Process Biochemistry 44 (2009) 499–503

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Process Biochemistry

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Short communication

Isolation and preliminary characterization of a nonbacteriocin antimicrobialcompound from Weissella paramesenteroides DFR-8 isolated from cucumber(Cucumis sativus)

Ajay Pal *, K.V. Ramana

Food Biotechnology Discipline, Defence Food Research Laboratory, Siddarthanagar, Mysore 570 011, India

A R T I C L E I N F O

Article history:

Received 28 July 2008

Received in revised form 14 November 2008

Accepted 13 January 2009

Keywords:

LAB

Nonbacteriocin

Biopreservation

Food-borne pathogens

Antimicrobials

RSM

A B S T R A C T

A non-proteinaceous antimicrobial substance (nonbacteriocin) produced by Weissella paramesenteroides

DFR-8, an isolate from cucumber (Cucumis sativus), was purified using cell adsorption–desorption and

gel permeation chromatography methods. A single peak observed in the purity check by analytical RP-

HPLC strongly indicates the homogeneity of the nonbacteriocin preparation. The active substance is

insensitive to proteolytic enzymes, lipase, amylase and catalase and shows a broad spectrum of activity

towards food-borne/spoilage pathogens including Gram-negative organisms. The non-proteinaceous

antimicrobial molecule has a molecular mass �2.5 kDa and is thermostable up to 121 8C at pH �4.0. A

central composite rotatable design (CCRD) was employed to study the interactive effect of temperature

and pH on antimicrobial activity and an equation was developed to deduce the residual activity of

inhibitory substance under any conditions of temperature and pH within the experimental domain. The

broad inhibitory spectrum and heat stability of the antimicrobial substance advocates its application as

food-biopreservative.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Lactic acid bacteria (LAB) are a fastidious group of aerotolerant,Gram-positive, non-sporing, non-motile, catalase negative andcarbohydrate fermenting food grade microorganisms that aregenerally regarded as safe [1]. These bacteria have been used in theproduction of fermented foods and feeds for many centuries.Although they are now also recognized for their health andnutritional benefits, these industrially important organisms wereprimarily used for the preservation of highly perishable foods ofplant and/or animal origin [2]. Increasing consumer demand for‘natural’ and ‘additive free’ products has led to greater interest inthe application of natural inhibitory substance as food preservativewhich could replace or reduce the use of chemical additives. It iswell known that LAB show antagonistic activity against otherbacteria including food spoilage and food-borne pathogens. Thereare several different mechanisms responsible for this inhibition. Inmost cases, the inhibition is caused by the formation of organicacids, diacetyl, hydrogen peroxide and/or by bacteriocin produc-tion [3].

* Corresponding author. Tel.: +91 821 2473686; fax: +91 821 2473468.

E-mail address: ajaydrdo@rediffmail.com (A. Pal).

1359-5113/$ – see front matter � 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2009.01.006

While the physiology, biochemistry and genetics of bacteriocinhave been investigated in detail [4,5], studies on nonbacteriocinantimicrobial compound produced by LAB are very few. During thepurification of bacteriocin from Weissella paramesenteroides, anisolate from cucumber (Cucumis sativus), using cell adsorption–desorption and gel permeation chromatography, we got anonbacteriocin antimicrobial compound. The aim of the presentstudy is to isolate and biochemically characterize this antimicro-bial compound to assess its potential in biopreservation of foods.

2. Materials and methods

2.1. The producer lactic acid bacterium

Weissella paramesenteroides DFR-8, the producer strain of antimicrobial

compound was isolated from cucumber (C. sativus). The strain was identified by

sequencing the 708 bases of 16S rDNA gene in forward direction followed by BLAST

homology search. The nucleotide sequences have been deposited with NCBI

database under accession number FJ390112.

