You are on page 1of 7

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 Naimah1 · Alaa Jabbar Abd Al‑Manhel1 · Manar Jabbar Al‑Shawi1 

Accepted: 26 September 2017

© Springer Science+Business Media, LLC 2017

Abstract  Saccharomyces boulardii was used for anti- Introduction

microbial peptides production. Separation process of pro-
duced antimicrobial peptides was conducted using ultrafil- Saccharomyces genus contains several yeasts including Sac-
tration technique through dialysis membranes with porous charomyces cerevisiae (used in bread, pastries wine, and
10 (MWCO) kDa. The inhibition activity was determined beer production), S. bayanus (used in wine making) and
against four bacterial isolates. As a result, higher inhibition S. boulardii (used as a probiotic yeast in food making as
zone against Bacillus cereus were 26, 29 and 33 mm after such yogurt and medicine) (Demain et al. 1998; Niamah
adding 50, 75 and 100 µL of concentrated peptide, respec- 2017). The dosage of S. boulardii has given rise to positive
tively. After that, peptide passed through the Sephadex G-50 health benefits by during regulation of gut microbial bal-
column to achieve purified peptide using gel filtration. The ance and prevention of many digestive disorders including
high activity of purified peptide was confirmed based on Clostridium difficile toxin, traveler’s diarrhea, Crohn’s dis-
the second peak reaching to 37 mm of bacterial inhibition ease, Helicobacter pylori infection and diarrhea associated
zone while other peaks did not show any inhibition against with antibiotics (Kelesidis and Pothoulakis 2012).
tested bacteria. Some of the important characteristics of S. boulardii was shown to produce many inhibition fac-
purified bioactive peptide were applied. Antimicrobial tors such as polyamines (Zaouche et al. 2000), protease ser-
peptides stability was studied and found to be stable at pH ine enzyme (Hedstrom 2002), Alkaline phosphomono ester-
range from 5 to 7 values studied in addition to its inhibi- ases (Buts et al. 2006) and short-chain fatty acids (Murzyn
tion activity reached to 100%. Regarding thermal stability, et al. 2010). S. boulardii has inhibition activity against many
it was observed that the peptide was fully activity at a both microorganisms such as Staphylococcus aureus, Escherichia
60–80 °C for 30 min. Moreover, molecular weight of a pep- coli, Klebsiella oxytoca, Yersinia enterocolitica, Clostridium
tide was identified using electrophoresis technique with SDS perfringens, Clostridium difficile, Salmonella sp., Shigella
measured at 5792 Dalton. sp., Candida albicans and Entamoeba hystolitica (Berni
Canani et al. 2011). The metabolic extract of S. boulardii has
Keywords  Saccharomyces boulardii · Antimicrobial inhibition activity against 26 isolates of bacteria; the highest
peptides · Ultrafiltration 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-
* Alaa Kareem Naimah tration process are usually used and sodium dodecyl sul-
fate–polyacrylamide gel electrophoresis (SDS-PAGE) can be
Department of Food Science, Agriculture College, Basrah used to confirm purification and molecular weight of active
University, Basra, Iraq peptide (Sah 2016).

