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A comparison of methods for the measurement of bacteriocin activity
ELSEVIER
Journal of Microbiological Methods 22 (1995) 95-108
A comparison Eugenio
of methods for the measurement bacteriocin activity
Parente, * Carla Brienza, Marcella Moles, Annamaria
of Ricciardi
Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali. Universitci della Basilicata; Via N. Sauro, 85, I-85100 Potenza, Italy
Received 8 June 1994; accepted 3 October 1994
Abstract
Agar diffusion (spot and well) and photometric (tube or microtiter) assays, both in the form of critical dilution and quantitative assays, were compared for 4 bacteriocins (enterocin 1146, lactococcin 140, leucocin FlO and nisin) against 2 indicator strains each. In the agar well diffusion assay (AWDA) a linear relationship existed between response (diameter or area of the zone of inhibition) and the logarithm of the dose while a non-linear equation was used to model the sigmoidal dose/response curve in photometric assays (PA). The dose/response curves were used to define titers of the standard solutions in arbitrary units and to develop quantitative assays for all the bacteriocins. With the exception of lactococcin 140, the PA provided estimates which were more reproducible than those obtained with the AWDA. The quantitative assays compared favourably with the classical critical dilution assays for bacteriocins, eliminating the need to dilute to extinction to estimate the titers of bacteriocin solutions, providing a continuous scale for activity and allowing the calculation of confidence intervals. Keywords:
Bacteriocin;
Agar well diffusion
assay; Critical dilution
assay; Photometric
assay
1. Introduction Bacteriocins are bacterial proteins which are inhibitory to microorganisms which are usually, but not always, closely related to the producer strain [l]. Because of their potential use as natural food preservatives and as phenotypic * Corresponding author. PZVX85.CINECA.IT.
Tel.:
+ 39-971-474384. Fax:
+ 39-971-471481. E-Mail PARENTE@
0167-7012/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0167-7012(94)00068-9
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markers for the construction of food-grade cloning vectors, bacteriocins produced by lactic acid bacteria have been the subject of intensive investigation in recent years [ 1.21. Quantitative measurements of bacteriocin activity are needed for most of the activities related to their characterization. Several methods have been used for the quantitation of the iantibiotic nisin [3]; because of its importance as food preservative, standard units and methods have been defined for this substance and a commercial standard preparation is available. Methods for the assay of bacteriocin activity are usually derived from those for antibiotics. Modifications (spot, well, disc diffusion) of the agar diffusion assay [4-61 and photometric [7-121 methods have been widely used. Methods based on conductance and bioluminescence have been used less frequently [13,14]. Since standard solution of bacteriocins are generally not available, critical dilution assays (CDA) are used with arbitrary units to express activity. The titer of the bacteriocin solution is defined as the reciprocal of the lowest amount of bacteriocin solution causing a definite inhibition of the indicator strain. This method is semi-quantitative, provides only a discontinuous scale for activity and always requires that a complete series of dilutions is carried out to estimate activity. Alhough CDA are easy to use and suffice for most applications, they are of little value when statistical comparisons on quantitative measurements of bacteriocin activity are to be carried out. When agar diffusion assays and photometric methods are based on a dose/response curve to estimate bacteriocin activity, a continuous scale for activity is obtained. Both agar diffusion and photometric methods for the quantitation of activity of antimicrobials have their drawbacks [15] and the lack of standard solutions make the construction of dose/response curves difficult. Even if several authors have used quantitative methods for the measurement of the activity of bacteriocins produced by lactic acid bacteria [7,8,10,16] the nature of the dose/response curve used is not clear and statements on the relative accuracy of methods or on the variability of the results are almost always lacking. Direct comparisons of methods for the quantitative measurement of bacteriocin activity are rare and of limited scope [11,17]. The objective of this study was to compare some of the most commonly used methods for the measurement of bacteriocin activity: agar diffusion assays (as spot or well diffusion) and photometric assays (in tubes and/or in microtiter plates) were compared for nisin and three partially purified bacteriocins both in the form of critical dilution assays and as quantitative methods based on dose/ response curves.
2. Materials and methods 2.1. Bacteriocins
Nisin (NIS) was obtained from Sigma as 2.5% powder and suspended in 0.02 N HCl to a final concentration of 2.50 pg ml-’ (10,000 IU ml -I). Partially purified
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of lactococcin 140 (LCN, produced by Lactococcus luctis subsp. enterocin 1146 (E1146, produced by Enterococcus fuecium DPC1146) and leucocin FlO (LFlO, produced by Leuconostoc carnosum FlO) were obtained by precipitating the bacteriocins from culture supernatants with ammonium sulfate (40% saturation, 4 h at 4°C); the pellet obtained after centrifugation (8000 g, 30 min.) was resuspended in 50 mM potassium phosphate buffer, pH 5.0, dialysed against the same buffer in 3,500 NMWCO membranes (Spectra-Pore, Spectrum, Los Angeles, California) and filter sterilized with Millipore (Bedford, Mass. USA) HV membranes (0.45 p). All bacteriocins were stored at - 20°C. preparations
factis 140NWC),
2.2. Indicator strains Lactococcus luctis subsp. lactis ATCC19435 and Lactococcus lactis subsp. cremoris CNRZ117 were used as indicator strains for LCN and NIS assay; Listeriu innocua BL86/26 and Lactobacillus sake NCFB2474 were used as indicators for
LFlO and E1146. CNRZ117, BL86/26 and NCFB2474 were obtained from Dr. T. Cogan, (National Dairy Products Research Centre, Fermoy, IRL); ATCC19435 was obtained from Prof. S. Coppola (Istituto di Microbiologia Agraria, Portici, Italy). All strains were maintained frozen at - 20°C in 25% glycerol. Working cultures were grown for 16 h at 30°C in Ml7 broth (Oxoid, Basingstoke, UK) + 0.5% glucose (GM17) for lactococci, MRS broth (Oxoid) for NCFB2474 and Tryptic Soy Broth (Oxoid) + 0.6% Yeast Extract (Oxoid) for BL86/26. The same media were used for their growth during bacteriocin assay, and bacteriological agar (Oxoid) was added to obtain top (0.6% w/v) and bottom (1.5% w/v) agar media for agar diffusion assays. Indicator strains cultures were always adjusted to OD,,, = 0.5 before inoculation of the media for bacteriocin assay.
2.3. Critical dilution assay (CDA) The method of Pucci et al. [4] was used. Serial two-fold dilutions were carried out in the same medium used for the growth of the appropriate indicator strain except for nisin, which was diluted in 0.02 N HCl. Ten ~1 of each dilution were spotted on the surface of the appropriate agar medium (20 ml in a 90 mm diameter Petri dish) which had been dried for 30 min. After diffusion (30 min. at room temperature) the plates were overlaid with 5 ml of semisolid agar medium inoculated (1% v/v) with a standardized suspension of the indicator strain. After incubation (16 h at 30°C) the plates were checked for zones of inibition. The critical dilution was defined as the highest dilution producing a definite inhibition zone. When a clear inhibition zone was followed by a hazy one the critical dilution was calculated as the average of the two dilutions. The titer of the bacteriocin solution, in AU ml-‘, was calculated as (lOOO/lO) D, where D is the dilution factor.