2.2. Bacterial strains and growth conditions

During isolation of antimicrobial compound, Staphylococcus aureus was used as

indicator strain. Once the compound was purified, its antimicrobial spectrum was

studied against other pathogenic microorganisms listed in Table 1. The pathogenic

strains namely Escherichia coli DFR-262 and Pseudomonas aeruginosa DFR-219 were

Defence Food Research Laboratory, Mysore, India, isolates while rest of the strains

used in study were obtained from Microbial Type Culture Collection (MTCC),

Chandigarh, India. The antimicrobial producing isolate was grown in MRS (de Mann

Table 1Antibacterial spectrum of nonbacteriocin antimicrobial compound.

Organism Zone of inhibition (mm)a Antimicrobial activity (AU/ml)a

Listeria monocytogenes MTCC 657 4.3 (4.0–4.5) 1274 (1205–1343)

Micrococcus luteus MTCC 2452 1.6 (1.4–2.0) 477 (416–600)

Staphylococcus aureus MTCC 737 4.4 (4.0–4.5) 1320 (1205–1343)

Aeromonas hydrophila MTCC 646 3.0 (2.8–3.6) 904 (823–1067)

Salmonella typhimurium MTCC 98 4.2 (3.6–4.5) 1250 (1066–1343)

Escherichia coli DFR 262 3.8 (2.8–4.5) 1117 (823–1343)

Vibrio parahaemolyticus MTCC 451 2.8 (2.0–3.6) 829 (600–1066)

Pseudomonas aeruginosa DFR 219 4.4 (4.0–4.5) 1320 (1205–1343)

Clostridium sporogenes 4.0 (3.6–4.5) 1182 (1066–1343)

Yersenia enterocolitica 3.3 (2.8–3.6) 985 (823–1066)

Bacillus subtilis 3.2 (2.8–3.6) 944 (823–1066)

a The values are means of 3 independent experiments carried out in duplicates; the range is given in parentheses.

A. Pal, K.V. Ramana / Process Biochemistry 44 (2009) 499–503500

Rogosa Sharpe) broth at 35 8C and the same was maintained as frozen stocks at

�20 8C on MRS soft agar (0.5%) slants overlaid with 50% glycerol. The pathogenic

strains were maintained on specified growth media.

2.3. Production and isolation of nonbacteriocin antimicrobial compound

The isolate, W. paramesenteroides DFR-8 was grown statically in MRS broth (1 l)

at 35 8C for �60 h. Extraction of extracellular nonbacteriocin antimicrobial

compound was carried out by using cell adsorption–desorption method as used

for bacteriocin extraction [6] with slight modifications. Briefly, pH of the culture

broth was adjusted to 6.5 by gradual addition of 4 M NaOH. It was stirred for 3 h at

room temperature to allow the antimicrobial compounds to adsorb on to the

producer cells, which were collected by centrifugation at 10,000 � g (4 8C) for

30 min. To further remove impurities, the cells were washed twice by resuspension

in 100 ml of 5 mM sodium phosphate buffer (pH 6.5), recentrifugation at 10,000 � g

(4 8C) and draining off the buffer washings. The cells were resuspended in 50 ml of

100 mM NaCl solution (pH 2.0) and kept for stirring in cold room (4 8C) for 18 h for

desorption of adsorbed compounds. The cells were removed by centrifugation at

10,000 � g for 15 min at 4 8C. The supernatant was concentrated, adjusted to pH 4.5

and subjected to gel permeation chromatography.

2.3.1. Gel permeation chromatography (GPC)

A column was packed with Sephadex G-25 (95 cm � 1.25 cm) and equilibrated

with 0.05 mM ammonium acetate buffer, pH 4.8. The same was used to elute the

sample. Void-volume was determined by passing 2 mg/ml solution of blue dextran

(2000 kDa) through the column. The extract obtained from cell adsorption–

desorption technique was loaded on to the column and fractions were collected

after void-volume at a flow rate of 0.5 ml/min at 4 min intervals. Absorbance was

measured at 280 nm using a Shimadzu spectrophotometer. Various fractions

around peaks, shoulders, and valleys were pooled separately, filter sterilized by

passing through 0.22 mm membrane filter and assayed for antimicrobial activity

using agar well diffusion assay [7] against S. aureus. Subsequently, the active

fractions were pooled, concentrated to 1 ml using vacuum flash evaporator

(Heidolph), adjusted to pH 4.5 and stored at low temperature for further studies. In

further experiments, the antibacterial activity was expressed as AU/ml and defined

as the reciprocal of the highest dilution showing a distinct zone of inhibition [8].