Int J Pept Res Ther

Bioactive peptides structures range from a 2 to 40 amino G-50 (Pharmacia, Sweden) was heated in 0.1 M citrate–phos-
acids. The peptide complex has a long chain of amino acids. phate buffer of pH 5.0 for 1 h at 90 °C. The prepared Sephadex
These bioactive peptides can be synthesized once described for G-50 was packed in glass column of 92 cm height and 2.5 cm
a large-scale manufacture. Synthesis of chemical, synthesis of diameter. Lyophilized metabolic extract of S. boulardii was
enzymatic and recombinant DNA technology are three main applied on the Sephadex column. The fractionation of meta-
mechanisms for bioactive peptide synthesis decide mainly by bolic extract was made using 0.1 M citrate–phosphate buffer
the size of peptide (Yazawa and Numata 2014; Sah et al. 2016). pH 5.0 and flow speed at 60 ml/h. 225 fractions were collected
The bioactive peptides have antibacterial, antifungal and (3 ml/tube) and recorded absorption of fractions by Spectro-
antioxidant properties (Jin et al. 2009; Lee et al. 2012; He photometer at 205 nm. The fractions were concentrated by
et al. 2013). The net positive charge of bioactive peptides Freeze-drier and tested their inhibition activity against bacte-
help an initial reaction with the negative charge of phospho- rial test (Agyei and Danquah 2011; Magaña et al. 2015).
lipids in bacterial outer membrane by electrostatic bound.
The bioactive peptide, after crossing during the membrane Antimicrobial Peptides Properties
of the bacterial cell and binding with cytoplasmic mem-
brane (negative charge). Inside the bacterial cell, this pep- Molecular Weight of Peptide
tides effect on DNA or RNA (Bahar and Ren 2013). The
study aimed to use the ultrafiltration process for produce The molecular weight of antimicrobial peptides from Sac-
antimicrobial peptides with small molecular weight from S. charomyces boulardii was estimated by 12% of polyacryla-
boulardii and studying the chemical and microbial proper- mide gel electrophoresis with Sodium dodecyl sulfate (SDS-
ties of these peptides. 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
Materials and Methods proteins were dissolved in sample buffer and transferred
onto slab vertical chamber (10 cm high, 10 cm wide and
Microbial Isolates 0.6 mm thick). After electrophoresis at 60 mA for 3–4 h.
The molecular weight of peptide was estimated after calcu-
Saccharomyces boulardii ATCC MYA-796TM was sup- lation the relative mobility (Rm) of antimicrobial peptides
plied from the Swanson laboratories/ Australia. Bacillus and proteins standard was determined through the equation
cereus, E. coli, Pseudomonas aeruginosa and Staphylococ- (Smith 1984; Ge et al. 2016).
cus aureus were obtained from Department of Food Science/
Rm = traveleddistance of peptide/traveled distance of dye
College of Agriculture/University of Basrah/Iraq.
pH Effect on Antimicrobial Peptides
Antimicrobial Peptides Production
Lyophilized antimicrobial peptides solutions preparation was
0.5 ml of old yeast (18 h) transferred to 100 ml of yeast dissolved in distilled water at a concentration of 100 mg/ml.
extract peptone glucose (YEPG) media (Himedia-India) and The solutions were adjusted with sterile 2N NaOH or 2N HCl
incubated at 30 °C for 24 h. After incubation, centrifuga- with different pH values between 2 to10. The final concentra-
tion at was carried out at 8000 rpm for 15 min and the cell tion of bioactive peptide was 5 mg/ml. After storage for 24 h
free supernatant of yeast was filtered (0.45 μl of Millipore at room temperature, these solutions were adjusted to pH 7.0
filter from Millipore company, UK) (Ali et al. 2012). The with 0.5 M sodium citrate buffer and assayed for inhibition
metabolic extract of yeast passed through ultrafiltration mem- activity with sample control by well diffusion agar method
branes (Millipore and Amicon, USA) are porous MWCO after adding 100 µL of antimicrobial peptides solution (Sch-
10 kDa. After ultrafiltration process, the metabolic extract ved et al. 1993). The inhibition activity percentage was cal-
of yeast was collected and lyophilized. Lyophilized of meta- culated by the following equation (Niamah et al. 2017)
bolic extract of yeast was tested against bacteria (Bacillus
cereus, E. coli, Pseudomonas aeruginosa and Staphylococcus Inhibition activity (%)
aureus) by well diffusion agar method after adding 50, 75 and = (inhibition zone of control − inhibition zone of sample/
100 µl of concentrated peptide to solid media (Niamah 2014). inhibition zone of control) × 100