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2.4. Agur Well Diffusion Assay (AWDA)
Twenty ml of the appropriate agar (1.5% w/v) medium were pipetted in 90 mm Petri dishes and inoculated (1% v/v) with a standardized indicator suspension. After drying for 30 min., six 6 mm wells were bored in each plate. Forty ~1 of 2-fold dilutions of bacteriocin were pipetted into each well and the plates were incubated at 30°C for 16 h. The diameter of the inhibition zones was measured using a calliper. Results were read either as a CDA (with the critical dilution being the last dilution which produced a zone larger than 6 mm and titer defined as described above) or as a quantitative assay. For the latter a standard curve was prepared using a series of 2-fold dilutions of bacteriocin solution. The response (R) was calculated either as the diameter or as the area of the inhibition zone corrected for the diameter or area of the well. The dose (d) was the amount of bacteriocin pipetted into each well (40 ~1 l/D). The dose/response curve had the following equation: R = a + b log(d) The critical dose (CD) was defined as the amount of bacteriocin solution ml) corresponding to a null inhibition zone and calculated by extrapolating dose/response curve. The titer, in AU ml-‘, was calculated as the reciprocal the CD. The standard curve, relating dose in AU to R, had a 0 intercept and same slope of Eq. 1. The activity in AU ml-’ of a sample was calculated follows: AU ml-’ = (1000/40) D 10’R’h’
(1) (in the of the as (2)
Three replicates, in different plates, were used for each dilution of the standard curve and for each sample. 2.5. Photometric assay (PA)
A standardized indicator culture was used to inoculate (5% v/v) the appropriate broth medium, which was cooled to 4°C and dispensed, in 2.4 ml amounts, in sterile test tubes. 100 ~1 of 2-fold dilutions of bacteriocin were pipetted into each tube and incubation was carried out at 30°C for 4 h (ATCC19435) or 6 h (other strains). 100 ~1 of sterile medium were added to control tubes. A standard curve was prepared using a series of 2-fold dilutions of bacteriocin solution. At the end of the incubation the absorbance of the culture was read at 600 nm using a Beckman DU-7 spectrophotometer with uninoculated medium as a blank. The response (R) was calculated as the ratio between the absorbance of a sample and that of the control (average of 5 replicates). The dose/response curve had the following equation: R = A + (1 - A)/(1 + EXP(B + C) log(d))
(3)
were the dose (d) was the amount of bacteriocin (ml ml-‘) in the assay tube. One Bacteriocin Unit (BU) was defined as the amount of bacteriocin needed to obtain
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a R = 0.5 and calculated for each standard solution from Eq. 3; the titer (T) in BU ml -’ was calculated as its reciprocal. The dose/response curve was recalculated as follows: R = a + (1 - a)/(1 + EXP(b + c) log(BU))
where BU = T d. The activity of an unknown following equation: BU ml-’ = (1000/40) D 10 <(ln((l-R)I(R-a))-6)/c)
(4) sample was obtained
from the
(3
were R is the response obtained for the sample, a, b and c the parameters of Eq. 4. Results were also scored as a critical dilution assay; the critical dilution was defined as the last dilution showing a visible inhibition compared to the control.The titer of the bacteriocin solution, in AU ml-‘, was calculated as (1000/25) D. The method was adapted for use in microtiter plates as follows: 10 ~1 of bacteriocin solution or of sterile growth medium (for blank and controls) were pipetted into the wells of sterile microtiter plates (Corning Cell Wells, Corning, NY); 240 ~1 of inoculated (control and bacteriocin wells) or uninoculated medium (blanks) were then pipetted into the wells. The plates were incubated at 30°C for 4 or 6 h and the absorbance at 620 nm was read with a Novapath Mini Reader (Biorad Laboraties, Hercules, CA). Results were scored as described above. 2.6. Statistics and graphics Statistical analyses were carried out with Systat 5.2.1 for the Macintosh, using procedures NONLIN for non-linear regression and MGLH for linear regression [lS]. Graphs were generated using Systat 5.2.1 [19] or CA-Cricket Graph III.
3. Results and discussion 3.1. Nature of the doselresponse
curves
While no dose/response curves are needed for critical dilution assays, some sort of mathematical relationship must be used for calculations of doses from responses in both the AWDA and the PA. In Agar Well Diffusion Assays for antibiotics and for some bacteriocins [3,15,16] a linear relationship between diameter or the area (the response) of the inibition zone and the logarithm of the dose is used; in Photometric Assays a linear relationship between the absorbance and the logarithm of the dose is used [3,15]. Unfortunately, purified bacteriocins are rarely available and doses are ususally expressed in arbitrary units. In agar diffusion assays for bacteriocins, titers in Arbitrary Units are calculated as the reciprocal of the critical dilution, which in turn is defined as the last dilution of a sample causing visible inhibition. In this study, three partially purified bacteriocins (enterocin 1146, leucocin FlO and lactococcin 140NWC) and nisin
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(250 pg ml ‘) were tested in an AWDA against 2 indicator organisms each. The dose/response curves relating the diameter or the area of the inhibition zone to the log of the dose (ml of bacteriocin pipetted in each well) for leucocin FlO and nisin are shown in Fig. 1 as an example while the parameters of the dose/response curves are shown in Table 1. Although high r2 and highly significant F values were obtained for most regressions, deviations from linearity were evident for some dose/response curves. Moreover, for El 146 and NIS the use of the
a
-d
log(dose)
-5
-4
-2
-3
-1
(ml)
-3
-2
-1
log(dose)(ml) Fig. 1. Dose/response curves for leucocin FlO (LFlO) and nisin (NE) tested against two indicator strains. 0 LFlO against Listeria innocua BL86/26; l LFlO against Lactobacillus sake NCFB2474; n NIS against Lactococcus lactis subsp. lactis ATCC19435; A NIS against Lact. lactis subsp. cremoris CNRZ117. (a) Response calculated as diameter of the zone of inhibition minus the diameter of the well. (b) Response calculated as area of the zone of inhibition minus the area of the well. Mean of three replicates with standard errors (vertical bars).
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Table 1 Parameters (estimate ‘- standard error) of the dose/response curves for the Agar Well Diffusion Assay Parameters were calculated using the equation: R = a + b log(d), where R is the response (diameter of the inhibition zone-diameter of the well (0) or area of the inhibition zone-area of the well, A) and d is the dose (amount of bacteriocin pipetted in the wells, ml). The estimated titer of each bacteriocin solution (AU ml-‘) is calculated as follows: AU ml-’ = 10-‘“-b’. Adjusted r2 and activities calculated from critical dilution asay (CDA) either in the well or spot format are also shown. Bacteriocin : Indicator”
R
a
b
r’
E1146:BL86/26
0
11.61 kO.18 181.06 2 4.15 18.39 k 0.31 328.33 + 7.64 10.74 2 0.22 161.58 * 2.48 9.69 -c 0.26 126.30 rt 2.98 21.00 -t 0.54 487.32 + 14.00 25.48? 0.39 604.42 + 8.32 5.55 k 0.14 70.73 + 2.70 17.94 k 0.33 339.26 f 9.16
2.83 -c 0.07 47.64 k 1.63 5.31 r 0.13 100.96 + 3.21 2.412 0.08 38.78 2 0.92 2.46 f 0.11 33.06 5 1.16 3.62 f 0.23 103.27 f 5.95 4.83 it 0.13 129.48 + 2.74 1.55 rt 0.06 20.74 2 1.14 4.00 IO.11 82.22 f 2.16
0.985* 12732 0.974+ 6318 0.988 2889 0.980 1787 0.972’ 28675 0.986* 14658 0.966* 8591 0.981 6619 0.929 635744 0.94O* 52372 0.979* 189164 0.986 46574 0.975: 3853 0.943* 2572 0.978* 30556 0.96O* 13375
AU ml-’
CDA (AU ml-‘) Well
E1146: NCFB2474 LFIO: BL86126 LFlO : NCFB2474 LCN : ATCC19435 LCN : CNRZl17 NIS : ATCC19435 NIS: CNRZ117
A 0 A 0 A 0 A 0 A 0 A 0 A 0 A
spot
3200
3200
3200
600
6400
9600
1600
2400
25600
25600
25600
25600
BOO 12800
800 9600
a El146 = enterocin 1146; LFlO = leucocin FlO; LCN = Iactococcin 140; NIS = nisin; BL86/26 = Listeria innocua BLg6/26; NCFB2474 = Lactobacilfus sake NCFB2474; ATCC19435 = Lactococcus lactis subsp. Ia& ATCC19435; CNRZ117 = Lactoc. cremoris subsp. cremoris CNRZ117. * Indicates the occurrence of deviations from the statistical assumptions for distribution of residuals in least square regression.
