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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-
alaakareem2002@hotmail.com
fate–polyacrylamide gel electrophoresis (SDS-PAGE) can be
1
Department of Food Science, Agriculture College, Basrah used to confirm purification and molecular weight of active
University, Basra, Iraq peptide (Sah 2016).

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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

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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
ing
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
nosa
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 
2.5
Absorption at 205 nm

1.5

0.5

0
0 50 100 150 200 250 300
Fractions numbers

* Indicator was Bacillus cereus

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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


(A)
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

Antimicrobial
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

3.9

3.8

3.7

3.6

3.5
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Relative mobility

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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
SDS-PAGE.
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
b
c
80
d

60
e
40 f

g
20

0
2 3 4 5 6 7 8 9 10
pH values

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

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Int J Pept Res Ther

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