African Journal of Biotechnology Vol. 10(11), pp.

2178-2185, 14 March, 2011

Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.1881
ISSN 16845315 2011 Academic Journals

Full Length Research Paper

Comparison of three methods for determination of

protein concentration in lactic acid bacteria for
proteomics studies
Ibrahim Mehmeti1, Fadime Kiran1,2 and Ozlem Osmanagaoglu2*
1
Department of Chemistry, Biotechnology and Food Science, University of Life Science, s, Norway.
2
Department of Biology, Faculty of Science, Ankara University, Tandogan, 06100, Ankara, Turkey.
Accepted 18 February, 2011

Finding the best method of cell lysis and extraction of protein from the lysed cells is the key step in
detection and identification of extra- and intra-cellular proteins in all applications of proteomics. To
develop an optimized protein extraction protocol, Enterococcus faecalis V583, Lactococcus lactis NIZO
0900 and Pediococcus pentosaceus OZF strains, respectively, belonging to each genus of
Enterococcus, Lactococcus and Pediococcus were used as a representative cells in a study of lactic
acid bacteria (LAB). This report covers the use and comparison of three different protein extraction
methods including sonication, centrifugation and rupture by glass beads (FastPrep) to get a better
understanding about which methods give better extract quality and higher amount of proteins when
applied to one dimensional (1D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) and for subsequent analysis by two dimensional (2D)-PAGE. The results clearly showed that, all
methods can be used to lyse LAB strains. However, a six fold greater amount of protein was obtained
when FastPrep was applied to lyse LAB cells. Our results also indicate that, this fast and easy
extraction method allows more spot-abundant polyacrylamide gels. More clear and consistent strips
were detected by SDS-PAGE when proteins were extracted by FastPrep. These results testify to the
suitability of FastPrep protein extraction protocols for 2D proteomic studies of representative strains of
LAB.

Key words: FastPrep, sonication, centrifugation, lactic acid bacteria (LAB).

INTRODUCTION

Lactic acid bacteria (LAB) are heterogeneous group of mentation and preservation, either as the natural micro-
gram-positive bacteria, which produce lactic acid as a flora or as starter cultures added under controlled
major end-product of their fermentative metabolism. conditions. Besides their technological roles, LAB make
Lactococcus, Streptococcus, Pediococcus, Leuconostoc, expire date of the food longer by inhibiting the growth of
Lactobacillus and Carnobacterium are the main genera of spoilage and pathogenic bacteria, by competing for
LAB. They play an important role in food and feed fer- nutrients and producing antimicrobial compounds such as
organic acids, carbon dioxide, ethanol, hydrogen pero-
xide and bacteriocins and therefore, thought to be poten-
tial biopreservativies (Klaenhammer, 1993). By definition,
bacteriocins are proteinaceous substances with bacteri-
*Corresponding author. E-mail: [email protected]. cidal or bacteriostatic activity against sensitive bacterial
edu.tr. Tel: +905324338662. Fax: 90-312-2232395. species (Klaenhammer, 1988; Daeschel, 1989; Piard and
Abbreviations: LAB, Lactic acid bacteria; SDS-PAGE, sodium
Desmazeaud, 1991; Stiles and Hastings, 1991; Piard and
dodecyl sulfate-polyacrylamide gel electrophoresis; MS, mass Desmazeaud, 1992; Klaenhammer, 1993). LAB and their
spectrometry; MRS, de Man, Rogosa and Sharpe; DTT, food products are thought to confer a variety of important
dithiothreitol; BSA, bovine serum albumine; IPG, immobilized nutritional and therapeutic benefits and have many docu-
pH gradient; TCA, trichloroacetic acid. mented health promoting or probiotic effects in human
 Mehmeti et al. 2179

