In vitro and in situ antimicrobial action and mechanism of glycinin and its basic subunit

International Journal of Food Microbiology 154 (2012) 19–29

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

In vitro and in situ antimicrobial action and mechanism of glycinin and its basic subunit Mahmoud Z. Sitohy a,⁎, Samir A. Mahgoub b, Ali O. Osman a a b

Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt Microbiology Department, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt

a r t i c l e

i n f o

Article history: Received 8 July 2011 Received in revised form 26 November 2011 Accepted 4 December 2011 Available online 13 December 2011 Keywords: Antimicrobial Cell killing Glycinin Basic subunit Listeria Salmonella

a b s t r a c t Glycinin, basic subunit and β-conglycinin were isolated from soybean protein isolate and tested for their antimicrobial action against pathogenic and spoilage bacteria as compared to penicillin. The three fractions exhibited antibacterial activities equivalent to or higher than penicillin in the next order; basic subunit > glycinin > β-conglycinin with MIC of 50, 100 and 1000 μg/mL, respectively. The IC50% values of the basic subunit, glycinin and β-conglycinin against Listeria\ monocytogenes were 15, 16 and 695 μg/mL, against Bacillus subtilis were 17, 20, and 612 μg/mL, and against S. Enteritidis were 18, 21 and 526 μg/mL, respectively. Transmission electron microscopy images of L. monocytogenes and S. Enteritidis exhibited bigger sizes and separation of cell wall from cell membrane when treated with glycinin or basic subunit. Scanning electron microscopy of B. subtilis indicated signs of irregular wrinkled outer surface, fragmentation, adhesion and aggregation of damaged cells or cellular debris when treated with glycinin or the basic subunits but not with penicillin. All tested substances particularly the basic subunit showed increased concentration-dependent cell permeation assessed by crystal violet uptake. The antimicrobial action of glycinin and basic subunit was swifter than that of penicillin. The cell killing efficiency was in the following descending order; basic subunit > glycinin > penicillin > β-conglycinin and the susceptibility of the bacteria to the antimicrobial agents was in the next order: L. monocytogenes > B. Subtilis > S. Enteritidis. Adding glycinin and the basic subunit to pasteurized milk inoculated with the three bacteria; L. monocytogenes, B. Subtilis and S. Enteritidis (ca. 5 log CFU/mL) could inhibit their propagation after 16–20 days storage at 4 °C by 2.42–2.98, 4.25–4.77 and 2.57–3.01 log and by 3.22–3.78, 5.65–6.27 and 3.35–3.72 log CFU/mL, respectively. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Plants produce a wide array of defense protein to control the attacks of microbial pathogens. As a result several classes of proteins with antibacterial and/or antifungal properties have been isolated, identified and recommended as antimicrobial agents (Kumarasamy et al., 2002). Plant antimicrobial cationic peptides or proteins (AMPs) which constitute a heterogeneous class of low molecular weight proteins, are important components of innate defense system directly interfering with the growth, multiplication and spread of microbial organisms (Garcia-Olmedo et al., 1998). The action of AMPs targets mainly the bacterial cell membranes (Zasloff, 2002) due to their positive net charge enabling the binding and permeation of negatively charged phospholipid membranes of bacteria (Shai, 2002). The frequent and massive use of antibiotics gave rise to multidrug resistant bacteria (Charpentier and Courvalin, 1999). Hence, the quest for new antibacterial drugs and agents is a continuous mission. A

⁎ Corresponding author. Tel.: + 20 552287567. E-mail address: [email protected] (M.Z. Sitohy). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.12.004

highly basic small protein (finotin) was purified from seeds of Clitoria ternatea and found to have a broad and potent inhibitory effect on the growth of various important fungal pathogens (Kelemu et al., 2004). Small seed basic proteins isolated from Robinia pseudoacacia L. Rozynskiana (Leguminosae), manifested in vitro antibacterial activity against seven bacteria (Talas-Oğraş et al., 2005). Puroindoline A and puroindoline B from plant seeds as well as lactoferrin and lysozyme were proved as in-vivo antimicrobial agents against Listeria monocytogenes (Palumbo et al., 2010). Antibacterial activities are not restricted to low molecular weight proteins but they can include high molecular weight ones e.g. achacin and aplysianin (56 and 320 KD, respectively) isolated from marine animals (Kamiya et al., 1986). Hydrophobic high molecular weight proteins (27 and 31 KD) isolated from fish were associated with strong pore-forming antibacterial activity (Ebran et al., 1999). Soybean storage proteins lie within the class of high molecular weight proteins. Globulins represent the majority of seed soybean proteins and can be subdivided into two main types according to their sedimentation coefficients: glycinin (11S) and β-conglycinin (7S). Glycinin has a molecular mass of 360 KD and is composed of 6 constituent subunits (A1aB2, A2B1a, AB, A5A4B3, A3B4 and A1bB2),