diameter of the zone of inhibition as a response provided a better fit than the area while the reverse was true for LFlO and LCN. A linear relationship between the logarithm of the dose and the absorbance is commonly used as a dose/repsonse curve for photometric assays [3,15]; however, we found that a sigmoidal relationship was more appropriate; Fig. 2 shows the dose/response curves for photometric assays in tubes. Similar results were obtained for assays in microtiter plates. The dose was the amount of bacteriocin pipetted in each tube while the response was defined as the ratio between the absorbance of a sample and that of a control. The following bounded monotonic function was used to model the relationship between dose and response, as previously described [ 171: R = A + (1 - A)/(1 + EXP(B + C log(d))) were the d is the dose. The parameters
of the dose/response
curves are shown in
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-4
-3 lcg(dose) (ml
Methods 22 (1995) 95-108
-2
ml-‘)
Fig. 2. Dose/response curves for the photometric assay in test tubes. 0 enterocin 1146 against Listeria innocua BL86126; A leucocin FlO against L.innocua BL86/26; 0 lactoccin 140NWC against Lactococcus lactis subsp. lactis ATCC19435; 0 nisin against Lact. lactis subsp. cremoris CNRZ117. Dose is the amount of bacteriocin pipetted in each tube (ml ml-‘); R is the response, the ratio of OD,,, of a sample with that of a control. Mean of five replicates 2 standard error (vertical bars).
Table 2. With a few exceptions the equation provided an excellent fit for dose/response data both in test tubes and in microtiter plates. When the parameter c (slope) was high (i.e. for LCN), the curve rose sharply from low (0.05-0.1) to high (0.9-0.95) responses within a very short dilution span. This significantly limits the useful dose range and the accuracy of the method (see below). 3.2. Comparison of the quantitative methods In this study, nisin was the only substance for which a standard solution of known activity was available. The estimates of nisin concentration obtained for a series of two-fold dilutions of a 250 pg ml-’ nisin sample (10,000 IU ml-‘) using the AWDA (with either diameter or area as the response) or the PA in tubes and Lactococcus lactis subsp. cremoris CNRZ117 as indicator are shown in Fig. 3. Both methods were less sensitive than the standard AWDA method for nisin [22] since the lowest dilution causing detectable inhibition had an activity of 20 IU ml-’ in the AWDA and 5 IU ml-’ in the PA. The AWDA was far from accurate: the marked deviation from linearity in the dose/response curves (Fig. 1) was reflected by differences in estimated activities obtained from different dilutions and the real nisin titre was within one standard error from the mean for only a few dilutions. This was not attributable to the large dose span used for the calculation of the standard curve, because the choice of smaller dose spans did not yield better estimates (not shown). The precision of the method was also unsatisfactory: the coefficient of variation (CV) was frequently higher than 20%
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Table 2 Parameters (estimate of:standard error) of the dose/response curves for the Photometric Assay Parameters were calculated using the equation: R = A + (1 - A) I( 1 + EXP(B + C. log(d)) where R is the response (ratio between the absorbance of a sample and that of the control) and d is the dose (amount of bacteriocin pipetted in the assay tube, ml ml-‘). A Bacteriocin Unit (BU) was defined as the amount of bacteriocin needed to obtain a R = 0.5. The amount of bacteriocin solution corresponding to 1 BU (critical dose, CD) was calculated for each standard solution from the equation. The titer (T) in BU ml-’ was calculated as the reciprocal of the CD. Adjusted r* and activities estimated from critical dilution assay (AU ml-‘) are also shown Bacteriocin: Indicator”
a
Photometric assay in test tubes E1146:BL86/26 0.036 ‘- 0.005 El 146 : NCFB2474 -0.021 r+0.040 LFlO:BL86126 0.034~0.086 LFlO : NCFB2474 0.053 + 0.018 LCN : ATCC19435 0.059 rt 0.014 LCN : CNRZl17 0.047 -e 0.013 NIS : ATCC19435 NIS : CNRZl17 :::8 t 0.014 Photometric assay in microtiter plates E1146:BL86/26 0.043 + 0.012 LFlO:BL86/26 0.039 i 0.005 LCN : ATCC19435 0.077 + 0.005
b 27.356 2 7.474 2 24.733 + 15.147 k 37.447 + 38.439 2
C
1.134 0.740 1.021 1.201 4.902 9.248
&a963 2 1.699
7.870 + 0.323 2.878 f 0.254 7.276~0.297 4.483 2 0.348 9/188 k 1.201 9.278 2 2.251
r2
BU ml-’
AU ml-’
0.998 0.917 0.996 0.980 0.977 0.936
2929 409 2451 2253 11541 13563
3200 16 3200 3200 12800 25600 n.a. 25600
;:&4 2 0.387 ::;82
30.735 2 1.532 9.490” 1.016 0.982 27.314” 0.741 8.9042 0.238 0.998 64.033 k 4.351 17.397 ” 1.154 0.995
:;:63 1696 1144 4688
1600 1600 3200
a El146 = enterocin 1146; LFlO = leucocin FlO; LCN = lactococcin 140; NIS = nisin; BL86/26 = Listeria innocua BL86126; NCFB2474 = Lactobacilus sake NCFB2474; ATCC19435 = Lactococcus lactis subsp. lactis ATCC19435; CNRZ117 = Lactoc. cremoris subsp. cremoris CNRZ117.
and only in a few cases was lower than 15% thus being higher than those for AWDA for nisin described in the literature [3]. In the PA, the useful dose range is limited to samples generating a response between 0.05 and 0.9; outside this range differences in response among different dilutions are not significant and usually generate biased estimates of activity or concentration (dilutions 6 and 7 in Fig. 3). Estimates of nisin concentration obtained with the PA for dilutions 8 to 11 were all within 15% of the true concentration of the sample with CV between 10 and 15% thus being more reliable than those obtained with the AWDA and comparing favourably to most methods of assay for nisin [3]. Partially purified bacteriocin solutions of unknown activity were used to obtain dose/response curves for enterocin 1146, leucocin FlO and lactococcin 140; the titer of each bacteriocin solution was calculated from the dose/response curve by using appropriate definitions for the critical dose and units (see methods) and expressed in AU ml-’ or BU ml-’ (Tables 1 and 2); since a standard solution was lacking no real evaluation of the accuracy of the methods was possible. However, a rough indication of accuracy, precision and useful dose range can be obtained by comparing the estimated activities for a series of two-fold dilutions of an independent sample of each partially purified bacteriocin. This is shown as an example for enterocin 1146 tested against Listeria innocua BL86/26 in Fig. 4. The deviation from linearity of the AWDA dose/response curves affects the estimates
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q
0
1
2
3
4
5
Methods 22 (1995) 95-108
AWDA(ci)
6
7
6
9
IO
11
b2(D)
Fig. 3. Comparison nisin (250 pg mlthe diameter (0). photometric assay standard error. D
among the estimates of activity obtained for different dilutions of a sample of ‘) using the Agar Well Diffusion Assay (AWDA. with response calculated either as or the area of the zone of inhibition corrected for the size of the well) or the in test tubes with Lactococcus lactis subsp. lactis CNRZ117 as indicator; mean k is the dilution factor.