(Ljungh and Wadstrm, 2006). Probiotics have been for scientists (Natarajan et al., 2005). An efficient protein
defined as living microorganisms which upon ingestion in extraction methodology is quite important for sample
adequate numbers exert positive health effects beyond preparation and for subsequent 2D-PAGE and MS analy-
inherent basic nutrition (FAO/WHO working group, 2001). sis. Cell lysis is the first step in protein extraction and
The beneficial contribution of the LAB to the food and purification protocol. In any proteomic experiment, finding
food-related industries is considerable. However, some the best method to lyse cells and/or extract proteins from
LAB can cause spoilage of a variety of foods, such as cells and the reliability of the methods is the most
meats, milk and milk products, vegetables, fruit juices, important step, as a reliable and comprehensive protein
sugary products, alcoholic beverages and products pre- extraction is the closest proteomic equivalent to a fully
served with vinegar (Sharpe and Pettipher, 1983). sequenced and annotated genome (Cilia et al., 2009).
Besides, it is usually accepted that with the exception of Any biological conclusions that are obtained from a pro-
some streptococci, LAB are rarely pathogenic to man and teomic study are only as strong as the data indicating
animals (Aguirre and Collins, 1993). Enterococcus that, the extracts are reproducible and rich in protein
faecalis which is one of the most studied species among diversity (Cilia et al., 2009). Many techniques including
LAB has been identified as causes of nosocomial infec- physical and detergent-based methods are available for
tions, causing an increasing incidence of endocarditis, cell disruption and protein extraction and have been used
bacteremia, urinary tract and neonatal infections (Schaberg by several researchers in their own works for different
et al., 1991; Moellering, 1992; Tailor et al., 1993; Dutka- purposes (Grabskia, 2009). However, there is only a few
Malen et al., 1995; Hancock and Gilmore, 2006). How- studies regarding comparison of these protein extraction
ever, this common intestinal microorganism (Devriese methods (Bhaduri and Demchick, 1983; De Mey et al.,
and Pot, 1995) is also used in some traditional food 2008; Abram et al., 2009). These techniques can vary
industries because of its beneficial role in the develop- widely in reproducibility and in representation of the total
ment of cheese aroma and stimulation of starter LAB proteome. Currently, although many methods have been
(Giard et al., 2001). Lactococcus lactis is commonly used developed and reported, there is no developed single
in industrial dairy fermentations, particularly in cheese protein extraction method. Besides, these methods have
making while Pediococcus pentosaceus is used in meats to be adapted, often two or more methods have to be
especially for sausage making and silage inoculants combined and further optimized to obtain the best possi-
(Hammes et al., 1990). ble yield and purity for different species of organisms, for
Since LAB have a couple of interesting properties of the type of sample (microbial cells or mammalian tissue)
great economic value, the growth of interest in LAB shows to be analyzed and for the interested proteins (soluble
an increase in the concern of their rational use. The cytosolic or highly insoluble membrane proteins). There-
characterization of these organisms using the tools of fore, the protein extraction method remains a challenge
proteomics as well as other approaches in systems biology for scientists in the accurate analysis of proteins
(genomics, transcriptomics, metabolomics) will generate (Natarajan et al., 2005).
the knowledgebase necessary to an understanding of In this study, we compared three different methods for
their biology. Compared with genomic studies, investiga- the extraction of proteins from LAB including sonication,
tions at the proteome level provide detailed information centrifugation and mechanic rupture by glass beads
such as protein abundance and posttranslational modifi- (FastPrep) to determine their efficiency in separating
cations. Proteomics is defined as the analysis of the proteins by SDS-PAGE and for subsequent analysis by
entire protein complement expressed in a cell or any 2D-PAGE.
biological sample at a given time under specific condi-
tions (Dierick et al., 2002). Proteomic technologies are
powerful tools to study the physiological response of MATERIALS AND METHODS
bacteria to various environmental stress conditions
(Renzone et al., 2005). A better understanding of the Bacterial strains, media and growth conditions
mechanisms of stress resistance should permit an under-
standing of the bases of the adaptive responses and Three representative strains belonging to LAB genera were
cross protection and to rationalize their exploitation in selected: E. faecalis V583, P. pentosaceus OZF (on which prote-
ome studies have been carrying out) and L. lactis NIZO 0900. E.
order to prepare LAB for industrial processes (Van de
faecalis V583 and P. pentosaceus OZF were grown in de Man,
Guchte et al., 2002). A proteomic study includes sodium Rogosa and Sharpe (MRS) broth at 37C, while GM 17 medium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- (M17 broth, pH 6.5, supplemented with 1.0% w/v glucose) was
PAGE), higher resolutions of two dimensional (2D)- used for growth of L. lactis NIZO 0900 at 30C. Cultures were
PAGE, tryptic digestion of proteins from gel and protein maintained in their appropriate broths as frozen stocks with 15%
identification by mass spectrometry (MS) by comparing (v/v) sterile glycerol and stored at -80C. Before experimental use,
they were subcultured twice in their appropriate medium and
the results with a data base (Haynes et al., 1998; Giard et temperatures. Three biological replicates (independent cultures) for
al., 2001; Angelika et al., 2004). Extraction of proteins is each protocol were made and samples in the late exponential
challenging and inconsistent and has long been an issue phase were used for each strain.
2180 Afr. J. Biotechnol.