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each of which consists of an acidic and a basic polypeptide, linked together by a disulfide bond (Kitamura and Shibasaki, 1975 and Nielsen, et al., 1989). The relative molecular masses of basic and acidic subunits are 20 and 34 KD, respectively (Kitamura et al., 1976). The subunit β-conglycinin is a trimeric glycosylated protein with a molecular mass of 150–200 KD (Ladin et al., 1987; Utsumi, 1992). The antimicrobial activities of these two main soybean protein subunits have never been investigated before due to the lack of direct traits of AMPs such as the cationic nature, low molecular weight and hydrophobicity. However, the basic subunit of glycinin is both hydrophobic and cationic and may be able to react with the bacterial cell wall and membrane in spite of its attachment to the acidic subunits. Based on previous reports that basic proteins or peptides can have antimicrobial activity (Dhatwalia et al., 2009), the objective of the current work was to specify the potential antimicrobial action of soybean subunits, particularly glycinin which has half of its subunits as basic polypeptides or the separated basic subunit against bacteria. These fractions were tested against L. monocytogenes and Salmonella enterica subsp enterica serovar Enteritidis and one spoilage bacterium (Bacillus subtilis) as compared to penicillin. L. monocytogenes causes epidemic listeriosis (Schuchat et al., 1991; Denny and McLauchlin, 2008). S. Enteritidis is the cause of salmonellosis, another foodborne disease (Scallan et al., 2011) and some strains of B. subtilis may occasionally cause food poisoning (Pavic et al., 2005). 2. Materials and methods 2.1. Materials Soybean (Glycine max L.) seeds were purchased from local market, Zagazig city, Sharkia, Egypt. L. monocytogenes Scott A and S. enterica subsp. enterica serovar Enteritidis PT4 strains used in this study were kindly obtained from Prof. George Nychas, Laboratory of Food Microbiology and Biotechnology, Department of Food Science and Technology, Agricultural University of Athens, Athens, Greece. B. subtilis was isolated from soil and identified according to Bergy's Manual systematic of bacteriology. For experimental use L. monocytogenes Scott A, S. Enteritidis and B. subtilis cultures were maintained on nutrient agar slopes at 4 °C and subcultured every 4 weeks. 2.2. Isolation of soybean protein subunits Soybean seeds were ground to pass through a 1 mm 2 sieve and the resulting powder was defatted using a mixed solvent of chloroform: methanol (3:1 v/v) for 8 h. Soybean protein isolate was separated using the procedure of Johnson and Brekke (1983). The total nitrogen was determined in soybean protein isolate according to AACC (2000) and multiplied by the conversion factor 6.25 to get the total protein content. Soybean protein isolate was used for the isolation of glycinin and β-conglycinin according to Nagano et al. (1992). Basic subunits were separated from the glycinin according to methods described by Damodaran and Kinsella (1982) with some modifications. Glycinin was dissolved in 30 mM Tris buffer (pH 8.0) containing 15 mM β-mercaptoethanol (at 0.5% w/v). The protein solution was heated to 90 °C for 30 min and then centrifuged at 10000 x g at 4 °C for 20 min. The precipitate (basic subunit) was washed twice with 30 mM Tris buffer (pH 8.0), suspended in distilled water, and freeze-dried. 2.3. Agar well-diffusion assay Soybean protein fractions were tested for antimicrobial activity by conventional well-diffusions methods against L. monocytogenes, S. Enteritidis and B. subtilis (Nanda and Saravanan, 2009; Zgoda and Porter, 2001) with some modifications. The pure cultures of bacterial strains were subcultured on MHB (Mueller Hinton broth) and

incubated on a rotary shaker at 200 rpm at 37 °C (L. monocytogenes and S. Enteritidis) or 28 °C (B. subtilis) for 24 h. An aliquot (0.1 mL) of the last culture was transferred into 10 mL MHB and incubated at 37 °C (L. monocytogenes and S. Enteritidis) or 28 °C (B. subtilis) for 24 h to reach a count of 1.05 × 10 9 CFU/mL. Each strain was spread uniformly onto individual plates using sterile cotton swabs. Wells of 6-mm diameter were made on Mueller Hinton Agar (MHA) plates using gel puncture. Aliquots (40 μL) of different protein concentrations (0, 25, 50, 100, 250, 500 and 1000 μg/mL) were transferred onto each well of all plates. After incubation at 37 °C (L. monocytogenes and S. Enteritidis) or 28 °C (B. subtilis) for 24 h, the different levels of zones of inhibition were measured using a transparent ruler and the diameter was recorded in mm to conclude the minimum inhibitory concentration (MIC). Penicillin was used as a positive control with the concentrations of 0, 25, 50, 100, 250 and 500 μg/mL. 2.4. Minimum inhibition concentration (MIC) Soybean protein isolate or its fractions (glycinin, β-conglycinin and the basic subunit) was tested for antimicrobial activity by conventional broth dilution assay against L. monocytogenes, S. Enteritidis and B. subtilis (Murray et al., 1995). Minimum inhibitory concentration (MIC) was evaluated using standard inoculums of 1 × 10 5 CFU/ mL (Rex et al., 2001). Serial dilutions of the test compounds, previously dissolved in sterilized distilled water, were prepared to final concentrations of 0, 25, 50, 100, 250, 500 and 1000 μg/mL of MHB. To each tube, 100 μL of the inoculum was added and incubated for 18–24 h at 37 °C (L. monocytogenes and S. Enteritidis) or at 28 °C (B. subtilis). At the end of incubation time, MIC was visually identified as the lowest concentration of the test compound which inhibits the visible growth and confirmed by measuring the OD600 of all treatments. Tests using sterilized distilled water as negative control and penicillin as positive control were carried out in parallel. All tests were performed in triplicate. 2.5. Rate of cell kill assay The rate of cell kill of the studied bacteria strains upon treatment with protein fractions and penicillin was determined as described by Culafic et al. (2005) with some modifications. Overnight growth cultures of the three studied bacteria were grown in MHB up to 2 × 10 9 CFU/mL. An aliquot (100 μL) of the last suspension was inoculated into fresh media and grown up to 10 5 CFU/mL (Rex et al., 2001). All cultures were treated with 100, 200 and 300 μg/mL of the antimicrobial agent (equivalent to 1×, 2 × and 3 × MIC). Control tubes were similarly prepared but without adding the antimicrobial protein. All treatments were incubated either at 37 °C (L. monocyotgenes and S. Enteritidis) or 28 °C (B. Subtilis) for 24 h and OD600 was measured using JENWAY 6405 UV/visible spectrophotometer (UK) at the end of incubation time. All the determinations were done in triplicates. 2.6. Crystal violet assay Alteration in membrane permeability was determined by crystal violet assay (Vaara and vaara, 1981). After overnight growth in MHB at 37 °C (L. monocyotgenes and S. Enteritidis) or 28 °C (B. Subtilis), the stationary phase cultures of the strains were adjusted to a concentration of 1 × 10 9 CFU/mL. An aliquot (1.5 mL) of the cell culture was centrifuged at 4500 × g for 15 min at 4 °C and the pellet was separated and washed three times before finally suspending in 1.5 mL peptone buffer solution (PBS) (0.1% w/v, pH 7.4). All antimicrobial agents were added to the cell suspension at 0, 50 and 100 μg/mL and incubated at 37 °C (L. monocyotgenes and S. Enteritidis) or 28 °C (B. Subtilis) for 30 min. To follow the kinetics of the permeation, all antimicrobial agents as well as EDTA were added to the cell suspension at 100 μg/mL and incubated at 37 °C (L. monocyotgenes

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and S. Enteritidis) or 28 °C (B. Subtilis) for 15, 30, 60,120 and 240 min. Respective controls were similarly prepared but without treatments. At the end of incubation intervals, the cells were harvested by centrifugation at 9300 ×g for 5 min and re-suspending in PBS (pH 7.4) containing 10 μg/mL of crystal violet. The cell suspensions were incubated for 10 min at 37 °C (L. monocyotgenes and S. Enteritidis) or 28 °C (B. Subtilis) and then centrifuged at 13400 ×g for 15 min before separating the supernatant and measuring its OD590 using JENWAY 6405 UV/visible spectrophotometer (UK). The OD value of the original crystal violet solution was recorded and considered as 100%. The percentage of crystal violet uptake of all the samples was calculated using the following formula:

OD value of the samples X 100= OD value of crystal violet solution

21

2.9. Scanning electron microscopy (SEM) Scanning electron microscopy (SEM) analysis was performed (Benli et al., 2008) to further explore the mode of action of the studied proteins on B. subtilis cell morphology. An aliquot of 0.1 mL of B. subtilis culture was inoculated into 10 mL MHB and cultivated at 28 °C with gentle agitation for 12 h. The Cells were collected at 4500 ×g for 15 min at 4 °C. Cells were washed three times and resuspended in PBS (pH 7.4) at the same volume. The antimicrobial agents (100 μg/mL) were added to the cell suspension and incubated at 28 °C with gentle agitation for 4 h. The control sample was prepared similarly but without treatments. Bacterial cells were recovered by centrifugation at 4500 × g for 15 min at 4 °C, washed with PBS (pH 7.4) and fixed in 2.5% glutaraldehyde in PBS. The fixed bacterial pellet was then dehydrated in graded alcohol series, dried and mounted onto stubs using double sided carbon tape, coated with thin layer of gold. All cell samples were examined in Scanning electron microscope (JEOL-SEM, Japan).

2.7. Cell lysis assay 2.10. SDS-PAGE of bacterial proteins The release of UV-absorbing material concentrations in the supernatant was measured by JENWAY 6405 UK, UV/visible spectrophotometer to indicate the rate of cell lysis (Zhou et al., 2008). The three bacterial strains were grown overnight at their optimal temperatures to reach a concentration of 1 × 10 9 CFU/mL. The Cells were collected after centrifugation at 4500 ×g for 15 min at 4 °C, washed three times and re-suspended in PBS (pH 7.4) at the same original volume. The antimicrobial agents were added (0, 50 and 100 μg/mL) to the cell suspensions and incubated at the optimal temperature (37 or 28 °C) for 30 min. To study the time course of cell lysis, the antimicrobial agents were added (100 μg/mL) to cell suspensions and incubated at optimal temperatures (37 or 28 °C) for 15, 30, 60,120 and 240 min. The control samples were prepared similarly without treatments. The cells were harvested at 13400 ×g for 15 min and OD260 value of the supernatant was taken as a percentage of the extracellular UV-absorbing materials released by cells which expresses cell lysis. 2.8. Transmission electron microscopy (TEM) L. monocytogenes and S. Enteritidis were grown on MHB and incubated at 37 °C to reach maximum level of 1 × 10 9 CFU/mL. Concentrations were then adjusted to 10 8 CFU/mL using PBS. The total protein, basic subunit, glycinin, β-conglycinin and penicillin (100 μg/mL) were added to the cell suspension and incubated at 37 °C for 4 h. The control samples were prepared similarly without treatments. Bacterial cells were collected after centrifugation at 4500 ×g for 10 min, fixed in glutaraldehyde (2.5% in 0.1 M phosphate buffer, pH 7.4) for 1 h, rinsed thrice for 10 min with 0.1 M phosphate buffer (pH 7.4) and post-fixed with 1% osmium tetraoxide for 2 h at 4 °C. After fixation, bacterial cells were rinsed thrice for 10 min with 0.1 M phosphate buffer (pH 7.4) and then dehydrated sequentially using 30%, 50%, 70% and 95% acetone for 15 min each. Next, the cells were dehydrated three times for 30 min with 100% acetone. Subsequently, cells were treated with propylene oxide twice for 10 min at 4 °C and sequentially infiltrated with a mixture of propylene oxide: Durcupan's ACM epoxy resin (3:1, 1:1 and 1:3) for 45 min. Polymerization of the resin to form specimen blocks was performed in an oven at 60 °C for 72 h. The specimen blocks were hand trimmed with a razor blade and sectioned with a diamond knife in a Reichert Ultracut R ultramicrotome (Leica, Wetzler, Germany). Thin sections (70–80 nm) were placed on 300 mesh copper grids. The sections were stained for 15–20 min in uranyl: ethyl alcohol (1:1), then washed three times with saline solution for 2 min and then incubated in a drop of Reynold's lead citrate and examined using a Transmission Electron Microscope (JEOL-TME-2100 F, Japan).

To follow the time-course of the antimicrobial action of antibacterial agents, SDS-PAGE of the bacterial proteins was carried out after different interval of incubation with the antimicrobial substance according to Vaara and vaara (1981). The cultures of L. monocyotgenes and S. Enteritidis strains were grown 1 × 10 9 CFU/mL after overnight incubation at 37 °C. An aliquot (0.1 mL) of the bacterial suspension was inoculated into 10 mL of fresh MHB and incubated at 37 °C for 24 h. Bacterial cells were separated by centrifugation at 13400 ×g for 10 min and re-suspended in saline solution (8.5 g NaCl/L) at the same original volume. The antibacterial agents were added to all cells suspension except control at 100 μg/mL and incubated at 37 °C for 15, 30, 60,120 or 240 min. An aliquot of 50 μL of the bacterial suspension was combined with 25 μl of the sample buffer pH-6.8 (1 M Tris–HCl, 50% glycerol, 10% SDS, 10% β-mercaptoethanol, 0.1% Bromophenol blue), heated at 100 °C for 10 min and loaded in 3 and 12% SDS-PAGE for stacking and resolving gel, respectively. After running at 10 mA on the stacking gel and 20 mA on the resolving gel protein bands were visualized on the gels by Coomassie Brilliant Blue R250 (Laemmli, 1970). 2.11. Pathogens inoculation in pasteurized milk The cultures of L. monocytogenes ScottA, S. Enteritidis PT4 and B. subtilis were activated by three successive transfers in tryptic soy broth (Biolife, Italy) at 37 or 28 °C for 24 h. Bacterial cells were harvested by centrifugation (10.000 ×g, 10 min, 4 °C), washed three times and resuspended in Ringer's solution (Lab M, Bury, UK), 10 mL final volume. A final inoculum was prepared by serially diluting in Ringer's solution to a level of 8 log CFU/mL. 2.12. Milk storage under refrigeration conditions Bovine raw milk (2.5 L) was immediately obtained after milking, maintained 1 h at 4 °C before pasteurization at 85 °C for 5 min. Pasteurized milk was distributed into 25 portions (100 mL), divided into five groups of five bottles each and transferred in sterile screwcapped bottles. The first group was not treated and served as negative control. The second group received penicillin (0.5%, w/v, equivalent to 5 mg/mL) and served as the positive control. The third, fourth and fifth group received glycinin, β-conglycinin and basic subunit (0.5%, w/v, equivalent to 5 mg/mL), respectively. All bottles were equally inoculated with 0.1 mL of a mixed culture of L. monocytogenes ScottA, B. subtilis and S. Enteritidis PT4 so that the final count of each becomes ca. 5 log CFU/mL (4.76, 4.94, and 4.60 CFU/mL, respectively). All bottles were preserved at 4 °C for 20 days where samples were

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withdrawn under aseptic conditions each four days for the microbiological assay. L. monocytogenes was counted on polymyxin–acriflavin– lithium chloride–ceftazidime–aesculin–mannitol agar (PALCAM agar, Biolife, 401604, Italy) after incubation for 24 h at 37 °C and confirmed according to ISO 11290. S. Enteritidis was counted on Xylose Lysine Deoxycholate (XLD agar, Merck, 1.05287, Ger.) for 24 h at 37 °C. B.subtilis was counted after heating the serial dilution of the milk samples at 80 °C for 15 min then plating on plate count agar and incubation for 24 h at 28 °C.

