 10 cm wide and 0.6 mm thick). After electrophoresis at 60 mA for 3–4 h. The molecular weight of peptide was estimated after calcu- lation the relative mobility (Rm) of antimicrobial peptides and proteins standard was determined through the equation (Smith 1984; Ge et al. 2016).

Rm = traveleddistance of peptide/traveled distance of dye

pH Effect on Antimicrobial Peptides

Lyophilized antimicrobial peptides solutions preparation was dissolved in distilled water at a concentration of 100 mg/ml. The solutions were adjusted with sterile 2N NaOH or 2N HCl with different pH values between 2 to10. The final concentra- tion of bioactive peptide was 5 mg/ml. After storage for 24 h at room temperature, these solutions were adjusted to pH 7.0 with 0.5 M sodium citrate buffer and assayed for inhibition activity with sample control by well diffusion agar method after adding 100 µL of antimicrobial peptides solution (Sch- ved et al. 1993). The inhibition activity percentage was cal- culated by the following equation (Niamah et al. 2017)

Inhibition activity ( %) = ( inhibition zone of control inhibition zone of sample/ inhibition zone of control) × 100

Heat Effect on Antimicrobial Peptides

To estimate the thermal stability of antimicrobial peptides from Saccharomyces boulardii, 3 ml (5 mg/ml) of peptide

Int J Pept Res Ther

was heated at 60, 80, 90, 100 and 121 °C for 30 min. Cooled and determined for antimicrobial activity by well diffusion agar method after adding 100 µl of bioactive peptide solu- tion with sample control and calculated inhibition activity percentage (Niamah 2010).

Statistical Analysis

All data were presented as mean ± SD (standard deviation), and were the results of at least three independent experi- ments with duplicate assays. All statistical analysis were per- formed using one-way analysis of variance (ANOVA table) followed by least significant difference (LSD) for mean com- parison. Statistical significance was established as p < 0.05 (Dean and Voss 1999).

Table 1 The inhibition activity of S. boulardii yeast extract against bacterial test (Mean ± SD)

Bacterial isolation

Gram

Diameter zones of inhibition

stain-

(mm)*

ing

50 μl

75 μl

100 μl

Bacillus cereus

+

26 ± 0.01 16 ± 0.04

29 ± 0.05 33 ± 0.06 19 ± 0.07 22 ± 0.03

E. coli

Pseudomonas aerugi-

18 ± 0.02

20 ± 0.05 22 ± 0.04

nosa

Staphylococcus aureus

+

21 ± 0.06

24 ± 0.03 27 ± 0.06

*No. of repeaters = 3

Results and Discussion

Isolation of Bioactive Peptide

The antimicrobial activity preparation obtained after ultra- filtration process. The extract of S. boulardii has inhibition activity against Gram positive and negative bacteria when tested against four bacterial isolates. Inhibition activity was highest against Bacillus cereus in the solid medium were 26, 29 and 33 mm after adding 50, 75 and100 µl of yeast extract while lowest diameter inhibition was 16, 19 and 22 mm of E. coli (Table 1). The ultrafiltration process that will lead to the removal of large molecules from the metabolic extract of S. boulardii. Usually, the small molecules such as peptide have antimicrobial activity. Antimicrobial peptides produced by several species and strains of probiotic microorganisms (bacteria and yeast) have possible uses such as biological food preservatives. In order to use them in the most effective methods it will be significant to purify the active compounds and estimate their chemical and physical and properties add to mode of inhibitory effect against food borne and food contamination bacteria. Recently the term appeared “Killer yeast” is meant by some yeast can produce glycoproteins or proteins which have inhibition activity against some bacteria and sensitive yeast (Hatoum et al. 2012; Buyuksirit and Kuleasan 2014).

The effect of antimicrobial peptides was related to inhibition

of the ATP, proteins and nucleic acids synthesis of microor- ganism. The antimicrobial peptides interred in microorgan- ism cells during worked pores in the cytoplasmic membrane (Bhunia et al. 1988).

Fig. 1 Optical absorption of four peak after filtration process of yeast extract. Asterisk indica- tor was Bacillus cereus

3 * 2.5 2 1.5 1 0.5 0 Absorption at 205 nm
3
*
2.5
2
1.5
1
0.5
0
Absorption at 205 nm

0

50

* Indicator was Bacillus cereus

100

150

Fractions numbers

200

250

300

Int J Pept Res Ther

Purification of Antimicrobial Peptides

Gel filtration chromatography of samples containing anti- microbial peptides of extract of S. boulardii, obtained after ultrafiltration and freeze drier process on a descending Sephadex G-50 column. The result was showed four peaks. Second peak has inhibition activity against bacterial test while other peaks did not have any mode active against bac- terial test. The zone inhibition of second peak was 37 mm of bacterial test and this peak contain 50 fractions (150 ml) (Fig. 1). The result agreed with Ali et al. (2012) who founded after filtration process by used Sephadex G-100 gel col- umn, the ethanol extract of S. boulardii had mode active against Staphylococcus aureus, E. coli, Candida albicans and Aspergillus niger.