Protein extraction SDS-PAGE

In this investigation, three different extraction methods including 15% acrylamide separating gel and 4% stacking gel were used for
sonication, centrifugation and mechanical rupture by glass beads SDS-PAGE and prestained protein marker-brand range (Bio-Labs)
(FastPrep) were used to extract proteins or lyse cells from 3 was used as a molecular weight standard. SDS-PAGE gels were
representative strains of LAB. Before the extraction of cells by use run at 80 V for 15 min and then, at 120 V for 2 h (Laemmli, 1970).
of each method, samples of 50 ml were taken from the late expo- At the end of electrophoresis, gels were visualized by staining with
nential phase. Pellets were obtained by centrifugation at 6000g for Coomassie brilliant blue according to Sambrook et al. (1992) and
10 min at 4C (SIGMA 3K30) and used in each method. destained for 2 h in a solution containing 7.5% methanol and 5%
glacial acetic acid.

Sonication method
2D-PAGE
Pellets were resuspended in 1 ml 20 mM TRIS buffer (pH 7.5)
containing 5 mM EDTA and 5 mM MgCl2. The cells were lysed by 2D gel electrophoresis was run to examine the quality of the
using transonic T460/H sonicator (John Morris Scientific Pty Ltd.) extracted proteins and was performed according to the method of
for 3 times, each 2 min in between which the cell suspension was OFarrell (1975) with some modifications. Briefly, 75 g crude
kept on ice for 3 min. The cell debris was removed by centrifugation protein extracts were solubilized in 450 l of a rehydration buffer
at 14000g for 10 min at 4C (Duch et al., 2002). The supernatant and applied on 24 cm IPG strips with a linear pH range of 4 to 7
containing extracted proteins was stored at -20C until further gradients (Bio-Rad) for the first dimension. IEF was carried out on a
analysis. Multiphor II electrophoresis unit (Pharmacia) at 50C with the
following program: 50 V in 30 min, 500 V for 1 h, 1000 V in 1 h and
10 000 V for 7 h (total 70 000 V h). In the first step, the IPGs were
Centrifugation method equilibrated for 15 min each in SDS equilibration buffer (6 M urea,
50 mM Tris-HCl, 30% Glycerol, 2% SDS and 0.01% bromophenol
Pellets were resuspended in 500 l of buffer containing 0.3% SDS, blue) containing 1% DTT followed by 15 min in an equilibration
200 mM dithiothreitol (DTT), 28 mM Tris HCl, 20 mM Tris. Cell buffer containing 5% iodoacetamide prior to second dimension
suspension containing microfuge tubes were stirred gently for 10 PAGE in 12% polyacrylamide gel in 375 mM Tris-HCl, 0.1% SDS,
min at 4C on a shaker (IKA-Werke KS260B), followed by removing 0.1% APS and 0.031% TEMED. Following electrophoresis, the gels
of cells by centrifugation at 14000g for 10 min at 4C. Supernatant were fixed for two hours in 7% acetic acid / 10% methanol and
was incubated at 100C for 5 min and then, chilled on ice. 24 l of stained with silver nitrate overnight; followed by destaining with 7%
buffer containing 24 mM Tris, 476 mM Tris-HCl, 50 mM MgCl2, 1 acetic acid / 10% methanol and imaged.
mg/ml DNaseI and 0.25 mg/ml RNaseA was then, added and
incubated again on ice for 15 min. The reaction was stopped by
addition of four volumes of ice-cold acetone and precipitation of Silver staining of 1D and 2D gels
proteins was allowed for 20 min on ice. The cell debris was
removed by centrifugation at 14000g for 10 min at 4C (Giard et Protein spots were visualized by staining with silver nitrate as
al., 2001). The supernatant containing protein extracts was stored described previously (Lauber et al., 2001) and run in triplicate for
at -20C until further analysis. each biological replicate to confirm the reproducibility of the protein
patterns. Gels were fixed overnight in 40% ethanol and 10% acetic
acid, washed two times for 20 min in 30% ethanol followed by 1 min
FastPrep method in thiosulphate reagent and incubated 20 min in 0.2% silver nitrate.
The gels were rinsed three times for 20 s with milli-Q water and for
Proteins were extracted by using the FP120 FastPrep bead-beater 10 to 30 s with 3% sodium carbonate, 0.05% p-formaldehyde. Gels
(BIO101/Savent) by alkaline-lysis protocol. In brief, the pellets were developed by incubation in sodium carbonate solution and the
resuspended in 400 l rehydration buffer including 8 M Urea, 2 M reaction was stopped in 0.5% glycine.
Thiourea, 0.5% CHAPS, 10 mM DTT and 0.1% immobilized pH
gradient (IPG). Cells were lysed by vortexing them with acid
washed glass beads with a diameter of 212 to 300 m (Biospec Statistical analysis
Products, Inc.) by use of FastPrep FP120 device at 6 m/s for 345
Statistical analysis was made using the Statistical Package for the
s at 4C, each 3 min in between which the cell suspension was kept
Social Sciences (SPSS) 15.0 software (SPSS Inc, Chicago, IL,
on ice for 1 min. The cell debris was removed by centrifugation at
USA). Data were analyzed by oneway analysis of variance
14000g for 10 min at 4C (Larsen et al., 2006). The supernatant
(ANOVA) followed by Tukeys posthoc test (GraphPad Prism v.3.0,
containing protein extracts was stored at -20C until further
GraphPad Software, San Diego, CA, USA) and shown as mean
analysis.
and standard deviation (S.D.). In all statistical analyses, p < 0.05
was taken as the level of significance.
Protein determination