β−conglycinin Glycinin Basic subunit Penicillin

32

2.13. Statistical analysis

L. monocytogenes

22

24

All experiments were performed in triplicates and results were expressed by the mean plus the standard error. Data were statistically analyzed using ANOVA variance analysis through the general linear models (GLM) procedure of the statistical analysis system software (SAS version 9.1, SAS Institute, Inc., 2003). Least significant differences were used to separate means at p b 0.05.

16

8

3. Results

B. subtilis

32

24

16

8

0

32

S. Enteritidis

The SDS-PAGE patterns of total and subunits of soybean proteins are shown in Fig. 1. The pattern of the total protein shows two main fractions (Glycinin & β-conglycinin) with their constituting subunits. The molecular masses of glycinin subunits are 21 and 34 KD, corresponding the basic and acidic subunits. The molecular mass of the β-conglycinin subunits ranges from 50 to 65 KD. Isolated glycinin showed pure band with the two main subunits, basic and acidic corresponding to the same previous found molecular masses (21 and 34 KD). Further isolation of these two subunits indicated good separation. It is pertinent to mention that this electrophoretic procedure shows only the constituting subunits since it was conducted in the presence of β-mercaptoethanol. Native electrophoresis showed the total molecular masses of every main subunit (glycinin and βconglycinin) corresponded to 150 and 360 KD, respectively (Data not shown). The pH-solubility curves of the three fractions show that glycinin has an isoelectric point of 6.5 while the basic subunits has it around pH 8.5 compared to pH 4.5 for soybean protein isolate (data not shown).

Inhibition zone diameter (mm)

0

3.1. Chemical specification of soybean protein subunits

24

3.2. Antimicrobial action Using agar well diffusion assay (Fig. 2), the three tested protein fractions induced inhibition zones against three tested bacteria

16

8 KD

St

SPI

Glycinin

BS

AS

0

50

100

250

500

1000

Substance concentration (μg/ml) Fig. 2. Agar well diffusion assay of the antimicrobial action of cationic soybean protein (0–1000 μg/mL) against L. monocytogenes ScottA, B. subtilis and S. Enteritidis PT4 as compared to penicillin.

Fig. 1. SDS-PAGE electrophoretic patterns of soybean protein isolate (SPI), and its fractions; glycinin, basic and acidic subunits (BS and AS).

(L. monocytogenes, B. subtilis and S. Enteritidis). The diameter of the inhibition zones increased proportionally with the increase of the substance concentration. The minimum inhibitory concentration (MIC) of glycinin was 100 μg/mL against the three bacteria indicating that the antibacterial action of the substances begins at very low

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concentration in the same level of penicillin. β-conglycinin showed very high MIC (1000 μg/mL) while the basic subunit recorded the least MIC (50 μg/mL) indicating its greater antimicrobial activity. The substance concentration effect (0–1000 μg/mL) of the three protein fractions against the growth of the three bacteria as compared to penicillin was also evaluated during 24 h of incubation at 37 °C using the conventional broth dilution assay (Fig. 3). Concentrationdependent effect can be observed in all cases. The substance concentration corresponding to 50% inhibition of L. monocytogenes (G+) (IC50%) was 15 μg/mL for penicillin against 16 and 15 μg/mL for glycinin and basic subunit while that of β-conglycinin was considerably higher (695 μg/mL). Close IC50% levels were observed against B. subtilis (G +), 20 μg/mL for glycinin and penicillin and 17 μg/mL for the basic subunit while a relatively higher value for β-conglycinin (612 μg/mL). The IC50% of penicillin against S. Enteritidis (G-) was relatively higher (85 μg/mL) than against the other two studied bacteria (G+)

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while glycinin and basic subunits recorded values within the same level (21 and 18 μg/mL, respectively) indicating that these two protein fractions are more potent against the (G-) Salmonella than penicillin. β-Conglycinin recorded a considerably higher value (526 μg/ mL). The time-growth curves (Fig. 4) of the three bacteria were followed during 24 h at 37 °C as influenced by the presence of the protein fractions (100 μg/mL). Similar reducing effects of glycinin, basic subunit and penicillin were monitored against the three bacteria starting after 6 h of incubation at 37 °C while β-conglycinin was still less effective. The antibacterial action of the three protein fraction starts at early groth stages of the three bacteria particularly with glycinin and the basic subunit. Penicillin was less effective against the growth curve of the G- (S. Enteritidis) than the G + (L. monocytogenes and B. subtilis) bacteria.

Control β-conglycinin Glycinin Basic subunit Penicillin

Penicillin β-Conglycinin Glycinin Basic subunit

1.4

L. monocytogenes

1.2

L. monocytogenes

1.2 1.0 0.8 0.6 0.4

0.9

0.6

0.3

0.2

0.0

0.0 0.4 1.0

0.4

OD600

B. subtilis

OD 600

0.6

B. subtilis

0.3

0.8

0.2

0.1 0.2 0.0

0.0 1.0 0.8 0.6 0.4

S.Enteritidis

S. Enteritidis

1.2

0.9

0.6

0.3 0.2 0.0

0.0 0

200

400

600

800

1000

Concentration (µg ml-1) Fig. 3. Broth tube dilution assay of the antimicrobial activity of soybean protein fractions; glycinin, β-conglycinin and basic subunit as compared to penicillin against L. monocytogenes Scott A, B. subtilis and S. Enteritidis PT4.

0

5

10

15

20

25

Incubation time (h) Fig. 4. Growth curve of L. monocytogenes Scott A, B. subtilis and S. Enteritidis PT4 during 24 h at 37 °C in the presence of 100 μg/mL of glycinin, β-conglycinin, basic subunit and penicillin.