Fig. 2 The molecular weight of antimicrobial peptides was determined by electrophoresis method. a SDS–polyacrylamide gel electrophoresis of antimi- crobial peptides and standard proteins. b Relative mobility of antimicrobial peptides and standard proteins

(A)

Lysozyme 14.4 kDa

Insulin 5.8 kDa

Glucagon 3.8 kDa

Molecular Weight of Antimicrobial Peptides

The purification of bioactive was followed at each step by polyacrylamide gel electrophoresis with Sodium dodecyl sulfate (SDS-PAGE). After elution of second peak from the gel filtration column, only a single band (Fig. 2). Relative mobility (Rm) of purification of bioactive was calculated and compared with Rm of standard proteins. The molecular weight of antimicrobial peptides was 5792 Dalton (Fig. 2). These results are consistent with those Palfree and Bussey (1979) who description of inhibitory substances produced from S. cerevisiae was single peptide chain with a molecu- lar weight of 11,470 Da and has an isoelectric point of 4.5. Pfeiffer and Radler (1982) reported the molecular weight of bioactive peptide produced by some yeast strains was 16 kDa.

bioactive peptide produced by some yeast strains was 16 kDa. Antimicrobial peptides samples (B) 4.2 4.1

Antimicrobial

peptides samples

(B) 4.2 4.1 y = -1.449x + 4.5058 R² = 0.9973 4 3.9 3.8 3.7
(B)
4.2
4.1
y = -1.449x + 4.5058
R² = 0.9973
4
3.9
3.8
3.7
3.6
3.5
Log. of molecular weight

0

0.1

0.2

0.3

0.4

0.5

Relative mobility

0.6

0.7

Int J Pept Res Ther

The molecular weight of antimicrobial peptides could be different because of media culture, extraction, purifica- tion and electrophoresis method. In this study, ultrafiltration process led to antimicrobial peptides production with small molecular weight to be about 5.8 kDa. Attempts to purify antimicrobial peptides from S. boulardii by concentration by ultrafiltration membranes during short time and efficient in the removal of contaminants present in the culture media however, a two-step procedure was applied in the purifi- cation of antimicrobial peptides. This purification by gel filtration procedure allowed us to obtain purity bioactive peptides as ruled by detecting single band of peptide after SDS-PAGE.

Effect of pH

Antimicrobial peptides from S. boulardii was found to be stable at pH values between 5 and 7. The inhibitory activity decreases when move away from positive or negative from those values and significant differences in inhibition activity were observed in pH values after 24 h at room temperature (Fig. 3). Bioactive peptide was stable in pH acidic values compared to pH alkaline values. Similar results have been found for peptide produced as bacteriocins by lactic acid bacteria (Niamah 2010), or pro- duced peptides from soybean by Alcalase enzyme and Prota- mex (Minh 2015). The stability of bioactive peptide in wide range of pH values, could be useful were bioactive peptide

Table 2 Effect of heat treatment on antimicrobial activity of bioac- tive peptide (Mean ± SD)

Heat treatment ()

Time (min)

Inhibition activity* (%)

60

30

100.0 a ± 0.57

80

30

100.0 a ± 0.33 93.3 b ± 0.88 86.6 c ± 0.57 55.8 d ± 0.88

90

30

100

30

121

30

Different superscript letters show significant differences between treatments

*Indicator was Bacillus cereus, No. of repeaters = 3

produced from S. boulardii used such as antimicrobial agent in food and dairy products.

Effect of Heat Treatment

Antimicrobial peptides was stable at thermal-processing temperatures in the range 60–80 °C. The inhibition activity was decreased significantly with heat treatment at 90 °C for 30 min. Half inhibition activity of bioactive peptide was loss at 121 °C for 30 min (Table 2). The reason of peptide resistance to thermal treatment is the chemical structure of peptides which contain primary structure of protein only. Similar results have been reported for bioactive peptide produced from different sources such as pediocin (Biswas et al. 1991), K 1 killer toxin (Bussey 1991).

Fig. 3 Effect of pH values on antimicrobial activity of bioac- tive peptide. Asterisk indicator was Bacillus cereus, No. of repeaters = 3

12 0 a a a 10 0 b c 80 d 60 e 40 f
12
0
a
a
a
10
0
b
c
80
d
60
e
40
f
g
20
0
2345678
9
10
inhibition activity percentage of bacterial test *

pH values

* Indicator was Bacillus cereus, No. of repeaters = 3.

Int J Pept Res Ther

Work is currently in progress to determine the amino acid consequence and antioxidant activity of antimicrobial pep- tides extract from probiotic yeast and to explain its mode of action against microorganism. By expanding our knowledge of these objects, we may be able to increase the effectiveness of antimicrobial peptides in food systems.

Conclusion

The study concluded that antimicrobial peptides isolated from probiotic yeast (Saccharomyces boulardii) by using ultrafiltration process 10(MWCO) kDa. The peptide produce has small molecular weight (5792 Da) and inhibition activ- ity against some bacterial isolate. The ultrafiltration process helped to remove high molecular weight proteins. The puri- fication process showed four peaks and only one peak has the inhibitory active. Antimicrobial peptides has stable wide range of pH levels and thermal processing temperatures in the range at 60–121 °C for 30 min.

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