Total cellular protein concentrations were determined by use of a RESULTS AND DISCUSSION
commercially colorimetric DC protein assay kit according to the
manufacturer protocol (Bio-Rad) with bovine serum albumine (BSA) For the one (1D) and two-dimensional (2D) polyacry-
as a standard and absorbance measurement at 750 nm. The kit is lamide gel electrophoresis techniques used in the initial
reported not to interfere with any chemicals used throughout
extraction protocols and therefore, is compatible with isoelectric experimental process of proteomics, the sample prepara-
focusing (IEF) (Bradford, 1976; Angelika et al., 2004). Samples and tion and/or high quality resolution of proteins are the most
standards were replicated three times. important point for quantification of proteins to optimize
 Mehmeti et al. 2181

Table 1. MeanSD of protein concentrations (g/l) of each strain obtained by

three different methods.

Method Sonication Centrifugation FastPrep

E. faecalis V583 1.33 0.01 1.25 0.01 4.85 0.05
L. lactis NIZO 0900 1.25 0.02 1.32 0.01 6.23 0.06
P. pentosaceus OZF 1.32 0.01 1,16 0.02 5.66 0.04
Values are mean S.D.(standard deviation) of results of three experiments.

over-expressed as well as under-expressed proteins proteomic research reports to obtain total cellular protein
between strains. It is required under the same experi- extracts from relatively small amounts of biomass (Wang
mental conditions, to compare different proteins that were et al., 2005; Dahl et al., 2007; Koskenniemi et al., 2009).
identified from spots showing consistent differences in There appear to be no reports in proteomics making a
intensity and/or abundance. Because of the differences comparison of the various protein extraction methods
between physical and chemical properties of proteins, it using industrially important strains of LAB. Some studies
is important to select an appropriate standard assay of focus on comparing protocols for the extraction of pro-
choice for a particular sample since different extraction teins from a large variety of organisms which are not
protocols may favor proteins from different strains. LAB. For example, in insects, a TCA-acetone extraction
Due to the increase in application of LAB in industries, method certainly performed well for the purpose of prepa-
the exploration of the stress resistance mechanisms ring quantitative 2D gel electrophoresis-based separa-
should let us to discover the fundamentals of the adaptive tions when compared with the use of phenol- and deter-
responses and cross protection and to modernize their gent-based methods and these experiments were able to
application to prepare LAB for industrial processing. assess the differences between the two aphid genotypes
Among these strains, E. faecalis and L. lactis are the LAB (Cilia et al., 2009). In plants, protein extraction has been
most commonly used in dairy fermentation, while P. problematic as standard protocols must contend with high
pentosaceus is being used in meat and vegetables concentrations of salt ions in plant tissues. Wang et al.
fermentation (Hammes et al., 1990). In this study we (2007) have developed an improved method for protein
compared three different protein extraction methods extraction from Salicornia europaea using borax, poly-
(sonication, centrifugation and FastPrep) for their compa- vinylpolypyrrolidone and phenol and this allowed removal
tibility with 1D- and 2D-PAGE analysis by use of 3 repre- of interfering compounds and salt ions. The comparative
sentative strains of LAB. In proteomics approaches, study of this method with several other protocols using
evaluation, standardization and selection of efficient NaCl-treated S. europaea shoots demonstrated that, this
methods are quite important for proteins analysis since method gave the best distinction of proteins on 2-DE gels
the presence of nonprotein impurities can critically affect (Wang et al., 2007). In another study, three different
the quality of 2D-PAGE separation by formation of protein extraction methods were compared for proteomic
artifactual spots and what is reported in this work is the analysis; the sucrose, Tris-HCl and trichloroacetic acid
relative efficiency or degree of extraction of the various (TCA)/acetone methods were all compared and the
methods sucrose extraction buffer was found to be the most
The concentrations of proteins extracted by three me- efficient and reliable method for extracting proteins from
thods are shown in Table 1. The results show that higher pine needles (Cai-yun et al., 2005). Sheoran et al. (2009)
amount of proteins were obtained when the cells were evaluated four protein extraction methods including
lysed with mechanic rupture using glass beads TCA/acetone, phenol, direct IEF buffer and Tris-HCl
(FastPrep). There were no significant differences bet- buffer, using tomato pollen for a proteome analysis. Their
ween the sonication and centrifugation methods of these results showed that the TCA-acetone and phenol protein
three strains (p > 0.05) when compared with FastPrep extraction methods were better than the two tested
method which was found significantly different when methods for tomato pollen proteome analysis (Sheoran et
compared with the other two methods (p < 0.05). As can al., 2009). For preparation of protein extracts from yeasts,
be seen in Table 1, although, all the methods had given there are many methods of extraction from lysis with
suitable amount of proteins, six times higher protein glass beads, to boiling in SDS-PAGE buffer or extraction
concentration, which is important for proteomics, espe- with NaOH and -mercaptoethanol. Kushnirov has repor-
cially before proceeding IEF part was obtained with ted that a mild alkali treatment followed by boiling in a
extraction by FastPrep. While applying the samples for standard electrophoresis loading buffer was efficient,
IEF, getting successful result depends on staining strate- easy and reliable for the electrophoretic analysis of diffe-
gies as well as different protein concentrations rather rent strains of Saccharomyces cerevisiae and of the
than protein amount (Angelika et al., 2004). Extraction of yeast Hansenula polymorpha DL-1 (Kushnirov, 2000).
proteins by FastPrep is also described for LAB in many For determination of protein fraction of microbial cells,
2182 Afr. J. Biotechnol.