obtained with this method from different dilutions. Estimated activities were within 20% and 40% of the theoretical activity when response was calculated from diameter or area of the inhibition zone, respectively. In both cases, CV were high, ranging from 12 to 24% for most dilutions. However, the theoretical activity of the sample was within the 95% confidence limits of mean for most dilutions (not shown). As described for nisin, only dilutions providing responses between 0.05 and 0.9 (dilutions 5 to 7 in Fig. 4) can be used to obtain reliable estimates of activity in the PA. For dilutions yielding a response within this range the estimated activity were within 5% of the theoretical activity for both methods and the CV was between 6 and 11% for the PA in tubes and lower than 6% for the PA in microtiter. Similar figures were obtained for all the other bacteriocins, methods and indicators tested (data not shown), with the PA performing better than the AWDA except for lactoccin 140, where the steep slope of the PA dose/response curve excessively reduced the useful dose range of the method. In the AWDA the response calculated from area occasionally provided better estimates those obtained from the diameter depending from the goodness of fit of the respective dose/response curves. The repeatability of the methods varied slightly depending on their precision; for example estimates of activity of a sample of enterocin 1146 obtained on 4 different days varied between 1542 and 1726 BU ml-’ (mean 1632 BU ml-‘, CV
E. Parente et al. I Journal of Microbiological
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Methods 22 (1995) 95-108
q n
AWDA(0)
AWDA
(A)
105
[
140 130 120 110 100 90 60 70 60 50 0
1
2
3
4
5
6
7
6
9
log,(D) Fig. 4. Comparison among the estimates of activity obtained for different dilutions of a sample of partially purified enterocin 1146 using the Agar Well Diffusion Assay (AWDA, with response calculated either as the diameter (O), or the area of the zone of inhibition corrected for the size of the well) or the Photometric Assay (in test tubes or in microtiter plates) with Listeria innocuo as indicator. Since estimated activity was different for each method the height of a bar (mean f standard error) is 100 ratio between activity estimated for each dilution and estimate obtained from the entire dose/response curve (see Tables 1 and 2). D is the dilution factor.
5.7%) for the PA in tubes and between 3641 and 4215 AU ml-’ (mean 4010 BU ml-‘, CV 8%) for the AWDA (area). The figures for accuracy, precision and repeatability obtained in this study for four bacteriocins tested with different quantitative methods against two indicators each are comparable with those obtained in a previous study [17]. Other authors [7,16] found that the PA or the AWDA were not repeatable over different days; in our experience, if the conditions of the assay are carefully standardized, estimates of activity obtained for a same sample over different days have a CV lower than 10% and 15% for PA and AWDA respectively.
3.3. Comparison
between quantitative and critical dilution assays
The results for the quantitative and critical dilution assays (CDA) are compared in Table 1 (agar diffusion assays) and 2 (photometric assays). With a few exceptions, in AWDA estimates of activities obtained with dose response curves were inflated in comparison with CDA, expecially when standard curves were based on diameters of the zones of inhibition. This depends on the definition of Arbitrary Units used for the quantitative assays, where the CD is calculated by
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extrapolating to 0 response the dose/response curve. If alternative definitions are used (for example dose needed to produce a 7 mm zone) estimates are much closer (not shown). Estimates obtained with quantitative photometric assays were remarkably close to those obtained with the corresponding CDA with the exception of El146 tested against NCFB2474; differences observed in other cases may be expected from differences used in the definition of BU and AU and in the assessment of the CD. Quantitative AWDA or PA have definite advantages over CDA. With quantitative assays, an estimate of activity can be obtained without the need of performing a complete series of dilutions for each sample: even in the worst situation the estimate obtained for a given dilution is within 40% of the theoretical activity of the sample, thus being at least as accurate as CDA, which, by definition, cannot differentiate two samples if their difference in activities is lower than 50%. Moreover, quantitative AWDA and PA methods provide a continuous, although still arbitrary, scale for activity and allow the calculation of confidence intervals for the estimates.
4. Conclusions The dose/response curves used in this study for the AWDA and the PA proved to be valid for 4 bacteriocins tested against 2 indicator strains each. In the AWDA, a good fit was obtained by using a linear relationship between either the area or diameter of the zone of inhibition and the logarithm of the dose; however, more or less severe deviations from linearity were evident in most instances and the response (diameter or area) which provided the best fit depended strongly on the bacteriocin / indicator combination. A sigmoidal dose /response curve was observed in all cases in the PA. Even when standard solutions of known activity were lacking, titers of the bacteriocin solutions in arbitrary units (AU or BU) were obtained directly from the dose/response curves which could in turn be used for the measurement of the activity of samples of unknown potency. PA provided estimates of activity which were more accurate and reproducible than those obtained with AWDA. In conclusion, both methods were more convenient than the classical critical dilution assays. However, the ultimate choice of a method for a given bacteriocin depends on a number of factors (sensitivity, accuracy, precision, ease of use, etc.). For example, the most sensitive indicator should be chosen to maximize the sensitivity of the method; PA are less time-consuming than AWDA both in the set-up of the assay and in the measurement of responses; however, if maximum accuracy is desired, their useful dose range is limited to dilutions providing a response between 0.1 and 0.9; moreover, the dose/response curve is markedly non-linear and appropriate software is required for the estimation of its parameters. On the other hand, AWDA, although less accurate, have a simpler dose/ response curve and a large useful dose range.
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107
Acknowledgements This research was supported by the National Research Council of Italy, Special Project RAISA, subproject 4, Paper 1825.
References [l] Klaenhammer, T.R. (1988) Bacteriocins of lactic acid bacteria. Biochimie 70, 337-349. [2] Nettles, C.G. and Barefoot, S.F. (1993) Biochemical and genetic characteristics of bacteriocins of food associated lactic acid bacteria. J. Food Prot. 56, 338-356. [3] De Vuyst, L. and Vandamme, E.J. (1994) Nisin, a lantibiotic produced by Lactococcus lack subsp. lactis: properties, biosynthesis, fermentation and applications. In: Bacteriocins of lactic acid bacteria (De Vuyst, L., and Vandamme. E.J., eds.), pp. 151-221, Blackie Academic and Professional, London. [4] Pucci, M.J.,Vedamuthu, E.R., Kunka, B.S. and Vanderbergh, P.A. (1988) Inhibition of Listeria monocytogenes by using bacteriocin PA-I produced by Pediococcus acidilactici PAC 1.0. Appi. Environ. Microbial. 54, 2349-2353. [5] Bhunia, A.K., Johnson, M.C. and Ray, B. (1988) Purification, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus acidilactici. J. Appl. Bacterial. 65, 261-268. [6] Harding, C. and Shaw, B.G. (1990) Antimicrobial activity of Leuconostoc gelidum against closely related species and Listeria monocytogenes. J. Appl. Bacterial. 69, 648-658. [7] Geis, A., Singh, J. and Teuber, M. (1983) Potential of lactic streptococci to produce bacteriocin. Appl. Environ. Microbial., 45, 202-211. [8] Mortvedt, C.I. and Nes, I.F. (1990) Plasmid-associated bacteriocinproduction by a Lactobacillus sake strain. J. Gen. Microbial. 136, 1601-1608. [9] Daba, H., Pandian, S., Gosselin, J.F., Simard, R.E., Huang, J. and Lacroix, C. (1991) Detection and activity of a bacteriocin produced by Leuconostoc mesenteroides. Appl. Environ. Microbial. 57, 3450-3455. [lo] Parente, E. and Hill, C. (1992). A comparison of factors affecting the production of two bacteriocins from lactic acid bacteria. J. Appl. Bacterial. 73,290-298. [ll] Skytta, E. and Mattila-Sandholm, T. (1991) A quantitative method for assessing bacteriocins and other food antimicrobials by automated turbidometry. J. Microbial. Methods 14, 77-88. [I2] Zezza, N., Pasini, G.. Lombardi, L., Mercenier, A., Spettoli, P., Zamorani, A., and Nuti, M.P. (1993) Production of a bacteriocin active against lactate-fermenting clostridia by Lactococcus lactis subsp. lactis immobilized incoated alginate beads. J. Dairy Res. 60, 581-591. [13] Giraffa. G., Neviani, E. and Veneroni, A. (1990) Use of conductance to detect bacteriocin activity. J. Food Prot. 53, 772-776. [14] Reid, F.L.; Ahmed, K.A.; Waites, W.M. and Stewart, G.S.A.B. (1990) The rapid detection of antimicrobials using bioluminescent lactic acid bacteria. FEMS Microbial. Rev. 87, P88. [15] Isaacson, D.M. and Kirschbaum, J. (1986) Assays of antimicrobial substances. In: Manual of industrial microbiology and biotechnology (Demain, A.L. and Solomon, N. A., eds.), pp. 410-435, American Society for Microbiology, Washington, D.C. [16] Rammelsberg, M. and Radler, F. (1990) Antibacterial polypeptides of Lactobacillus species. J. Appl. Bacterial. 69. 177-184. [17] Parente, E., Ricciardi. A. and Villani,V (1993) Evaluation of two methods for the measurement of bacteriocin activity. In: Biotechnology and molecular biology of lactic acid bacteria for the improvement of foods and feeds quality (Zamorani, A., Manachini, P.L., Bottazzi, V. and Coppola, S., eds.), pp. 318-327, Istituto Poligrafico e Zecca dello Stato, Rome.