Purification of Antimicrobial Peptides Heat Effect on Antimicrobial Peptides

Sephadex G-50 gel used in the purification of antimicrobial To estimate the thermal stability of antimicrobial peptides
peptides from Saccharomyces boulardii. 30 g of Sephadex 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 Results and Discussion
and determined for antimicrobial activity by well diffusion
agar method after adding 100 µl of bioactive peptide solu- Isolation of Bioactive Peptide
tion with sample control and calculated inhibition activity
percentage (Niamah 2010). The antimicrobial activity preparation obtained after ultra-
filtration process. The extract of S. boulardii has inhibition
Statistical Analysis activity against Gram positive and negative bacteria when
tested against four bacterial isolates. Inhibition activity was
All data were presented as mean ± SD (standard deviation), highest against Bacillus cereus in the solid medium were 26,
and were the results of at least three independent experi- 29 and 33 mm after adding 50, 75 and100 µl of yeast extract
ments with duplicate assays. All statistical analysis were per- while lowest diameter inhibition was 16, 19 and 22 mm of
formed using one-way analysis of variance (ANOVA table) E. coli (Table 1). The ultrafiltration process that will lead to
followed by least significant difference (LSD) for mean com- the removal of large molecules from the metabolic extract
parison. Statistical significance was established as p < 0.05 of S. boulardii. Usually, the small molecules such as peptide
(Dean and Voss 1999). 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
Table 1  The inhibition activity of S. boulardii yeast extract against be significant to purify the active compounds and estimate
bacterial test (Mean ± SD)
their chemical and physical and properties add to mode of
Bacterial isolation Gram Diameter zones of inhibition inhibitory effect against food borne and food contamination
stain- (mm)* bacteria. Recently the term appeared “Killer yeast” is meant
50 μl 75 μl 100 μl by some yeast can produce glycoproteins or proteins which
have inhibition activity against some bacteria and sensitive
Bacillus cereus + 26  ±  0.01 29  ±  0.05 33  ±  0.06
yeast (Hatoum et al. 2012; Buyuksirit and Kuleasan 2014).
E. coli − 16  ±  0.04 19  ±  0.07 22  ±  0.03
The effect of antimicrobial peptides was related to inhibition
Pseudomonas aerugi- − 18  ±  0.02 20  ±  0.05 22  ±  0.04
of the ATP, proteins and nucleic acids synthesis of microor-
Staphylococcus aureus + 21  ±  0.06 24  ±  0.03 27  ±  0.06
ganism. The antimicrobial peptides interred in microorgan-
ism cells during worked pores in the cytoplasmic membrane
*No. of repeaters = 3 (Bhunia et al. 1988).

Fig. 1  Optical absorption of
four peak after filtration process
3 *
of yeast extract. Asterisk indica-
tor was Bacillus cereus 
Absorption at 205 nm



0 50 100 150 200 250 300
Fractions numbers

* Indicator was Bacillus cereus

Int J Pept Res Ther

Purification of Antimicrobial Peptides Molecular Weight of Antimicrobial Peptides

Gel filtration chromatography of samples containing anti- The purification of bioactive was followed at each step by
microbial peptides of extract of S. boulardii, obtained after polyacrylamide gel electrophoresis with Sodium dodecyl
ultrafiltration and freeze drier process on a descending sulfate (SDS-PAGE). After elution of second peak from the
Sephadex G-50 column. The result was showed four peaks. gel filtration column, only a single band (Fig. 2). Relative
Second peak has inhibition activity against bacterial test mobility (Rm) of purification of bioactive was calculated
while other peaks did not have any mode active against bac- and compared with Rm of standard proteins. The molecular
terial test. The zone inhibition of second peak was 37 mm weight of antimicrobial peptides was 5792 Dalton (Fig. 2).
of bacterial test and this peak contain 50 fractions (150 ml) These results are consistent with those Palfree and Bussey
(Fig. 1). (1979) who description of inhibitory substances produced
The result agreed with Ali et al. (2012) who founded from S. cerevisiae was single peptide chain with a molecu-
after filtration process by used Sephadex G-100 gel col- lar weight of 11,470 Da and has an isoelectric point of 4.5.
umn, the ethanol extract of S. boulardii had mode active Pfeiffer and Radler (1982) reported the molecular weight
against Staphylococcus aureus, E. coli, Candida albicans of bioactive peptide produced by some yeast strains was
and Aspergillus niger. 16 kDa.