the use of many different extraction and analysis used an extraction buffer that is directly used as a
methods is known. For Escherichia coli six different rehydration buffer for polyacrylamide strips used IEF (8 M
protein extraction protocols were tested and compared. urea, 2 M thiourea, 0.5% CHAPS, 10 mM DTT and 0.1%
Comparison was based on the reliability of the methods pH 3-10 ampholytes). Koskenniemi et al. (2009) and
and it was found that cell lysis using the BugBuster McLeod et al. (2010) used only 10 mM Tris-HCl buffer,
protein extraction reagent (Bugbuster) gave the best pH 7.5 and their disruption time was different from ours. If
results (De Mey et al., 2008). there is no FastPrep cell disrupter in laboratory, a method
Methods employing sonication have been used in combining the use of centrifugation with glass beads has
many studies involving the extraction of proteins from been described (Giard et al., 2001) and is more efficient
LAB. While there seem to be as many different lysis than employing just centrifugation alone.
buffer preparations as their are reports in the literature, a Although, the extraction method by FastPrep showed a
consistent application of sonication has been shown in higher efficiency and higher quality in extracting proteins
which samples are generally sonicated three times and compared with the two other methods to check these re-
the cycle involves 2 min of sonication with 3 min rest coveries and to clarify the results of protein concen-
intervals with sample kept on ice. In our study we used trations, proteins have been applied to SDS-PAGE. It can
TrisHCl buffer, pH 7.5, containing 5 mM EDTA and 5 mM be clearly seen from the Coomassie brilliant blue (Figure
MgCl2. In studying the proteome of intestinal probiotic 1) and silver nitrate (data not shown) stained SDS-PAGE
Lactobacillus reuteri strain grown under acid stress gel images, that higher amounts of proteins are obtained
conditions, Lee and Pi (2010) used a Tris-based lysis only when proteins extracted by FastPrep method were
buffer (62.5 mM Tris-HCl, pH 6.8) combined with mec- used (lanes 4, 5 and 6 in Figure 1) as compared to the
hanical disruption by ultrasonication, much as we have other lines in which proteins extracted by two other
done in our study. In contrast, others have used different methods were loaded (lanes 1, 2 and 3, in which the
lysis buffers such as denaturing buffers for 2-DE (7 M proteins extracted by centrifugation is loaded while lanes
urea, 2 M thiourea, 4% (w/v) CHAPS and 50 mM DTT) 7, 8 and 9 are the lanes of proteins extracted by soni-
containing complete protease inhibitors (Yuan et al., cation). However, the lack of protein bands in Figure 1
2006; Wang et al., 2009). In some studies, the cells were does not mean that the other two methods did not work. It
directly sonicated without any use of a special lysis buffer means that protein concentration applied to SDS-PAGE
(De Angelis et al. 2001; Di Cagno et al. 2007). is very low because when pooled, supernatants of the
Centrifugation is another technique used during a samples were concentrated by using speed vacuum
protein extraction method. Of course before the use of a centrifuge to a final volume of 10 l and applied to SDS-
centrifugation technique, the membrane or cell wall must PAGE, visible protein bands were obtained (data not
be broken. The details of the membrane lysis step can be shown). Therefore, when the FastPrep method is un-
different in each protocol as different chemical means available and one needs to use either sonication or cen-
(lysozyme, DTT, etc.) are employed. The details of the trifugation for extracting proteins for proteomics studies, it
centrifugation step also vary, but the treatment with cold is recommended to concentrate cells by use of vacuum
acetone on ice as the last step is generally same in many centrifuge following extraction protocols. Proteins
protocols. In our study, two different buffers were used extracted by FastPrep method have also been applied to
and the final step of the protocol was an ice-cold acetone 2D-PAGE for further confirmation (Figure 2). In this
precipitation for 20 min on ice. Koistinen et al. (2007) and application, only E. faecalis V583 and P. pentosaceus
Plumed-Ferrer et al., (2008) used centrifiguation steps in OZF strains were used. When the proteins of E. faecalis
their proteomic research of the Lactobacillus plantarum V583 and P. pentosaceous OZF extracted by FastPrep
strain; they wash frozen bacterial pellets, treat with were separated by 2D-PAGE, more than 400 protein
lysozyme at 37C for 45 min, followed by DNase spots, with isoelectric points (pI) ranging from 4.0 to 7.0
treatment, with a final centrifugation step after which the and molecular weights (MW) from 0 to 100 kDa, were
supernatant is mixed ice-cold acetone to precipitate the observed. The little disturbance of vertical and horizontal
proteins. This protocol is quite useful for laboratories that streaks as well as regular and reproducible protein spots
may not be equipped with a sonicator, FastPrep cell dis- were seen in the figure (2D gel illustrating the intracellular
ruptor or a pressure cell disrupter. proteins of P. pentosaceus OZF is not given here).
The use of the FastPrep method requires a special The FastPrep method showed higher protein resolu-
instrument. In our study, cells were lysed with glass tion and spot intensity of all proteins. In addition, proteins
beads in a FastPrep cell disruptor. This method has been with less abundance and high molecular weights were
used in many proteomics studies involving LAB (Wang et resolved clearly and detected strongly on 2D gel when
al., 2005; Dahl et al., 2007; Koskenniemi et al., 2009). FastPrep method was used. Besides, differentially
What often varies in the FastPrep method is the type of expressed proteins were detected by presence of clear
buffer used, the ratio of the volume (as an amount) of and well resolved protein spots on 2DE (unpublished
glass beads to the volume of the pelleted cell mass and data). This confirmed that, the method with FastPrep
the disruption time for homogenization. In our study, we extraction was an efficient and reliable method for lysing
 Mehmeti et al. 2183