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[18] Systat (1992) Statistics, Version 5.2 Edition. Systat Inc., Evanston IL, 724 pp. [19] Systat (1992) Graphics, Version 5.2 Edition. Systat Inc., Evanston IL. 600 pp. [ZO] Tramer, J. and Fowler, G.G. (1964) Estimation of nisin in foods. J. Sci. Food 522-528.
Agric.
15
Journal of Microbiological Methods 22 (1995) 95-108
A comparison Eugenio
of methods for the measurement bacteriocin activity
Parente, * Carla Brienza, Marcella Moles, Annamaria
of Ricciardi
Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali. Universitci della Basilicata; Via N. Sauro, 85, I-85100 Potenza, Italy
Received 8 June 1994; accepted 3 October 1994
Abstract
Agar diffusion (spot and well) and photometric (tube or microtiter) assays, both in the form of critical dilution and quantitative assays, were compared for 4 bacteriocins (enterocin 1146, lactococcin 140, leucocin FlO and nisin) against 2 indicator strains each. In the agar well diffusion assay (AWDA) a linear relationship existed between response (diameter or area of the zone of inhibition) and the logarithm of the dose while a non-linear equation was used to model the sigmoidal dose/response curve in photometric assays (PA). The dose/response curves were used to define titers of the standard solutions in arbitrary units and to develop quantitative assays for all the bacteriocins. With the exception of lactococcin 140, the PA provided estimates which were more reproducible than those obtained with the AWDA. The quantitative assays compared favourably with the classical critical dilution assays for bacteriocins, eliminating the need to dilute to extinction to estimate the titers of bacteriocin solutions, providing a continuous scale for activity and allowing the calculation of confidence intervals. Keywords:
Bacteriocin;
Agar well diffusion
assay; Critical dilution
assay; Photometric
assay
1. Introduction Bacteriocins are bacterial proteins which are inhibitory to microorganisms which are usually, but not always, closely related to the producer strain [l]. Because of their potential use as natural food preservatives and as phenotypic * Corresponding author. PZVX85.CINECA.IT.
Tel.:
+ 39-971-474384. Fax:
+ 39-971-471481. E-Mail PARENTE@
0167-7012/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0167-7012(94)00068-9
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I Journal of Microbiological Methods 22 (19%) 95-108
markers for the construction of food-grade cloning vectors, bacteriocins produced by lactic acid bacteria have been the subject of intensive investigation in recent years [ 1.21. Quantitative measurements of bacteriocin activity are needed for most of the activities related to their characterization. Several methods have been used for the quantitation of the iantibiotic nisin [3]; because of its importance as food preservative, standard units and methods have been defined for this substance and a commercial standard preparation is available. Methods for the assay of bacteriocin activity are usually derived from those for antibiotics. Modifications (spot, well, disc diffusion) of the agar diffusion assay [4-61 and photometric [7-121 methods have been widely used. Methods based on conductance and bioluminescence have been used less frequently [13,14]. Since standard solution of bacteriocins are generally not available, critical dilution assays (CDA) are used with arbitrary units to express activity. The titer of the bacteriocin solution is defined as the reciprocal of the lowest amount of bacteriocin solution causing a definite inhibition of the indicator strain. This method is semi-quantitative, provides only a discontinuous scale for activity and always requires that a complete series of dilutions is carried out to estimate activity. Alhough CDA are easy to use and suffice for most applications, they are of little value when statistical comparisons on quantitative measurements of bacteriocin activity are to be carried out. When agar diffusion assays and photometric methods are based on a dose/response curve to estimate bacteriocin activity, a continuous scale for activity is obtained. Both agar diffusion and photometric methods for the quantitation of activity of antimicrobials have their drawbacks [15] and the lack of standard solutions make the construction of dose/response curves difficult. Even if several authors have used quantitative methods for the measurement of the activity of bacteriocins produced by lactic acid bacteria [7,8,10,16] the nature of the dose/response curve used is not clear and statements on the relative accuracy of methods or on the variability of the results are almost always lacking. Direct comparisons of methods for the quantitative measurement of bacteriocin activity are rare and of limited scope [11,17]. The objective of this study was to compare some of the most commonly used methods for the measurement of bacteriocin activity: agar diffusion assays (as spot or well diffusion) and photometric assays (in tubes and/or in microtiter plates) were compared for nisin and three partially purified bacteriocins both in the form of critical dilution assays and as quantitative methods based on dose/ response curves.
2. Materials and methods 2.1. Bacteriocins
Nisin (NIS) was obtained from Sigma as 2.5% powder and suspended in 0.02 N HCl to a final concentration of 2.50 pg ml-’ (10,000 IU ml -I). Partially purified
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I Journal of Microbiological Methods 22 (1995) 95-108
97
of lactococcin 140 (LCN, produced by Lactococcus luctis subsp. enterocin 1146 (E1146, produced by Enterococcus fuecium DPC1146) and leucocin FlO (LFlO, produced by Leuconostoc carnosum FlO) were obtained by precipitating the bacteriocins from culture supernatants with ammonium sulfate (40% saturation, 4 h at 4°C); the pellet obtained after centrifugation (8000 g, 30 min.) was resuspended in 50 mM potassium phosphate buffer, pH 5.0, dialysed against the same buffer in 3,500 NMWCO membranes (Spectra-Pore, Spectrum, Los Angeles, California) and filter sterilized with Millipore (Bedford, Mass. USA) HV membranes (0.45 p). All bacteriocins were stored at - 20°C. preparations
factis 140NWC),
2.2. Indicator strains Lactococcus luctis subsp. lactis ATCC19435 and Lactococcus lactis subsp. cremoris CNRZ117 were used as indicator strains for LCN and NIS assay; Listeriu innocua BL86/26 and Lactobacillus sake NCFB2474 were used as indicators for
LFlO and E1146. CNRZ117, BL86/26 and NCFB2474 were obtained from Dr. T. Cogan, (National Dairy Products Research Centre, Fermoy, IRL); ATCC19435 was obtained from Prof. S. Coppola (Istituto di Microbiologia Agraria, Portici, Italy). All strains were maintained frozen at - 20°C in 25% glycerol. Working cultures were grown for 16 h at 30°C in Ml7 broth (Oxoid, Basingstoke, UK) + 0.5% glucose (GM17) for lactococci, MRS broth (Oxoid) for NCFB2474 and Tryptic Soy Broth (Oxoid) + 0.6% Yeast Extract (Oxoid) for BL86/26. The same media were used for their growth during bacteriocin assay, and bacteriological agar (Oxoid) was added to obtain top (0.6% w/v) and bottom (1.5% w/v) agar media for agar diffusion assays. Indicator strains cultures were always adjusted to OD,,, = 0.5 before inoculation of the media for bacteriocin assay.