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
Lysozyme 14.4 kDa

Insulin 5.8 kDa peptides samples

Glucagon 3.8 kDa

(B) 4.2
y = -1.449x + 4.5058
4.1 R² = 0.9973
Log. of molecular weight





0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Relative mobility

Int J Pept Res Ther

The molecular weight of antimicrobial peptides could Table 2  Effect of heat treatment on antimicrobial activity of bioac-
be different because of media culture, extraction, purifica- tive peptide (Mean ± SD)
tion and electrophoresis method. In this study, ultrafiltration Heat treatment (℃) Time (min) Inhibition activity* (%)
process led to antimicrobial peptides production with small
60 30 100.0a  ± 0.57
molecular weight to be about 5.8 kDa. Attempts to purify
80 30 100.0a ± 0.33
antimicrobial peptides from S. boulardii by concentration
90 30 93.3b ± 0.88
by ultrafiltration membranes during short time and efficient
100 30 86.6c ± 0.57
in the removal of contaminants present in the culture media
121 30 55.8d ± 0.88
however, a two-step procedure was applied in the purifi-
cation of antimicrobial peptides. This purification by gel Different superscript letters show significant differences between
filtration procedure allowed us to obtain purity bioactive treatments
peptides as ruled by detecting single band of peptide after *Indicator was Bacillus cereus, No. of repeaters = 3
produced from S. boulardii used such as antimicrobial agent
Effect of pH in food and dairy products.

Antimicrobial peptides from S. boulardii was found to be Effect of Heat Treatment

stable at pH values between 5 and 7. The inhibitory activity
decreases when move away from positive or negative from Antimicrobial peptides was stable at thermal-processing
those values and significant differences in inhibition activity temperatures in the range 60–80 °C. The inhibition activity
were observed in pH values after 24 h at room temperature was decreased significantly with heat treatment at 90 °C for
(Fig. 3). Bioactive peptide was stable in pH acidic values 30 min. Half inhibition activity of bioactive peptide was
compared to pH alkaline values. loss at 121 °C for 30 min (Table 2). The reason of peptide
Similar results have been found for peptide produced as resistance to thermal treatment is the chemical structure of
bacteriocins by lactic acid bacteria (Niamah 2010), or pro- peptides which contain primary structure of protein only.
duced peptides from soybean by Alcalase enzyme and Prota- Similar results have been reported for bioactive peptide
mex (Minh 2015). The stability of bioactive peptide in wide produced from different sources such as pediocin (Biswas
range of pH values, could be useful were bioactive peptide et al. 1991), ­K1 killer toxin (Bussey 1991).

Fig. 3  Effect of pH values on 120

antimicrobial activity of bioac-
tive peptide. Asterisk indicator a a a
inhibition activity percentage of bacterial test *