Figure 1. Representative Coomassie brilliant blue stained SDS-

PAGE illustrating the intracellular proteins of three representative
strains of LAB. The lanes (1 to 9) contains extracts of P.
pentosaceus OZF (lanes 1, 4, 7); E. faecalis V583 (lanes 2, 5, 8)
and L. lactis NIZO 0900 (lanes 3, 6, 9) obtained by centrifugation
(lanes 1 to 3), FastPrep (lanes 4 to 6) and sonication (lanes 7 to 9);
M, prestained broad range protein marker (Bio Labs).

7 pI 4 pI
High
100 kDa

MW

Low
0 kDa

Figure 2. Representative 2D-PAGE illustrating the intracellular proteins of E. faecalis V583

at pI 4 to 7 after lysing cells by FastPrep. See higher protein resolution and spot intensity.
2184 Afr. J. Biotechnol.

and/or extracting proteins of LAB for proteomic protein purification (Chapter 18). Method Enzymol. 463: 285-303.
Hammes WP, Bantleon A, Min S (1990). Lactic acid bacteria in meat
approach and reproducible amounts of bacterial proteins
fermentation. FEMS Microbiol. Rev. 87: 165-174.
can successfully be extracted. Pictures were excellent Hancock LE, Gilmore MS (2006). Pathogenecity of Enterococci. In:
enough to be used in alignment for statistical analyses Gram positive pathogens. Fischetti VA, Novick RP, Ferretti JJ,
and spots well-resolved for MALDI TOF analyses (Figure Portnoy DA, Rood JI (eds). Washington DC, ASM Press, pp. 299-
2). 311.
Haynes PA, Gygi SP, Figeys D, Aebersold R (1998). Proteome
analysis: Biological assay or data archive? Electrophoresis, 19:
1862-1871.
REFERENCES Klaenhammer TR (1988). Bacteriocins of lactic acid bacteria. Biochimie,
70: 337-349.
Abram F, Gunnigle E, OFlaherty V (2009). Optimisation of protein Klaenhammer TR (1993). Genetics of bacteriocins produced by lactic
extraction and 2-DE for metaproteomics of microbial communities acid bacteria. FEMS Microbiol. Rev. 12: 39-86.
from anaerobic wastewater treatment biofilms. Electrophoresis, 30: Koistinen KM, Plumed-Ferrer C, Lehesranta SJ, Karenlampi SOK,
4149-4151. Wright AV (2007). Comparison of growth-phase-dependent cytosolic
Aguirre M, Collins MD (1993). Lactic acid bacteria and human clinical proteomes of two Lactobacillus plantarum strains used infoodand
infection. J. Appl. Bacteriol. 75: 95-107. feed fermentations. FEMS Microbiol Lett. 273: 12-21.
Angelika G, Walter W, Michael JD (2004). Current two-dimensional Koskenniemi K, Koponen J, Kankainen M, Savijoki K, Tynkkynen S, de
electrophoresis technology for proteomics. Proteomics, 4: 3665- Vos VM, Kalkkinen N, Varmanen P (2009). Proteome analysis of
3685. Lactobacillus rhamnosus GG using 2-D DIGE and Mass
Bhaduri S, Demchick PH (1983). Simple and rapid method for disruption Spectrometry shows differential protein production in laboratory and
of bacteria for protein studies. Appl. Environ. Microbiol. 46(4): 941- industrial-type growth media. J. Proteome Res. 8: 4993-5007.
943. Kushnirov VV (2000). Rapid and reliable protein extraction from yeast.
Bradford MM (1976). A rapid and sensitive method for the quantification Yeast, 16: 857-860.
of microgram quantities of protein utilizing the principle of protein- Laemmli UK (1970). Cleavage of structural proteins during assembly of
dye binding. Anal. Biochem. 72: 248-254. the head of bacteriophage T4. Nature, 227: 680-685.
Cai-yun H, Jian-guo Z, Ai-guo D, Ji-yan Y, Dao-shan Z (2005). Larsen N, Boye M, Siegumfeldt H, Jakobsen M (2006). Differential
Comparison of methods for protein exraction from pine needles. expression of proteins and genes in the lag phase of Lactococcus
Forestry Studies in China, 7(4): 20-23. lactis subsp. lactis grown in synthetic medium and reconstitued skim
Cilia M, Fish T, Yang X, Mclaughlin M, Thannhauser TW, Gray S milk. Appl. Environ. Microbiol. 72: 1173-1179.
(2009). A comparision of protein extraction methods suitable for gel- Lauber WM, Carroll JA, Dufield DR, Kiesel JR, Radabaugh MR, Malone
based proteomic studies of aphid proteins. J. Biomol. Tech. 20(4): JP (2001). Mass spectrometry compatibility of two-dimensional gel
201-215. protein stains. Electrophoresis, 22: 906-918.
Daeschel MA (1989). Antimicrobial substances from lactic acid bacteria Lee K, Pi KB (2010). Effect of transient acid stress on the proteome of