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3.3. TEM and SEM TEM (transmission electron microscope) images of L. monocytogenes and S. Enteritidis were taken after treating with protein fractions at 100 μg/mL for 4 h at room temperature (Fig. 5). Different signs of cell wall and membrane deformation could be monitored. The most deformation signs were most evident with glycinin and basic subunit manifested by cell wall and cell membrane disintegration, loss of regular cellular shapes, bigger cell sizes and separation of cell wall from cell membrane. Cells treated with β-conglycinin were least affected and showed normal outer shape. Cells treated with penicillin showed less signs of deformations which were generally more evident in case of the (G +) L. monocytogenes than in case of (G-) S. Enteritidis. SEM examination was used to follow the morphological changes of an overnight culture of B. subtilis (10 5 CFU/mL) induced by the treatment with soybean protein fractions or penicillin for 4 h at room temperature (Fig. 6). Control cells were morphologically regular and typical. Glycinin-treated cells showed signs of irregular wrinkled outer surface, fragmentation, adhesion and aggregation of damaged cells or cellular debris. More evident irregular wrinkled outer surface, fragmentation, cellular damage and adhesion were noticeable by the treatment with the basic subunit. Pencillin treated B. Subtilis did not show clear signs of irregularity of the outer-surface but rather reduced cellular sizes when comparing with control at different magnification powers. Similar results were obtained for the other two studied bacteria (data not shown). 3.4. Crystal violet uptake and cell lysis

Control

β-Conglycinin

3.5. Cell killing efficiency The cell kill efficiency of three concentrations (100, 200 and 300 μg/mL) against a standard inoculums of bacteria (10 5 CFU/mL) was determined (Fig. 9). It can be observed that the killing efficiency was in the following descending order; basic subunit > glycinin > penicillin > β-conglycinin. There was a concentration effect but the slope of increasing was very little indicating that the least concentration is quite enough for an important antimicrobial activity. The susceptibility of the bacteria to the antimicrobial agents was in the next order: L. monocytogenes > B. Subtilis > S. Enteritidis. Glycinin

Basic subunit

Penicillin

S. Enteritidis

L. monocytogenes

Crystal violet uptake by the bacterial cells after treatment with the antimicrobial agents was used to assess cell permeation (Fig. 7). All

tested substances showed increased concentration-dependent cell permeation where the basic subunit was the most acting. Penicillin permeation effect on S. Enteritidis (G-) was less than on L. monocytogenes (G+). Glycinin was either equal to or better than penicillin in permeation effect. The most effective substance concentration was in the range 50–100 μg/mL. Maximum permeation rates were associated with the basic subunit followed by glycinin which was in the same level of penicillin or even higher in case of S. Enteritidis. The effect of this treatment was conceived after 30 min of treating the bacterial cells with the substance. So, it is intriguing to follow the effect of the substances (100 μg/mL) on the cellular membrane during longer period (30–240 min) of treatment (Fig. 8). It can be noticed that prolonging the contact time up to 60 min could increase the permeation of the bacterial cell membranes treated with glycinin or the basic subunits. Cells treated with penicillin reached the maximum crystal violet permeation after a longer period (120 min). The kinetic of cell lysis (Fig. 8) simulated that of crystal violet uptake, i.e. more rapid cell lysis with glycinin and the basic subunit than with penicillin (Fig. 8). Further time prolongation did not produce any additive effect.

Fig. 5. Transmission Electron Microscopy (TEM) of L. monocytogenes Scott A and S. Enteritidis PT4 treated with 1 MIC (100 μg/mL) of glycinin, β-conglycinin, basic subunit and penicillin for 4 h at room temperature.

Glycinin

Basic subunit

Penicillin

20 000 X

10 000 X

Control

Fig. 6. Scanning Electron Microscopy (SEM) of B. subtilis treated with 100 μg/mL of glycinin, β-conglycinin, basic subunit and penicillin for 4 h at room temperature.

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Total protein EDTA Penicillin β-Conglycinin Glycinin Basic subunit

L. monocytogenes

90 80 70 60 50

fraction and then inoculated with 10 5 CFU/mL of L. monocytogenes, B. Subtilis and S. Enteritidis and stored at 4 °C for 30 days. The presence of glycinin and basic subunit could evidently preclude the proliferation of the three inoculated bacteria in milk while β-conglycinin was less effective. The basic subunits were the most effective. The bacterial load of L. monocytogenes, B. Subtilis and S. Enteritidis, was significantly (p b 0.05) respectively inhibited by 2.42–2.98, 4.25–4.77 and 2.57–3.01 log CFU/mL in glycinin-treated milk after 16–20 days of storage at 4 °C. Higher levels of bacterial inhibition (3.22–3.78, 5.65–6.27 and 3.35–3.72 log CFU/mL, respectively) were recorded when milk was combined with 0.5% w/v basic subunit while limited inhibition extents were observed with β-conglycinin treatment. The basic subunit was more effective than penicillin in counteracting the three studied bacteria and 11S was even more effective than penicillin against B. Subtilis and S. Enteriditis, particulary the latter one. 4. Discussion

90

80

B. subtilis

Crystal violet uptake %

25

70

60

50

90

S. Enteritidis

80 70 60 50

0

25

50

75

100

Concentration (μg ml-1) Fig. 7. Crystal violet uptake by bacteria cells treated with glycinin, β-conglycinin, basic subunit and penicillin (0, 25, 50, 75 and 100 μg/mL) for 30 min at 28 °C (B. subtilis) or at 37 °C (L. monocytogenes Scott A and S. Enteritidis PT4).

3.6. Bacterial proteins Kinetics of bacterial protein changes was followed through sequential chronological SDS-PAGE profiling (Fig. 10). L. monocytogenes showed early (after 120 min) fading out of most of the bacterial protein bands when treated with glycinin or basic subunit. With penicillin the changes in the bacterial protein was less noticeable while with total soybean protein the SDS profile was nearly typical during the whole incubation time (4 h). Similar changes were also noticed with the two other tested bacteria referring to a common mode of action against different bacteria. 3.7. Antibacterial action in food system The action of glycinin, β-coglycinin and basic subunit was testified in milk during its storage at 4 °C for 20 days (Fig. 11). Milk pasteurized at 85 °C for 5 min was combined with 0.5% (w/v) of protein