2.3. Critical dilution assay (CDA) The method of Pucci et al. [4] was used. Serial two-fold dilutions were carried out in the same medium used for the growth of the appropriate indicator strain except for nisin, which was diluted in 0.02 N HCl. Ten ~1 of each dilution were spotted on the surface of the appropriate agar medium (20 ml in a 90 mm diameter Petri dish) which had been dried for 30 min. After diffusion (30 min. at room temperature) the plates were overlaid with 5 ml of semisolid agar medium inoculated (1% v/v) with a standardized suspension of the indicator strain. After incubation (16 h at 30°C) the plates were checked for zones of inibition. The critical dilution was defined as the highest dilution producing a definite inhibition zone. When a clear inhibition zone was followed by a hazy one the critical dilution was calculated as the average of the two dilutions. The titer of the bacteriocin solution, in AU ml-‘, was calculated as (lOOO/lO) D, where D is the dilution factor.
98
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2.4. Agur Well Diffusion Assay (AWDA)
Twenty ml of the appropriate agar (1.5% w/v) medium were pipetted in 90 mm Petri dishes and inoculated (1% v/v) with a standardized indicator suspension. After drying for 30 min., six 6 mm wells were bored in each plate. Forty ~1 of 2-fold dilutions of bacteriocin were pipetted into each well and the plates were incubated at 30°C for 16 h. The diameter of the inhibition zones was measured using a calliper. Results were read either as a CDA (with the critical dilution being the last dilution which produced a zone larger than 6 mm and titer defined as described above) or as a quantitative assay. For the latter a standard curve was prepared using a series of 2-fold dilutions of bacteriocin solution. The response (R) was calculated either as the diameter or as the area of the inhibition zone corrected for the diameter or area of the well. The dose (d) was the amount of bacteriocin pipetted into each well (40 ~1 l/D). The dose/response curve had the following equation: R = a + b log(d) The critical dose (CD) was defined as the amount of bacteriocin solution ml) corresponding to a null inhibition zone and calculated by extrapolating dose/response curve. The titer, in AU ml-‘, was calculated as the reciprocal the CD. The standard curve, relating dose in AU to R, had a 0 intercept and same slope of Eq. 1. The activity in AU ml-’ of a sample was calculated follows: AU ml-’ = (1000/40) D 10’R’h’
(1) (in the of the as (2)
Three replicates, in different plates, were used for each dilution of the standard curve and for each sample. 2.5. Photometric assay (PA)
A standardized indicator culture was used to inoculate (5% v/v) the appropriate broth medium, which was cooled to 4°C and dispensed, in 2.4 ml amounts, in sterile test tubes. 100 ~1 of 2-fold dilutions of bacteriocin were pipetted into each tube and incubation was carried out at 30°C for 4 h (ATCC19435) or 6 h (other strains). 100 ~1 of sterile medium were added to control tubes. A standard curve was prepared using a series of 2-fold dilutions of bacteriocin solution. At the end of the incubation the absorbance of the culture was read at 600 nm using a Beckman DU-7 spectrophotometer with uninoculated medium as a blank. The response (R) was calculated as the ratio between the absorbance of a sample and that of the control (average of 5 replicates). The dose/response curve had the following equation: R = A + (1 - A)/(1 + EXP(B + C) log(d))
(3)
were the dose (d) was the amount of bacteriocin (ml ml-‘) in the assay tube. One Bacteriocin Unit (BU) was defined as the amount of bacteriocin needed to obtain
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99
Methods 22 (1995) 95-108
a R = 0.5 and calculated for each standard solution from Eq. 3; the titer (T) in BU ml -’ was calculated as its reciprocal. The dose/response curve was recalculated as follows: R = a + (1 - a)/(1 + EXP(b + c) log(BU))
where BU = T d. The activity of an unknown following equation: BU ml-’ = (1000/40) D 10 <(ln((l-R)I(R-a))-6)/c)
(4) sample was obtained
from the
(3
were R is the response obtained for the sample, a, b and c the parameters of Eq. 4. Results were also scored as a critical dilution assay; the critical dilution was defined as the last dilution showing a visible inhibition compared to the control.The titer of the bacteriocin solution, in AU ml-‘, was calculated as (1000/25) D. The method was adapted for use in microtiter plates as follows: 10 ~1 of bacteriocin solution or of sterile growth medium (for blank and controls) were pipetted into the wells of sterile microtiter plates (Corning Cell Wells, Corning, NY); 240 ~1 of inoculated (control and bacteriocin wells) or uninoculated medium (blanks) were then pipetted into the wells. The plates were incubated at 30°C for 4 or 6 h and the absorbance at 620 nm was read with a Novapath Mini Reader (Biorad Laboraties, Hercules, CA). Results were scored as described above. 2.6. Statistics and graphics Statistical analyses were carried out with Systat 5.2.1 for the Macintosh, using procedures NONLIN for non-linear regression and MGLH for linear regression [lS]. Graphs were generated using Systat 5.2.1 [19] or CA-Cricket Graph III.
3. Results and discussion 3.1. Nature of the doselresponse
curves
While no dose/response curves are needed for critical dilution assays, some sort of mathematical relationship must be used for calculations of doses from responses in both the AWDA and the PA. In Agar Well Diffusion Assays for antibiotics and for some bacteriocins [3,15,16] a linear relationship between diameter or the area (the response) of the inibition zone and the logarithm of the dose is used; in Photometric Assays a linear relationship between the absorbance and the logarithm of the dose is used [3,15]. Unfortunately, purified bacteriocins are rarely available and doses are ususally expressed in arbitrary units. In agar diffusion assays for bacteriocins, titers in Arbitrary Units are calculated as the reciprocal of the critical dilution, which in turn is defined as the last dilution of a sample causing visible inhibition. In this study, three partially purified bacteriocins (enterocin 1146, leucocin FlO and lactococcin 140NWC) and nisin
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Methods 22 (1995) 95-108
(250 pg ml ‘) were tested in an AWDA against 2 indicator organisms each. The dose/response curves relating the diameter or the area of the inhibition zone to the log of the dose (ml of bacteriocin pipetted in each well) for leucocin FlO and nisin are shown in Fig. 1 as an example while the parameters of the dose/response curves are shown in Table 1. Although high r2 and highly significant F values were obtained for most regressions, deviations from linearity were evident for some dose/response curves. Moreover, for El 146 and NIS the use of the
a
-d
log(dose)
-5
-4
-2
-3
-1
(ml)
-3
-2
-1
log(dose)(ml) Fig. 1. Dose/response curves for leucocin FlO (LFlO) and nisin (NE) tested against two indicator strains. 0 LFlO against Listeria innocua BL86/26; l LFlO against Lactobacillus sake NCFB2474; n NIS against Lactococcus lactis subsp. lactis ATCC19435; A NIS against Lact. lactis subsp. cremoris CNRZ117. (a) Response calculated as diameter of the zone of inhibition minus the diameter of the well. (b) Response calculated as area of the zone of inhibition minus the area of the well. Mean of three replicates with standard errors (vertical bars).