for use as food preservatives. Food Technol. 43: 164-167. intestinal probiotic Lactobacillus reuteri. Biochemistry (Moscow),
Dahl JL, Tengra FK, Dutton D, Yan J, Andacht TM, Coyne L, Windell V, 75(4): 558-564.
Garza AG (2007). Identification of major sporulation proteins of Ljungh A, Wadstrm T (2006). Lactic acid bacteria as probiotics. Curr.
Myxococcus xanthus using a proteomic approach. J. Bacteriol. Issues Intestinal Microbiol. 7: 73-90.
189(8): 3187-3197. McLeod A, Zagorec M, Champomier-Verg MC, Naterstad K, Axelsson L
De Angelis M, Bini L, Pallini V, Cocconcelli PS, Gobbetti M (2001). The (2010). Primary metabolism in Lactobacillus sakei food isolates by
acid-stress response in Lactobacillus sanfranciscensis CB1. proteomic analysis. BMC Microbiol. 10: 120-130.
Microbiology, 147: 1863-1873. Moellering RC (1992). Emergence of Enterococcus as a significant
De Mey M, Lequeux GJ, Maertens J, De Muynck CI, Soetaer WK, pathogen. Clin. Infect. Dis. 14: 1173-1178.
Vandamme EJ (2008). Comparison of protein quantification and Natarajan S, Xu C, Caperna TJ, Garrett WM (2005). Comparison of
extraction methods suitable for E. coli cultures. Biologicals, 36: 198- protein solubilization methods suitable for proteomic analysis of
202. soybean seed proteins. Anal. Biochem. 342: 214-220.
Devriese LA, Pot B (1995). The genus Enterococcus. In: The lactic acid O'Farrell PH (1975). High resolution two-dimensional electrophoresis of
bacteria, vol. 2. The genera of lactic acid bacteria. Wood BJB, proteins. J. Biol. Chem. 250: 4007-4021.
Holzapfel WH (eds). Blackie Academic, London, pp. 327-367. Piard JC, Desmazaud M (1991). Inhibition factors produced by lactic
Di Cagno R, De Angelis M, Limitone A, Minervini F, Simonetti MC, acid bacteria. 1. Oxygen metabolites and catabolism end-products.
Buchin S, Gobbetti M (2007). Cell-cell communication in sourdough Lait, 71: 525-541.
lactic acid bacteria: A proteomic study in Lactobacillus Piard JC, Desmazeaud M (1992). Inhibiting factors produced by lactic
sanfranciscensis CB1. Proteomics, 7: 2430-2446. acid bacteria. 2. Bacteriocins and other antibacterial substances.
Dierick JF, Dieu M, Remacle J, Raes M, Roepstorff P, Toussaint O Lait, 72: 113-142.
(2002). Proteomics in experimental gerontology. Exp. Gerontol. 37: Plumed-Ferrer C, Koistinen KM, Tolonen TL, Lehesranta SJ,
721-734. Karenlampi SO, Makimattila E, Joutsjoki V, Virtanen V, Wright AV
Duch O, Trmoulet F, Glaser P, Labadie J (2002). Salt stress proteins (2008). Comparative study of sugar fermentation and protein
induced in Listeria monocytogenes. Appl. Environ. Microbiol. 68(4): expression patterns of two Lactobacillus plantarum strains grown in
1491-1498. three different media. Appl. Environ. Microbiol. 74(17): 5349-5358.
Dutka-Malen S, Evers S, Courvalin P (1995). Detection of glycopeptide Renzone G, DAmbrosio C, Arena S, Rullo R, Ledda L, Ferrara L,
resistance genotypes and identification to the species level of Scalonia A (2005). Differential proteomic analysis in the study of
clinically relevant enterococci by PCR. J. Clin. Microbiol. 33: 24-27. prokaryotes stress resistance. Ann. Ist. Super Sanit. 41(4): 459-
FAO/WHO working group (2001). Report of a Joint FAO/WHO expert 468.
consultation on evaluation of health and nutritional properties of Sambrook J, Fritsch EF, Maniatis T (1992). Molecular Cloning: A
probiotics in food including powder milk with live lactic acid bacteria. Laboratory Manual.
World Health Organization and Food and Agriculture Organization of Schaberg DR, Culver DH, Gaynes RP (1991). Major trends in the
the United Nations, London, Ontario, Canada. microbial etiology of nosocomial infections. Am. J. Med. 912(Suppl.
Giard JC, Rince LJM, Rince A, Pichereau V, Benachour A, Leboeuf C, 3B): 72S-75S.
Flahaut S, Auffray Y, Hartke A (2001). The stress proteome of Sharpe ME, Pettipher GL (1983). Food spoilage by lactic acid bacteria.
Enterococcus faecalis. Electrophoresis, 22: 2947-2954. In: Rose AH (ed). Academic Press, Inc., New York, Food Microbiol.
Grabskia AC (2009). Advances in preparation of biological extracts for p. 200.
 Mehmeti et al. 2185