The SDS-PAGE patterns of total and subunits of soybean proteins in the presence of β-mercaptoethanol indicating bands with molecular masses of 21 and 34 KD corresponding the basic and acidic subunits is in accordance with the available literature (Nielsen et al., 1989). The subunits of β-conglycinin were in the range of 50 to 65 kD as reported by (Ladin et al., 1987; Utsumi, 1992). These subunits are arranged in high molecular structures (150 and 360 KD) as revealed by native-PAGE. In spite of the high molecular weight of glycinin it can dissociate into trimers under different environmental conditions of ionic strength, pH, denaturant and high temperatures (Utsumi et al., 1997) and the new molecular species may be more reactive and accessing the targeted bacteria. Moreover, the dimensions of glycinin are 11 × 11 × 7.5 nm (Badley et al., 1975), i.e. smaller than the pores dimensions in the bacterial cell wall (Scherrer et al., 1977) permitting their passive passage to cell membrane. In spite of the complexity of glycinin structure (12 subunits), the basic subunit may be oriented to target the cell membranes. Alternatively, the giant protein molecules may exert their action by introducing only a reactive part of its multi combined structure through the cell wall to interact with the cell membrane. Solubility curves indicated that glycinin (pI 6.5) is more basic than the original protein mixture (pI 4.5) while the basic subunit (pI 8.5) is still more basic than glycinin. Glycinin is composed of hydrophobic basic polypeptide and hydrophilic acidic polypeptides (Kuipers and Gruppen, 2008). Basic subunits may have higher potentiality since they have more positive charges and can also undergo strong hydrophobic interactions (German et al., 1982). Based on the amino acid sequence data, the highest charges separation of the glycinin subunits are in following A3B4 > A5A4B3 > AB where the net charges for the basic fragments are +14, + 10 and + 9, respectively encountered by − 14, −16 and −9 for the attached acidic fragments, respectively (Adachi et al., 2000; Cho and Nielsen, 1989; Scallon et al., 1987). So the basic fragments of these glycinin subunits may be the most active ones either in the bound or free state. The closeness of the antibacterial action of glycinin and the basic subunits from penicillin G refers to their considerably high activity as this antibiotic was reported mostly active toward Gram positive bacteria (Jeremy et al., 2011). The superiority of basic subunits over glycinin in the antimicrobial action confirms the importance of the basic nature to the antimicrobial action reported previously (Niyonsaba et al., 2009). Although glycinin may not have a net positive charge but half of its constituting subunits are basic and its pI (6.5) is rather near form neutral. β-Conglycinin manifested antimicrobial action only at very high concentration due to lacking distinctive positive charge on its molecular composition. The lower effectiveness of antibacterial action of penicillin against S. Enteritidis is in accordance with previous reports (Thykaer and

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M.Z. Sitohy et al. / International Journal of Food Microbiology 154 (2012) 19–29

Total protein Penicillin β-Conglycinin Glycinin Basic subunit

Crystal violet assay 90

Cell lysis assay

75

L. monocytogenes

2.0 1.5

60

1.0

45 0.5 30

75

B. subtilis

2.0 1.5

OD260

60 45

1.0 0.5

30 0.0 90

2.0

75

S. Enteritidis

Crystal violet uptake (%)

0.0 90

1.5

60

1.0

45 0.5 30 0.0 0

60

120

180

240

0

60

120

180

240

Incubation time (min) Fig. 8. Kinetic of crystal violet uptake and bacterial cell lysis of as treated with 100 μg/mL of glycinin, β-conglycinin, basic subunit and penicillin for 0, 60, 120 and 240 min at 28 °C (B. subtilis) or at 37 °C (L. monocytogenes Scott A and S. Enteritidis PT4).

Nielsen, 2003 and Ridley et al., 2004) and may be attributed to the emergence of a common pool of R-factors plasmid (Tripathy et al., 2007) or the presence of the outer cell membrane in Gram-bacteria which may reduce penicillin permeation. Conversely, the observed higher antibacterial action of glycinin and the basic subunit than penicillin against the growth of S. Enteritidis may be a result of the exclusive specificity of R-factors plasmid towards penicillin. It also may be envisaged that the action of penicillin is induced internally after penetrating the cell barrier and subsequently affecting the biological activities ending with reduced cellular sizes. The increase in the permeation of the bacterial cells by treatment with glycinin and basic subunit confirms the TEM data showing greatly disrupted cell membranes. Deformation signs on cellular membrane and wall of L. monocytogenes and S. Enteritidis revealed by TEM and SEM (B. Subtilis) may refer to a direct action of the glycinin and basic subunits. This may show that one of the main mechanisms of the natural basic proteins may occur through their direct action the cell wall and cell membrane mediated by the amphipathic nature of the protein fractions. These basic proteins involve both basic and hydrophobic domains in their constituting structure. Both these two traits may participate in empowering protein molecules with specific reactivity to react with bacterial cell wall and membrane. Penicillin treated cells do not show equivalent magnitude of cellular wall or membrane damage indicating that it does not have similar direct action on cell wall and membrane and it rather exerts its action through inhibiting bacterial cell wall synthesis (Ghooi and Thatte, 1995; Rai et al., 2003).

The inactivity of the whole soybean protein in spite of the antimicrobial activity of its isolated fractions (glycinin and basic subunit) may be a result of counteracting actions of other mixture constituents of the whole protein canceling the biological activities of each other's. Glycinin fraction is distinct from the whole protein fraction by its higher isoelectric point (pI 6.5) which renders it more reactive. The basic subunit isolated from glycinin is more basic (pI 8.5) and more hydrophobic and hence its antibacterial action is expectedly higher. At its free form it is more active than in its bound form. However, the effect of the natural basic proteins is not restricted to cell membranes and may be extended to influence bacterial protein synthesis. The kinetics of this effect shows that it starts very early referring to a direct rapid action of the basic proteins on the bacterial proteins through interactions with cell wall and membrane. The corresponding late effects associated with penicillin action are due to its action is rather on protein synthesis. Previous reports referred to the rapid action of natural antimicrobial proteins constituting the innate defense system (Yount and Yeaman, 2005). These conclusions were further supported by the results of the permeation of crystal violet stain which indicate that glycinin and basic subunit added to cellular emulsion were associated with higher rates of stain permeation than penicillin in a concentrationdependent mode. The coordination between the kinetics of crystal violet uptake and cell lysis refers to high association between the two phenomena and indicates that increasing cell permeation is precedent or simultaneous step in the cell lysis and death.

M.Z. Sitohy et al. / International Journal of Food Microbiology 154 (2012) 19–29

27

β-Conglycinin Glycinin Basic subunit Penicillin

L. moncytogenes 80

60

40

Relative cell kill rate %

20

80

B.subtilis

X Data

60

40

20

S.Enteritidis 80

60

40

20

1

2

3

MIC Fig. 9. Relative kill rate of bacteria (OD = 0.5 and 108 CFU/mL) when treated with 100, 200 and 300 μg/mL of glycinin, β-conglycinin, basic subunit and penicillin for 24 h at 28 °C (B. subtilis) or at 37 °C (L. monocytogenes Scott A and S. Enteritidis PT4).