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E. Parente et al. I Journal of Microbiological Methods 22 (1995) 95-108
Table 1 Parameters (estimate ‘- standard error) of the dose/response curves for the Agar Well Diffusion Assay Parameters were calculated using the equation: R = a + b log(d), where R is the response (diameter of the inhibition zone-diameter of the well (0) or area of the inhibition zone-area of the well, A) and d is the dose (amount of bacteriocin pipetted in the wells, ml). The estimated titer of each bacteriocin solution (AU ml-‘) is calculated as follows: AU ml-’ = 10-‘“-b’. Adjusted r2 and activities calculated from critical dilution asay (CDA) either in the well or spot format are also shown. Bacteriocin : Indicator”
R
a
b
r’
E1146:BL86/26
0
11.61 kO.18 181.06 2 4.15 18.39 k 0.31 328.33 + 7.64 10.74 2 0.22 161.58 * 2.48 9.69 -c 0.26 126.30 rt 2.98 21.00 -t 0.54 487.32 + 14.00 25.48? 0.39 604.42 + 8.32 5.55 k 0.14 70.73 + 2.70 17.94 k 0.33 339.26 f 9.16
2.83 -c 0.07 47.64 k 1.63 5.31 r 0.13 100.96 + 3.21 2.412 0.08 38.78 2 0.92 2.46 f 0.11 33.06 5 1.16 3.62 f 0.23 103.27 f 5.95 4.83 it 0.13 129.48 + 2.74 1.55 rt 0.06 20.74 2 1.14 4.00 IO.11 82.22 f 2.16
0.985* 12732 0.974+ 6318 0.988 2889 0.980 1787 0.972’ 28675 0.986* 14658 0.966* 8591 0.981 6619 0.929 635744 0.94O* 52372 0.979* 189164 0.986 46574 0.975: 3853 0.943* 2572 0.978* 30556 0.96O* 13375
AU ml-’
CDA (AU ml-‘) Well
E1146: NCFB2474 LFIO: BL86126 LFlO : NCFB2474 LCN : ATCC19435 LCN : CNRZl17 NIS : ATCC19435 NIS: CNRZ117
A 0 A 0 A 0 A 0 A 0 A 0 A 0 A
spot
3200
3200
3200
600
6400
9600
1600
2400
25600
25600
25600
25600
BOO 12800
800 9600
a El146 = enterocin 1146; LFlO = leucocin FlO; LCN = Iactococcin 140; NIS = nisin; BL86/26 = Listeria innocua BLg6/26; NCFB2474 = Lactobacilfus sake NCFB2474; ATCC19435 = Lactococcus lactis subsp. Ia& ATCC19435; CNRZ117 = Lactoc. cremoris subsp. cremoris CNRZ117. * Indicates the occurrence of deviations from the statistical assumptions for distribution of residuals in least square regression.
diameter of the zone of inhibition as a response provided a better fit than the area while the reverse was true for LFlO and LCN. A linear relationship between the logarithm of the dose and the absorbance is commonly used as a dose/repsonse curve for photometric assays [3,15]; however, we found that a sigmoidal relationship was more appropriate; Fig. 2 shows the dose/response curves for photometric assays in tubes. Similar results were obtained for assays in microtiter plates. The dose was the amount of bacteriocin pipetted in each tube while the response was defined as the ratio between the absorbance of a sample and that of a control. The following bounded monotonic function was used to model the relationship between dose and response, as previously described [ 171: R = A + (1 - A)/(1 + EXP(B + C log(d))) were the d is the dose. The parameters
of the dose/response
curves are shown in
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E. Parente et al. I Journal of Microbiological
-4
-3 lcg(dose) (ml
Methods 22 (1995) 95-108
-2
ml-‘)
Fig. 2. Dose/response curves for the photometric assay in test tubes. 0 enterocin 1146 against Listeria innocua BL86126; A leucocin FlO against L.innocua BL86/26; 0 lactoccin 140NWC against Lactococcus lactis subsp. lactis ATCC19435; 0 nisin against Lact. lactis subsp. cremoris CNRZ117. Dose is the amount of bacteriocin pipetted in each tube (ml ml-‘); R is the response, the ratio of OD,,, of a sample with that of a control. Mean of five replicates 2 standard error (vertical bars).
Table 2. With a few exceptions the equation provided an excellent fit for dose/response data both in test tubes and in microtiter plates. When the parameter c (slope) was high (i.e. for LCN), the curve rose sharply from low (0.05-0.1) to high (0.9-0.95) responses within a very short dilution span. This significantly limits the useful dose range and the accuracy of the method (see below). 3.2. Comparison of the quantitative methods In this study, nisin was the only substance for which a standard solution of known activity was available. The estimates of nisin concentration obtained for a series of two-fold dilutions of a 250 pg ml-’ nisin sample (10,000 IU ml-‘) using the AWDA (with either diameter or area as the response) or the PA in tubes and Lactococcus lactis subsp. cremoris CNRZ117 as indicator are shown in Fig. 3. Both methods were less sensitive than the standard AWDA method for nisin [22] since the lowest dilution causing detectable inhibition had an activity of 20 IU ml-’ in the AWDA and 5 IU ml-’ in the PA. The AWDA was far from accurate: the marked deviation from linearity in the dose/response curves (Fig. 1) was reflected by differences in estimated activities obtained from different dilutions and the real nisin titre was within one standard error from the mean for only a few dilutions. This was not attributable to the large dose span used for the calculation of the standard curve, because the choice of smaller dose spans did not yield better estimates (not shown). The precision of the method was also unsatisfactory: the coefficient of variation (CV) was frequently higher than 20%
E. Parente et al. / Journal of Microbiological Methodr 22 (1995) 95-108
103
Table 2 Parameters (estimate of:standard error) of the dose/response curves for the Photometric Assay Parameters were calculated using the equation: R = A + (1 - A) I( 1 + EXP(B + C. log(d)) where R is the response (ratio between the absorbance of a sample and that of the control) and d is the dose (amount of bacteriocin pipetted in the assay tube, ml ml-‘). A Bacteriocin Unit (BU) was defined as the amount of bacteriocin needed to obtain a R = 0.5. The amount of bacteriocin solution corresponding to 1 BU (critical dose, CD) was calculated for each standard solution from the equation. The titer (T) in BU ml-’ was calculated as the reciprocal of the CD. Adjusted r* and activities estimated from critical dilution assay (AU ml-‘) are also shown Bacteriocin: Indicator”
a
Photometric assay in test tubes E1146:BL86/26 0.036 ‘- 0.005 El 146 : NCFB2474 -0.021 r+0.040 LFlO:BL86126 0.034~0.086 LFlO : NCFB2474 0.053 + 0.018 LCN : ATCC19435 0.059 rt 0.014 LCN : CNRZl17 0.047 -e 0.013 NIS : ATCC19435 NIS : CNRZl17 :::8 t 0.014 Photometric assay in microtiter plates E1146:BL86/26 0.043 + 0.012 LFlO:BL86/26 0.039 i 0.005 LCN : ATCC19435 0.077 + 0.005
b 27.356 2 7.474 2 24.733 + 15.147 k 37.447 + 38.439 2
C
1.134 0.740 1.021 1.201 4.902 9.248
&a963 2 1.699
7.870 + 0.323 2.878 f 0.254 7.276~0.297 4.483 2 0.348 9/188 k 1.201 9.278 2 2.251
r2
BU ml-’
AU ml-’
0.998 0.917 0.996 0.980 0.977 0.936
2929 409 2451 2253 11541 13563
3200 16 3200 3200 12800 25600 n.a. 25600
;:&4 2 0.387 ::;82
30.735 2 1.532 9.490” 1.016 0.982 27.314” 0.741 8.9042 0.238 0.998 64.033 k 4.351 17.397 ” 1.154 0.995
:;:63 1696 1144 4688
1600 1600 3200
a El146 = enterocin 1146; LFlO = leucocin FlO; LCN = lactococcin 140; NIS = nisin; BL86/26 = Listeria innocua BL86126; NCFB2474 = Lactobacilus sake NCFB2474; ATCC19435 = Lactococcus lactis subsp. lactis ATCC19435; CNRZ117 = Lactoc. cremoris subsp. cremoris CNRZ117.