2.3.2. Purity check and molecular mass determination

Purity of antimicrobial compound was tested by injecting 20 ml sample into an

analytical RP-HPLC (Waters 600 analytical HPLC system, Milford, MA, USA)

equipped with an analytical column (C18, 4.6 mm � 250 mm, pore size 80 A). The

solvents used were solvent A (0.1% trifluoroacetic acid (TFA) in water) and solvent B

(0.1% TFA in acetonitrile) [9]. A 40 min linear gradient from 100% A/0% B–70% A/30%

B at a flow rate of 0.7 ml/min was used for sample elution, which was monitored in

the range 210–400 nm. The approximate molecular mass of the nonbacteriocin

antimicrobial compound was determined by gel permeation chromatographic

technique. Briefly, a column was packed with sephadex G-25 (95 cm � 1.25 cm)

and a calibration curve was prepared by using proteins of known molecular masses

(myoglobin (17.0 kDa), a-lactalbumin (14.2 kDa), bovine lung aprotinin (6.5 kDa),

bovine insulin chain B (3.5 kDa) and bradykinin (1.06 kDa)). The molecular mass of

nonbacteriocin was deduced from its elution volume. Ammonium acetate buffer

(0.05 mM, pH 4.8) was used throughout the elution.

2.4. Effect of various hydrolytic enzymes on antimicrobial activity

The antimicrobial moiety was subjected to hydrolysis by various hydrolytic

enzymes (3 mg/ml), one at a time. In all the cases, the residual antimicrobial activity

was evaluated. To determine if the lactic acid participate in the antimicrobial

activity, the sample was subjected to lactate dehydrogenase treatment (3 mg/ml).

The remaining antimicrobial activity in the sample was tested as mentioned earlier.

2.5. Effect of organic solvents on antimicrobial activity

Resistance of antimicrobial compound to various organic solvents was studied

after 4 h exposure to organic solvents (50%) at 35 8C.

2.6. Antimicrobial spectrum

The antimicrobial spectrum of the purified nonbacteriocin compound against a

large number of food-borne/spoilage Gram-positive and -negative bacteria was

studied using 60 ml appropriately diluted sample employing agar well diffusion

assay [7] and the results were further expressed in AU/ml (8).

2.7. Combined effect of temperature and pH on the antimicrobial activity

Temperature and pH are the most important parameters that affect the stability/

activity of any antimicrobial compound. Preliminary studies suggested the

individual effects of pH and temperature on the stability of nonbacteriocin

antimicrobial compound. However, our interest was to examine the thermostability

of active compound as influenced by pH and in order to predict antimicrobial

activity under any conditions of temperature and pH in the experimental domain,

response surface methodology (RSM) was used.

A central composite rotatable design (CCRD) based on five levels and two

variables (temperature and pH) was used to study the combined influence of

temperature and pH on the stability of antimicrobial compound. The design

consisted of 13 experiments with four factorial points, four axial points with

a = 1.441 and five center points for replication. In developing the regression

equation the test factors are coded according to the following equation:

xi ¼Xi � X0

dXi

where, xi is the dimensionless coded value of the ith independent variable; Xi the

natural value of the ith independent variable; X0 the natural value of the ith

independent variable at the center point and dXi the step change value. Once the

experiments were performed, the experimental results were fitted with a 2nd order

polynomial function:

Y ¼ b0 þ b1x1 þ b2x2 þ b11x21 þ b22x2

2 þ b12x1x2

where, Y is the predicted response; b0 the intercept; b1, b2 the linear co-efficient;

b11, b22 the squared co-efficient and b12 the interaction co-efficient.