was Bacillus cereus, No. of

repeaters = 3 100

40 f


2 3 4 5 6 7 8 9 10
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 Lactobacillus paracasei HD1-7 isolated from Chinese sauerkraut
consequence and antioxidant activity of antimicrobial pep- juice. Scientific reports, p 6
Hatoum R, Labrie S, Fliss I (2012) Antimicrobial and probiotic prop-
tides extract from probiotic yeast and to explain its mode of erties of yeasts: from fundamental to novel applications. Front
action against microorganism. By expanding our knowledge Microbiol 3:421. doi:10.3389/fmicb.2012.00421
of these objects, we may be able to increase the effectiveness He R, Alashi A, Malomo SA, Girgih AT, Chao D, Ju X, Aluko RE
of antimicrobial peptides in food systems. (2013) Antihypertensive and free radical scavenging properties of
enzymatic rapeseed protein hydrolysates. Food Chem 141:153–
159. doi:10.1016/j.foodchem.2013.02.087
Hedstrom L (2002) Serine protease mechanism and specificity. Chem
Conclusion Rev 102:4501–4524. doi:10.1021/cr000033x
Jin Z, Shinde PL, Yang YX, Choi JY, Yoon SY, Hahn T-W, Lim
HT, Park YK, Hahm KS, Joo JW (2009) Use of refined potato
The study concluded that antimicrobial peptides isolated (Solanum tuberosum L. cv. Gogu valley) protein as an alterna-
from probiotic yeast (Saccharomyces boulardii) by using tive to antibiotics in weanling pigs. Livestock Sci 124:26–32.
ultrafiltration process 10(MWCO) kDa. The peptide produce doi:10.1016/j.livsci.2008.12.003
has small molecular weight (5792 Da) and inhibition activ- Kelesidis T, Pothoulakis C (2012) Efficacy and safety of the probiotic
Saccharomyces boulardii for the prevention and therapy of gastro-
ity against some bacterial isolate. The ultrafiltration process intestinal disorders. Therapeutic Adv Gastroenterol 5(2):111–125.
helped to remove high molecular weight proteins. The puri- doi:10.1177/1756283X11428502
fication process showed four peaks and only one peak has Lee JK, Gopal R, Seo CH, Cheong HS, Park YK (2012) Isolation and
the inhibitory active. Antimicrobial peptides has stable wide purification of a novel deca-antifungal peptide from potato (Sola-
num tuberosum L. cv. Jopung) against. Int J Mol Sci 13:4021–
range of pH levels and thermal processing temperatures in 4032. doi:10.3390/ijms13044021
the range at 60–121 °C for 30 min. Magaña MD, Segura-Campos M, Dávila-Ortiz G, Betancur-Ancona
D, Chel-Guerrero L (2015) ACE-I inhibitory properties of hydro-
lysates from germinated and ungerminated Phaseolus lunatus pro-
teins. Food Sci Technol (Campinas) 35(1):167–174
References Minh NP (2015) Alcalase and protamex hydrolysis of bioac-
tive peptides from soybean. Bull Environ Pharmacol Life Sci
Agyei D, Danquah MK (2011) Industrial-scale manufacturing of 4(7):132–143
pharmaceutical-grade bioactive peptides. Biotechnol Adv Murzyn A, Krasowska A, Stefanowicz P, Dziadkowiec D, Łukaszewicz
29(3):272–277 M (2010) Capric acid secreted by S. boulardii inhibits C. albicans
Ali MAE, Abdel-Fatah OM, Janson JC, Elshafei AM (2012) Antimi- filamentous growth, adhesion and biofilm formation. PLoS ONE
crobial potential of Saccharomyces boulardii extracts and frac- 5(8):e12050. doi:10.1371/journal.pone.0012050
tions. J Appl Sci Res 8(8):4537–4543 Niamah AK (2010) Production of pediocin like bacteriocin from a local
Bahar AA, Ren D (2013) Antimicrobial Peptides Pharmaceuticals. isolate of Pediococcus acidilactici and using it as foods preserva-
6(12):1543–1575. doi:10.3390/ph6121543 tive. Ph.D. thesis, College of Agriculture, University of Basrah,