Sheoran IS, Ross ARS, Olson DJH, Sawhney VK (2009). Compatibility Wang Y, Delettre J, Guillot A, Corrieu G, Beal C (2005). Influence of
of plant protein extraction methods with mass spectrometry for cooling temperature and duration on cold adaptation of Lactobacillus
proteome analysis. Plant Sci. 176: 99-104. acidophilus RD758. Cryobiology, 50: 294-307.
Stiles ME, Hastings JW (1991). Bacteriocin production by lactic acid Wang X, Li X, Deng X, Han H, Shi W, Li Y (2007). A protein extraction
bacteria: potential for use in meat preservation. Trends Food Sci. method compatible with proteomic analysis for the euhalophyte
Technol. 2: 247-251. Salicornia europaea. Electrophoresis, 28: 3976-3987.
Tailor SAN, Bailey EM, Rybak MJ (1993). Enterococcus, an emerging Wang X, He X, Jiang Z, Wang J, Chen X, Liu D, Wang F, Guo Y, Zhao
pathogen. Ann. Pharmacother. 27: 1231-1242. J, Liu F, Huang L, Yuan J (2009). Proteomic analysis of the
Van de Guchte M, Serror P, Chervaux C, Smokvina T, Ehrlich SD, Enterococcus faecalis V583 strain and clinical isolate V309 under
Maguin E (2002). Stress responses in lactic acid bacteria. A. van vancomycin treatment. J. Proteome Res. 9: 1772-1785.
Leeuw. J. Microb. 82: 187-216.
Yuan J, Zhu L, Liu X, Li T, Zhang Y, Ying T, Wang B, Wang J, Dong H,
Feng E, Li Q, Wang J, Wang H, Wei K, Zhang X, Huang C, Huang
P, L Huang L, Zeng M, Wang H (2006). A proteome reference map
and proteomic analysis of Bifidobacterium longum NCC2705. Mol.
Cell. Proteomics, 5: 1105-1118.