Samples of nonbacteriocin antimicrobial compound were adjusted at

different pH values with 5.0 N H3PO4 or NaOH and incubated for 15 min at

temperatures as defined in the experimental matrix. After incubation, the

samples were adjusted to pH 4.5 and monitored for residual activity (% RA). The

average of the RA of 3 experiments carried out in duplicate was taken as the

dependent variable or response. Controls consisted of untreated antimicrobial

samples.

2.7.1. Data analysis

Design expert 7.1.4 (Stat-Ease, Inc., Minneapolis, USA) was used for the

regression analysis of the experimental data obtained. The quality of fit of the

polynomial model equation was expressed by the co-efficient of determination, R2,

and its statistical significance was checked by Fisher’s F-test. The significance level

of each regression co-efficient was determined by Student’s t-test. The level of

significance was given as value of p value.

2.8. Storage stability of nonbacteriocin compound

Purified samples were stored at �20, 4 and 37 8C and at regular time intervals,

the samples were withdrawn to determine the residual activity.

A. Pal, K.V. Ramana / Process Biochemistry 44 (2009) 499–503 501

3. Results and discussion

3.1. Production and isolation of nonbacteriocin antimicrobial

compound

The concentrate obtained from cell adsorption–desorptionmethod was subjected to size exclusion chromatographic elution(Fig. 1) and fractions were collected after the elution of void-volume. Among the fractions collected, the fractions 33–41 werefound active against S. aureus. The active fractions were pooled andconcentrated to 1 ml. During the purity check step, a single peakwas observed in the range 210–400 nm, which was subsequentlyextracted at 280 nm (Inset Fig. 1) indicating the absence of otherimpurities and purity and homogeneity of the antimicrobialpreparation. The purified preparation has molecular mass�2.5 kDa, as determined by gel permeation chromatographictechnique (graph not shown).

3.2. Effect of various hydrolytic enzymes on antimicrobial activity

The active fraction from gel filtration was assayed for itssensitivity to various hydrolytic enzymes and the antimicrobialactivity was found relatively intact after treatment with all theproteolytic enzymes (protease, trypsin, proteinase K and pepsin),lipase, catalase and amylase. On finding resistant to variousproteolytic enzymes, it was termed as nonbacteriocin. Further-more, the active compound could not be quantified by Biuretmethod of protein estimation and also could not be visualized onSDS-PAGE confirming its non-proteinaceous nature. But because itabsorbs UV light at 280 nm, it is quite evident that the compoundunder study is a low molecular weight aromatic compound. Inaddition, the resistance to other hydrolytic enzymes allowed alipid or glycosidic nature of active molecule to be excluded. Also,the antimicrobial activity of nonbacteriocin produced by organismin this study was not due to hydrogen peroxide or acidity, asactivity was not lost after treatment with catalase and as activitywas observed at pH 4.5–5.0 eliminating effect of acids. Moreover,the sample was subjected to LDH treatment to remove lactic acid ifany, and the antimicrobial activity was found unchanged

Fig. 1. The elution profile of nonbacteriocin antimicrobial compound on Sephadex G-25

purity check of nonbacteriocin compound by analytical RP-HPLC, peak retention time =

confirming that lactic acid did not participate in the activity ofsample and the sole activity is due to nonbacteriocin molecule. Theinsensitivity to proteolytic enzymes, lipase, catalase, and unrelat-edness to lactic acid effects are features that are common to thoseshown by very few antimicrobial compounds reported from LAB[10,11].

The effect of organic solvents, at a final concentration of 50%, onantimicrobial activity of nonbacteriocin was also studied. Thenonbacteriocin molecule retained almost 100% activity afterexposure at 35 8C for 4 h to acetone while the maximum loss inactivity was brought by isopropanol (activity retention 60%).Treatment with organic solvents such as chloroform, isobutanol,methanol and toluene also brought down antimicrobial activity ofnonbacteriocin to 85, 85, 75 and 70%, respectively. The slight loss inactivity observed might be attributed to removal of bound watermolecules accompanied by change in conformation of thenonbacteriocin molecule.