and only in a few cases was lower than 15% thus being higher than those for AWDA for nisin described in the literature [3]. In the PA, the useful dose range is limited to samples generating a response between 0.05 and 0.9; outside this range differences in response among different dilutions are not significant and usually generate biased estimates of activity or concentration (dilutions 6 and 7 in Fig. 3). Estimates of nisin concentration obtained with the PA for dilutions 8 to 11 were all within 15% of the true concentration of the sample with CV between 10 and 15% thus being more reliable than those obtained with the AWDA and comparing favourably to most methods of assay for nisin [3]. Partially purified bacteriocin solutions of unknown activity were used to obtain dose/response curves for enterocin 1146, leucocin FlO and lactococcin 140; the titer of each bacteriocin solution was calculated from the dose/response curve by using appropriate definitions for the critical dose and units (see methods) and expressed in AU ml-’ or BU ml-’ (Tables 1 and 2); since a standard solution was lacking no real evaluation of the accuracy of the methods was possible. However, a rough indication of accuracy, precision and useful dose range can be obtained by comparing the estimated activities for a series of two-fold dilutions of an independent sample of each partially purified bacteriocin. This is shown as an example for enterocin 1146 tested against Listeria innocua BL86/26 in Fig. 4. The deviation from linearity of the AWDA dose/response curves affects the estimates
104
E. Parente et al. I Journal of Microbiological
q
0
1
2
3
4
5
Methods 22 (1995) 95-108
AWDA(ci)
6
7
6
9
IO
11
b2(D)
Fig. 3. Comparison nisin (250 pg mlthe diameter (0). photometric assay standard error. D
among the estimates of activity obtained for different dilutions of a sample of ‘) using the Agar Well Diffusion Assay (AWDA. with response calculated either as or the area of the zone of inhibition corrected for the size of the well) or the in test tubes with Lactococcus lactis subsp. lactis CNRZ117 as indicator; mean k is the dilution factor.
obtained with this method from different dilutions. Estimated activities were within 20% and 40% of the theoretical activity when response was calculated from diameter or area of the inhibition zone, respectively. In both cases, CV were high, ranging from 12 to 24% for most dilutions. However, the theoretical activity of the sample was within the 95% confidence limits of mean for most dilutions (not shown). As described for nisin, only dilutions providing responses between 0.05 and 0.9 (dilutions 5 to 7 in Fig. 4) can be used to obtain reliable estimates of activity in the PA. For dilutions yielding a response within this range the estimated activity were within 5% of the theoretical activity for both methods and the CV was between 6 and 11% for the PA in tubes and lower than 6% for the PA in microtiter. Similar figures were obtained for all the other bacteriocins, methods and indicators tested (data not shown), with the PA performing better than the AWDA except for lactoccin 140, where the steep slope of the PA dose/response curve excessively reduced the useful dose range of the method. In the AWDA the response calculated from area occasionally provided better estimates those obtained from the diameter depending from the goodness of fit of the respective dose/response curves. The repeatability of the methods varied slightly depending on their precision; for example estimates of activity of a sample of enterocin 1146 obtained on 4 different days varied between 1542 and 1726 BU ml-’ (mean 1632 BU ml-‘, CV
E. Parente et al. I Journal of Microbiological
T
Methods 22 (1995) 95-108
q n
AWDA(0)
AWDA
(A)
105
[
140 130 120 110 100 90 60 70 60 50 0
1
2
3
4
5
6
7
6
9
log,(D) Fig. 4. Comparison among the estimates of activity obtained for different dilutions of a sample of partially purified enterocin 1146 using the Agar Well Diffusion Assay (AWDA, with response calculated either as the diameter (O), or the area of the zone of inhibition corrected for the size of the well) or the Photometric Assay (in test tubes or in microtiter plates) with Listeria innocuo as indicator. Since estimated activity was different for each method the height of a bar (mean f standard error) is 100 ratio between activity estimated for each dilution and estimate obtained from the entire dose/response curve (see Tables 1 and 2). D is the dilution factor.
5.7%) for the PA in tubes and between 3641 and 4215 AU ml-’ (mean 4010 BU ml-‘, CV 8%) for the AWDA (area). The figures for accuracy, precision and repeatability obtained in this study for four bacteriocins tested with different quantitative methods against two indicators each are comparable with those obtained in a previous study [17]. Other authors [7,16] found that the PA or the AWDA were not repeatable over different days; in our experience, if the conditions of the assay are carefully standardized, estimates of activity obtained for a same sample over different days have a CV lower than 10% and 15% for PA and AWDA respectively.
3.3. Comparison
between quantitative and critical dilution assays
The results for the quantitative and critical dilution assays (CDA) are compared in Table 1 (agar diffusion assays) and 2 (photometric assays). With a few exceptions, in AWDA estimates of activities obtained with dose response curves were inflated in comparison with CDA, expecially when standard curves were based on diameters of the zones of inhibition. This depends on the definition of Arbitrary Units used for the quantitative assays, where the CD is calculated by
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extrapolating to 0 response the dose/response curve. If alternative definitions are used (for example dose needed to produce a 7 mm zone) estimates are much closer (not shown). Estimates obtained with quantitative photometric assays were remarkably close to those obtained with the corresponding CDA with the exception of El146 tested against NCFB2474; differences observed in other cases may be expected from differences used in the definition of BU and AU and in the assessment of the CD. Quantitative AWDA or PA have definite advantages over CDA. With quantitative assays, an estimate of activity can be obtained without the need of performing a complete series of dilutions for each sample: even in the worst situation the estimate obtained for a given dilution is within 40% of the theoretical activity of the sample, thus being at least as accurate as CDA, which, by definition, cannot differentiate two samples if their difference in activities is lower than 50%. Moreover, quantitative AWDA and PA methods provide a continuous, although still arbitrary, scale for activity and allow the calculation of confidence intervals for the estimates.
4. Conclusions The dose/response curves used in this study for the AWDA and the PA proved to be valid for 4 bacteriocins tested against 2 indicator strains each. In the AWDA, a good fit was obtained by using a linear relationship between either the area or diameter of the zone of inhibition and the logarithm of the dose; however, more or less severe deviations from linearity were evident in most instances and the response (diameter or area) which provided the best fit depended strongly on the bacteriocin / indicator combination. A sigmoidal dose /response curve was observed in all cases in the PA. Even when standard solutions of known activity were lacking, titers of the bacteriocin solutions in arbitrary units (AU or BU) were obtained directly from the dose/response curves which could in turn be used for the measurement of the activity of samples of unknown potency. PA provided estimates of activity which were more accurate and reproducible than those obtained with AWDA. In conclusion, both methods were more convenient than the classical critical dilution assays. However, the ultimate choice of a method for a given bacteriocin depends on a number of factors (sensitivity, accuracy, precision, ease of use, etc.). For example, the most sensitive indicator should be chosen to maximize the sensitivity of the method; PA are less time-consuming than AWDA both in the set-up of the assay and in the measurement of responses; however, if maximum accuracy is desired, their useful dose range is limited to dilutions providing a response between 0.1 and 0.9; moreover, the dose/response curve is markedly non-linear and appropriate software is required for the estimation of its parameters. On the other hand, AWDA, although less accurate, have a simpler dose/ response curve and a large useful dose range.
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Acknowledgements This research was supported by the National Research Council of Italy, Special Project RAISA, subproject 4, Paper 1825.
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