Conclusively, the first step of antimicrobial action of the natural basic proteins may be electrostatic interactions between the positively charged cationic proteins and the negatively charged regions of cell wall or cell membrane. Cell membrane negative charges emerge from teichoic acid component while that of cell membrane originates from phospholipid constituent (Tsuruta, 2009; Murzyn et al., 2005). This may be accompanied by hydrophobic interaction between the hydrophobic regions of the cationic proteins and corresponding regions of bacterial cell membrane, e.g. the hydrophobic core of cell membrane. The oscillating random Brownian movement of the cell wall-attached giant molecules will cause the stretching of the cell membrane or cell wall producing bigger-sized pores. This will subsequently induce pore channel formations, cell wall and cell membrane disintegration. As a consequence cell permeability will considerably increase engendering ultimately cell emptiness, lysis and death. The antimicrobial action of glycinin and its basic subunit against the pathogenic and spoilage bacteria was proved in-situ in milk

Fig. 10. SDS-PAGE pattern of L. monocytogenes Scott A, B. subtilis and S. Enteritidis PT4 treated with 100 μg/mL of glycinin, basic subunit and penicillin during 2 h of incubation at 37 °C.

system, where the basic subunit was more effective than penicillin. This powerful effectiveness was associated with the basicity of the protein fraction that can serve as safe antimicrobial agent in preserving food systems. The actions of glycinin and basic subunits are rather bactericidal since the bacterial count was less than the starting level in the bacterial growth curve of the treated milk after 20 days of storage at 4 °C. Additional indications from the cell killing tests as well as the results of EM analysis confirm the bactericidal action of these proteins. The higher or equal effectiveness of these substances from that of penicillin supports this conclusion. Pasteurized milk can be re-contaminated during storage or handling, so adding this small extent of the natural protein (0.5%) may protect it from post-contamination enhancing its safety and storage quality. Glycinin and basic subunit may be used to counteract the microbes through inducing external effect on the microbes if being accessed either in food or in the gastro-intestinal tract. These

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M.Z. Sitohy et al. / International Journal of Food Microbiology 154 (2012) 19–29

Control β-conglycinin Glycinin Basic subunit Penicillin

L. monocytogenes

8

6

4

2

B. subtilis

Log CFU ml-1

8

6

4

2

S. Enteritidis

8

6

4

2 0

4

8

12

16

20

Incubation time (days) Fig. 11. Antibacterial action of glycinin, β-conglycinin and basic subunit against the proliferation of L. monocytogenes Scott A, B. subtilis and S. Enteritidis PT4 inoculated into milk during 20 days of storage at 4 °C as compared to penicillin.

substances can be used alone or as adjuvant in combination with some antibiotic since they have two different pathways of action. References AACC, 2000. Crude protein-Micro Kjeldahl method, Approved methods of the AACC, 10th ed. : AACC method, Vol-II, 46-13. Adachi, M., Katsube, T., Masuda, T., Utsumi, S., 2000. cDNA of glycinin A3B4 subunit. EMBL/GenBank/DDBJ databases. Badley, R.A., Atkinson, D., Hauser, H., Oldani, D., Green, J.P., Stubbs, J.M., 1975. The structure, physical and chemical properties of the soy bean protein glycinin. Biochimica et Biophysica Acta (BBA) Protein Structure 412, 214–228. Benli, M., Yigit, N., Geven, F., Guney, K., Bingol, U., 2008. Antimicrobial activity of endemic Crataegus tanacetifolia (Lam.) Pers and observation of the inhibition effect on bacterial cells. Cell Biochemistry and Function 26, 844–851. Charpentier, E., Courvalin, P., 1999. Antibioticresistancein Listeria spp. Antimicrobial Agents and Chemotherapy 43, 2103–2108. Cho, T.-J., Nielsen, N.C., 1989. The glycinin Gy3 gene from soybean. Nucleic Acids Resarch 17, 4388.