3.3. Antimicrobial spectrum

The antimicrobial spectrum of the nonbacteriocin compoundshowed its activity against food-borne pathogens and spoilageorganisms, including Gram-negative organisms (Table 1), whichare normally resistant to the bacteriocins of LAB. The antimicrobialactivity was observed maximum against S. aureus (1320 AU/ml), P.

aeruginosa (1320 AU/ml), L. monocytogenes (1274 AU/ml), S.

typhimurium (1250 AU/ml), C. sporogenes (1182 AU/ml) and E. coli

(1117 AU/ml) while minimum activity was found against M. luteus

(477 AU/ml). Considering a broad spectrum of antibacterialactivity against important Gram-positive and negative foodpoisoning and spoilage bacteria, such nonbacteriocin compoundwould have wider application in food-system.

3.4. Combined effect of temperature and pH on the

antimicrobial activity

Computation of the individual effect of temperature and pH onan antimicrobial sample is a general procedure. Our interest was todetermine the thermostability of nonbacteriocin compound as a

(the active fractions (33–41) are indicated by bar (—)). Inset is the chromatogram of

3.39 min).

Table 3Estimated regression co-efficients for %RA.

Term Constant Temp pH Temp2 pH2 Temp � pH

Co-efficient 84.70 �4.50 �18.60 0.37 �5.51 �2.75

t value 181.75 �12.20 �50.49 0.93 �13.93 �5.27

p value <0.0001 <0.0001 <0.0001 0.382 <0.0001 <0.0001

Fig. 2. Surface graph showing % residual activity of nonbacteriocin with respect to

temperature and pH.

A. Pal, K.V. Ramana / Process Biochemistry 44 (2009) 499–503502

function of pH. For this purpose the interaction effects between thetwo variables were studied using statistical experimental design.The experimental results were fitted to a quadratic model that willenable the predictions of the output response (residual activity,%RA) under any given condition of pH and temperature. Experi-ments were performed with various combinations of temperatureand pH as given in Table 2. The application of RSM yielded thefollowing regression equation, which is an empirical relationshipbetween %RA, and the test variables in coded units:

%RA ¼ 84:70� 4:50 Temp� 18:60 pHþ 0:37 Temp2 � 5:51 pH2

� 2:75 Temp� pH

The significance of each co-efficient was determined byStudent’s t-value and p values, which are given in Table 3. Thesmaller the value of p value and larger the t-value, the moresignificant is the corresponding co-efficient. Student’s t-test showsthat all the linear co-efficients, pH quadratic co-efficient andinteraction co-efficient were highly significant (p<0.0001) while,the temperature quadratic co-efficient was not significant. Highsignificance of these terms indicates that they can act as limitingfactors and even small variations in their values will alter %RA to aconsiderable extent. The sign and magnitude of co-efficientsindicate the effect of the variable on the response. Negative sign ofthe co-efficient at linear level indicated decrease in response valuewith an increase in level of variable. Comparison of predicted andexperimental values (Table 2) revealed good correspondencebetween them, implying that empirical model derived from RSMcan be used to adequately describe the relationship between thevariables and the response.

The co-efficient of determination of the quadratic model, R2,calculated as 0.998 implies that the fitted model explains nearly99.8% of total variation in the residual activities. Further proof ofthe high significance of the model obtained for %RA of nonbacter-iocin is the plot (not shown) representing predicted versusexperimental value where the plot is very close to y = x

(r2 = 0.9975).At the same time, a relatively lower value of the co-efficient of

variance (C.V.% = 1.28) indicates better precision and reliability ofthe experiments carried out. Statistical testing of the model wascarried out by Fisher’s statistical test for analysis of variance. If themodel is a good predicator of the experimental results, thecalculated F values should be several times greater than tabulated F

value. In this case, the F value of 585.77 was greater than tabulatedF5, 7, 1% (7.46) corresponding to a probability of <0.0001, whichimplies that model is significant and there is a quadraticrelationship between the independent variables and response

Table 2Experimental design and results (values in parenthesis indicate the actual values).