Berni Canani R, Cucchiara S, Cuomo R, Pace F, Papale F (2011) Sac- 177p. doi:10.13140/RG.2.2.31314.35529
charomyces boulardii: a summary of the evidence for gastroen- Niamah AK (2014) Determination, identification of bioactive com-
terology clinical practice in adults and children. Eur Rev Med pounds extracts from yellow banana peels and used in vitro as
Pharmacol Sci 15(7):809–822 antimicrobial. Int J Phytomed 6(4):625–632
Bhunia AK, Johnson MC, Ray B (1988) Purification, characterization Niamah AK (2017) Physicochemical and microbial characteristics of
and antimicrobial spectrum of a bacteriocin produced by Pedio- yogurt added with Saccharomyces boulardii. Curr Res Nutr Food
coccus acidilactici. J Appl Microbiol 65(4):261–268 Sci.
Biswas SR, Ray P, Johnson MC, Ray B (1991) Influence of growth Niamah AK, Al-Manhel AJA, Al-Shawi MJ (2017) Study of inhibi-
conditions on the production of a bacteriocin pediocin AcH, by tory spectrum of metabolic extract from Saccharomyces bou-
Pediococcus acidilactici H. Appl Environ Microbiol 57:1265-1 lardii yeast against some food related bacteria. Pak J Food Sci
267 27(1):26–32
Bussey H (1991) ­K1 killer toxin, a pore forming protein from yeast. Palfree RG, Bussey H (1979) Yeast killer toxin: purification and char-
Mol Microbiol 5(10):2339–2343 acterisation of the protein toxin from Saccharomyces cerevisiae.
Buts J-P, Dekeyser N, Stilmant C, Delem E, Smets F, Sokal E (2006) Eur J Biochem 93(3):487–493
Saccharomyces boulardii produces in rat small intestine a novel Pfeiffer P, Radler F (1982) Purification and characterization of extracel-
protein phosphatase that inhibits Escherichia coli endotoxin by lular and intracellular killer toxins of Saccharomyces cerevisiae-
dephosphorylation. Pediatric Res, 60:24–29. doi:10.1203/01. strain 28. J Gen Microbiol 128:2699–2706
pdr.0000220322.31940.29 Sah BNP (2016) Identification of bioactive peptides produced in syn-
Buyuksirit T, Kuleasan H (2014) Antimicrobial agents produced biotic yogurt having anticancer properties. Ph.D. thesis, Victoria
by yeasts. Int J Biol Biomol Agric Food Biotechnol Eng University, Melbourne, Australia
8(10):1114–1117 Sah BNP, Vasiljevic T, McKechnie S, Donkor ON (2016) Antibacterial
Dean A, Voss D (1999) Design and analysis of experiments, Springer, and antiproliferative peptides in synbiotic yogurt—Release and
New York, p 740 stability during refrigerated storage. J Dairy Sci 99(6):4233–4242.
Demain AL, Phaff HJ, Kurtzman CP (1998) The industrial and agricul- doi:10.3168/jds.2015-10499
tural significance of yeasts. In: Kurtzman CP, Fell JW (eds) The Smith BJ (1984) SDS poly acrylamide gel electrophoresis of proteins
yeasts, a taxonomic study, 4th edn., Elsevier Science, Amsterdam In: Walker JM (ed) Methods in molecular biology, The Humana
Ge J, Sun Y, Xin X, Wang Y, Ping W (2016) Purification and par- press, New York, pp 41–55
tial characterization of a novel bacteriocin synthesized by

Int J Pept Res Ther

Schved F, Lalazar A, Henis Y, Juven BJ (1993) Purification, partial Zaouche A, Loukil C, De Lagausie P, Peuchmaur M, Macry J, Fitoussi
characterization and plasmid-linkage of pediocin SJ-1, a bacte- F, Bernasconi P, Bingen E, Cezard J (2000) Effects of oral Sac-
riocin produced by Pediococcus acidilactici. J Appl Microbiol charomyces boulardii on bacterial overgrowth, translocation, and
74(1):67–77 intestinal adaptation after small-bowel resection in rats. Scand J
Yazawa K, Numata K (2014) Recent advances in chemoenzymatic Gastroenterol 35:160–165. doi:10.1080/003655200750024326
peptide syntheses. Molecules, 19(9):13755–13774. doi:10.3390/