Culafic, D.M., Gacic, B.V., Vukcevic, J.K., Stankovic, S., Simic, D., 2005. Comparative study on the antibacterial activity of volatiles from sage (Salvia officinalis L.). Archives of Biological Sciences 57, 173–178. Damodaran, S., Kinsella, J.E., 1982. Effect of conglycinin on the thermal aggregation of glycinin. Journal of Agricultural and Food Chemistry 30, 812–817. Denny, J., McLauchlin, J., 2008. Human Listeria monocytogenes infections in Europe — an opportunity for improved European surveillance. Euro Surveillance 13, 1–5. Dhatwalia, V.K., Sati, O.P., Tripathi, M.K., Kumar, A., 2009. Isolation, characterization and antimicrobial activity at diverse dilution of wheat puroindoline protein. World Journal of Agricultural Science 5, 297–300. Ebran, N., Juliena, S., Orangeb, N., Saglioc, P., Lemaîtrea, C., Molle, G., 1999. Pore-forming properties and antimicrobial activity of proteins extracted from epiderma mucus of fish. Comprative Biochemistry and Physiology 122, 181–189. Garcia-Olmedo, F., Molina, A., Alamillo, J.M., Rodriguez-Palenzuela, P., 1998. Plant defense peptides. Biopolymers 47, 479–491. German, B., Damodaran, S., Kinsella, J.E., 1982. Thermal dissociation behavior of soy proteins. Journal of Agricultural and Food Chemistry 30, 807–817. Ghooi, R.B., Thatte, S.M., 1995. Inhibition of cell wall synthesis— is this the mechanism of action of Penicillins. Medical Hypotheses 44, 127–131. Jeremy, J.M., Sondra, C.F., Anthony, C.J.L., 2011. Longer-duration uses of tetracyclines and penicillins in U.S. food-producing animals: Indications and microbiologic effects. Environment International 37, 991–1004. Johnson, E.A., Brekke, J., 1983. Functional properties of acylated pea protein isolates. Journal of Food Science 48, 722–725. Kamiya, H., Muramoto, K., Yamazaki, M., 1986. Aplysianin-A, an antibacterial and antineoplastic glycoprotein in the albumen gland of a sea hare, Aplysia kurodai. Experientia 42, 1065–1067. Kelemu, S., Cardona, C., Segura, G., 2004. Antimicrobial and insecticidal protein isolated from seeds of Clitoria ternatea, a tropical forage legume. Plant Physiology and Biochemistry 42, 867–873. Kitamura, K., Shibasaki, K., 1975. Isolation and some physicochemical properties of the acidic subunits of soybean 11S globulin. Agricultural and Biological Chemistry 39, 945–951. Kitamura, K., Takagi, T., Shibasaki, K., 1976. Subunit structure of soyabean 11S globulin. Agricultural and Biological Chemistry 40, 1837–1844. Kuipers, B.J., Gruppen, H., 2008. Identification of strong aggregating regions in soy glycinin upon enzymatic hydrolysis. Journal of Agricultural and Food Chemistry 56, 3818–3827. Kumarasamy, Y., Fergusson, M., Nahar, L., Sarker, S.D., 2002. Biological activity of moschamindole from Centaurea moschata. Pharmceutical Biology 40, 307–310. Ladin, B.F., Tierney, M.L., Meinke, D.W., Hosa' ngadi, P., Veith, M., Beachy, R.N., 1987. Developmental regulation of β-conglycinin in soybean axes and cotyledons. Plant Physiology 84, 35–41. Laemmli, U.K., 1970. Cleavage of structural proteins during properties of acidic subunits of soyabean 11S globulin. Agricultural and Biological Chemistry 39, 945–951. Murray, P.R., Baron, E.J., Pfallar, M.A., Tenover, F.C., Yolke, R.H., 1995. 6th edn. Manual of Clinical Microbiology, Vol-6. ASM, Wash-ington DC, pp. 214–215. Murzyn, K., Rog, T., Pasenkiewicz-Gierula, M., 2005. Phosphatidylethanolamine-phosphatidylglycerol bilayer as a model of the inner bacterial membrane. Biophysical Journal 88, 1091–1103. Nagano, T., Hirotsuka, M., Mori, H., Kohyama, K., Nishinari, K., 1992. Dynamic viscoelastic study on the gelation of 7S globulin from soybeans. Journal of Agricultural and Food Chemistry 40, 941–944. Nanda, A., Saravanan, M., 2009. Biosynthesis of silver nanoparticles from Staphylococcus aureus and its antimicrobial activity against MRSA and MRSE. Nanomedicine 5, 452–456. Nielsen, et al., 1989. Characterization of the glycinin gene family in soybean. The Plant Cell 1, 313–328. Niyonsaba, F., Nagaoka, I., Ogawa1, H., Okumura, K., 2009. Multifunctional antimicrobial proteins and peptides: natural activators of immune systems. Current Pharmaceutical Design 15, 2393–2413. Palumbo, D., Iannaccone, M., Porta, A., Capparelli, R., 2010. Experimental antibacterial therapy with puroindolines, lactoferrin and lysozyme in Listeria monocytogenesinfected mice. Microbes and Infection 12, 538–545. Pavic, S., Brett, M., Petric, N., Lastre, D., Smoljanovic, M., Atkinson, M., 2005. An outbreak of food poisoning in a kindergarten caused by milk powder containing toxigenic Bacillus subtilis and Bacillus licheniformis. Archiv für Lebensmittelhygiene 56, 20–22. Rai, A.K., Rai, S.B., Rai, D.K., 2003. Quantum chemical studies on the conformational structure of bacterial peptidoglycans and action of penicillin on cell wall. Journal of Molecular Structure 626, 53–61. Rex et al, 2001. Antifungal susceptibility testing: practical aspects and current challenges. Clinical Microbiology Reviews 14 (4), 643–658. Ridley, A.M., Sharma, M., Stapleton, K., 2004. Molecular epidemiology of antibiotic resistance in Salmonella enterica serotypes of veterinary and public health interest in the UK. Abstracts/Infection Genetics and Evolution 4, 253–292. Scallan et al, 2011. Foodborne illness acquired in the United States—major pathogens. Emerging Infectious Diseases 17 (1), 7–15. Scallon, B.J., Dickinson, C.D., Nielsen, N.C., 1987. Characterization of a null-allele for the Gy4 glycinin gene from soybean. Molecular and General Genetics 208, 107–113. Scherrer, R., Berlin, E., Gerhardt, P., 1977. Density, porosity, and structure of dried cell walls isolated from Bacillus megaterium and Saccharomyces cerevisiae. Journal of Bacteriology 129, 1162–1164. Schuchat, A., Swaminathan, B., Broome, C.V., 1991. Epidemiology of human listeriosis. Clinical Microbiology Reviews 4, 169–183.

M.Z. Sitohy et al. / International Journal of Food Microbiology 154 (2012) 19–29 Shai, Y., 2002. Mode of action of membrane active antimicrobial peptides. Biopolymers 66, 236–248. Talas-Oğraş, T., İpekçi, Z., Bajroviç, K., Gözükırmızı, N., 2005. Antibacterial activity of seed proteins of Robinia pseudoacacia. Fitoterapia 76, 67–72. Thykaer, J., Nielsen, J., 2003. Metabolic engineering of b-lactam production. Metabolic Engineering 5, 56–69. Tripathy, S., Kumar, N., Mohanty, S., Samanta, M., Mandal, R.N., Maiti, N.K., 2007. Characterisation of Pseudomonas aeruginosa isolated from freshwater culture systems. Microbiological Research 162, 391–396. Tsuruta, T., 2009. Biosorption of some heavy metals using microorganisms. Current Top Biotechnology 5, 21–28. Utsumi, S., 1992. In: Kinsella, J.E. (Ed.), Plant food protein engineering. : Advances in Food and Nutrition Research, Vol. 36. Academic Press, San Diego, CA, pp. 89–208. Utsumi, S., Matsumura, Y., Mori, T., 1997. Structure-function relationships of soy protein. In: Damodaran, S., Paraf, A. (Eds.), Food Proteins and Their Applications. Marcel Dekker, New York, pp. 257–291.

29

Vaara, M., Vaara, T., 1981. Outer membrane permeability barrier disruption by polymyxin in polymyxin-susceptibility and resistant Salmonella typhimurium. Antimicrobial Agent and Chemotherapy 19, 578–583. Yount, N.Y., Yeaman, M.R., 2005. Immunocontinuum: perspectives in antimicrobial peptide mechanisms of action and resistance. Protein and Peptide Letters 12, 49–67. Zasloff, M., 2002. Antimicrobialpeptides of multicellularorganisms. Nature 415, 389–395. Zgoda, I.R., Porter, J.R., 2001. A convenient microdilution method for screening natural products against bacteria and fungi. Pharmaceutical Biology 39, 221–225. Zhou, K., Zhou, W.L.I.P., Liu, G., Zhang, J., Dai, Y., 2008. Mode of action of pentocin 31–1: an antilisteria bacteriocin produced by Lactobacillus pentosus from Chinese traditional ham. Food Control 19, 817–822.