Run number Coded level

x1 x2

1 �1 (80) �1 (5)

2 +1 (114) �1 (5)

3 �1 (80) +1 (9)

4 +1 (114) +1 (9)

5 �a (72.96) 0 (7)

6 +a (121.04) 0 (7)

7 0 (97) �a (4.17)

8 0 (97) +a (9.83)

9 0 (97) 0 (7)

10 0 (97) 0 (7)

11 0 (97) 0 (7)

12 0 (97) 0 (7)

13 0 (97) 0 (7)

a Relative deviation ð%Þ ¼ Predicted %RA�observed %RAPredicted %RA � 100.

variables. The Fisher’s F-test with a very low probability (<0.0001)demonstrates a high significance for the regression model andconfirms the adequacy of the quadratic model. The ‘Lack of Fit F-value’ of 1.74 (Table F3, 4, 1% = 16.7) implies that ‘Lack of Fit’ is notsignificant relative to the pure error. Non-significant ‘Lack of Fit’demonstrates that the source of variation contained in theresiduals (i.e. difference between the predicted value and theexperimental value) was due to experimental error and not Lack ofFit. Fig. 2 shows the surface graph of residual activity (%RA) ofnonbacteriocin after 15 min of treatment. A maximum residualactivity (100%RA) was observed up to autoclaving temperaturewhen pH was kept �4.0. Further increase in the pH value resultedin decreased thermostability of antimicrobial compound i.e. thenonbacteriocin compound is thermostable at low pH and as the pHincreases, the thermostability decreases. Most of the inhibitorycompounds described in the literature are unstable at and above

Responses (%RA) Relative deviationa

Observed Predicted (%)

100 99.91 �0.09

96.0 96.41 0.42

67.5 68.21 1.04

52.5 53.71 2.25

92.0 91.35 �0.71

80.0 79.07 �1.17

100 99.99 �0.01

48.5 47.39 �2.34

84.5 84.70 0.23

83.5 84.70 1.18

86.0 84.70 �1.53

85.0 84.70 �0.35

84.5 84.70 0.23

A. Pal, K.V. Ramana / Process Biochemistry 44 (2009) 499–503 503

neutral pH but the nonbacteriocin produced by W. paramesenter-

oides also maintains significant activity at the alkaline pH also. Anon-proteinaceous thermostable antimicrobial compound hasbeen studied earlier in Lactobacillus casei strain of vegetable origin[11]. These data are important with regard to using nonbacteriocinin food and should be considered when thermal treatments areincluded in food.

3.5. Storage stability of nonbacteriocin

The residual activity of nonbacteriocin compound when storedat different temperature with respect to time was monitoredperiodically up to 5 months. It was observed that compound understudy retained full antibacterial activity when stored at �20 and4 8C for more than 3 months while 45% activity was lost whenstored at 37 8C for same time. After 5 months of storage at 37 8C,only 30% activity was retained while 100% activity was retainedwhen stored at �20 8C for the same time.

4. Conclusion

LAB produce a number of antimicrobials such as lactic acid,acetic acid, hydrogen peroxide, CO2 and bacteriocin to inhibitpathogenic and spoilage microorganisms and this property of LABhas been exploited by mankind since years in extending andenhancing the self-life and safety of food products. Bacteriocins ofLAB like nisin, pediocin, plantaricin, acidophilin etc. have beenstudied in details by various groups of researchers but studies onnonbacteriocin molecules are still rare. To our knowledge, this isthe first study on the isolation and characterization of anonbacteriocin compound from W. paramesenteroides. The activecompound secreted by the W. paramesenteroides DFR-8 displayed,in vitro, a broad spectrum of activity against Gram-positive andGram-negative bacteria. Resistance to the various hydrolyticenzymes and heat treatments suggests that the active moleculeis a low molecular mass, nonproteinaceous compound. Furtherwork is in progress to study the structure of the broad-spectrum

nonbacteriocin antimicrobial compound and to determine itsgenetic determinants for immunity as well as production.

Acknowledgement

The authors are grateful to Dr. A.S. Bawa, Director, Defence FoodResearch Laboratory, Mysore, for providing all the necessaryfacilities, constant guidance and encouragement during thisinvestigation.

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