Inremutionat Journal of Food Science nrid Technology ( 1990) 25, 5912-595

An enzyme immunoassay for nisin

M . B . FALAHEE”, M. R. ADAMS, J . W . DALE & B. A . MORRIS?, University of Surrey, Guildford, Surrey, UK

Summary

The development of an enzyme-linked immunosorbent assay (ELISA) for nisin using polyclonal antiserum is described. The method has a limit of detection of 1.9 x i.u. ml-’ and correlates well with the bioassay. Its use in the analysis of commercial processed cheese samples is described and the results obtained compared with thosc from the bioassay.

Keywords

ELISA, food preservative.

Introduction

Nisin is a 34 amino acid peptide antibiotic (mol. wt 3510) produced by some strains of Lactococcus luctis and active against a rangc of Gram-positive bacteria (Hurst, 19x1 ). Its heat and acid stability and the lack of clinical use, which would prevent application as a food preservative, has led to its widespread adoption by the food industry (E234). This is particularly so in canned foods and processed cheeses where it inhibits the growth of Grain-positive spore formers. At present it is permitted up to various maximum prescribed levels in inore than 50 countries. The current analytical procedure for nisin employs a bioassay in which the inhibition of a test organism is measured (Tramer & Fowler, 1964). Here we report the development of an enzyme-linked irnmunosorbent assay (ELISA) and its application to commercial cheese samples.

Materials and methods

Pure nisin (calibrated against the international reference preparation and containing 37 x lo6 i.u. g-’) was obtained from Aplin & Barrett Ltd, Trowbridge, UK. Horseradish peroxidase (type VI) and o-phenylene diamine were supplied by Sigma Chemical Co., Poole, UK. Chemicals for phosphate-buffered saline were from BDH, Poole, I J K . ELISA microtitration plates (Nunc Immunoplate I ; Gibco, Paisley, UK) were washed (Titertek S8/S12 plate washer; Flow Laboratories, Rickmansworth, UK) and read with a Dynatech MR600 plate reader (Billingshurst, UK).

The bioassay medium contained (g 1- ‘) Bacteriological peptone (Oxoid, Basing- stoke, UK), 1; Lab-lemco (Oxoid), 3; NaCl (BDH), 3; yeast extract (Oxoid), 1.5; raw

Authorb’ addrcsscs: Department of Microbiology and tDepartment of Biochemistry. Univcrsity uf

*Currespondent. Surrey. Guildford, Surrcy, GU2 SXH, UK.

Nisin ELISA 59 1

cane sugar (muscovado) 1 (J Sainsbury, London, UK); agar no. 1 (Oxoid). 10. The pH of the medium was adjusted to 7.5 f 0.1 prior to sterilization (20 min, 1.035 Bar).

MicrococcusJlauus (NClB 8166) was maintained on slopes of the assay medium, and stored at 4-TC for a maximum of 14 days before subculturing.

Processed cheese samples were commercial brands purchased locally.

Antiserum production Antiserum was raised in a Suffolk sheep with nisin (1 mg) conjugated to egg

albumen using the glutaraldehyde condensation technique (Reichin et al. , 1968). The conjligate was emulsified with non-ulcerative Freund's adjuvant (Guildhay Antisera, Guildford, UK) at a ratio adjuvant:immunogen, 2:l and injected intramuscularly. Serum used for assay development was obtained after the first boost at 9 months. IgG was prepared from whole serum by the method of Hurn & Chantler (1980). Immunospecific antibody was isolated by affinity chromatography.

AfJinity chromatography Nisin (200 mg) was dissolved in 5 mlO.l M acetate buffer (pH 4.5) and mixed with 1 g

activated glass (gift, P. Kwasowski) at room temperature for 4h. Solid glycine was added to yield a final concentration of 0.1 M, and mixing continued at room temperature for 4 h. The matrix was left at 4°C overnight before packing into a column. The column was washed through with an excess of distilled water followed by 0.1 M glycine-HC1 (pH 2.2) and distilled water. The column was equilibrated with phosphate-buffered saline (PBS).

The IgG fraction was applied to the column, which was washed through with PBS until all non-specific protein had been removed. Nisin specific antibody was eluted in I .5 ml fractions with 0.1 M glycine-HCI (pH 2.8). Fractions were neutralized with 2M Tris (pH 7.0). The concentration of anti-nisin IgG was calculated using the equation:

A2SO X dilution factor 1.34

(IgG) mg ml-' =

Fractions containing specific antibody were pooled and dialysed at 4°C against several changes of PBS.

ELISA protocol Antinisin IgG was conjugated to horseradish peroxidase as described in Beyzavi and

co-workers (1987). Microtitration plates were prewashed by soaking in coating buffer (0.1 M carbonate-

bicarbonate, pH 9.6) for 15 min. Wells were coated with affinity purified antinisin IgG (5 pg ml-') in coating buffer (0.2 ml well-'). After a 2-h incubation at 37°C the plates were washed three times with phosphate-buffered saline (pH 7.4) containing 0.1 % gelatin and 0.05% Tween 20 (PBSGT). Subsequently they were blocked for 1 h with casein buffer (NaCI, 9; Tris, 1.2; casein, 5 (g I - ' ) , pH 7.6) at 37°C and washed three times with PBSGT. Samples (0.2 ml well-') were added and incubated for 16h at 4°C. After washing three times with PBSGT, 0.2 ml of the horseradish peroxidase conjugate (diluted 1 : 25000 [vh] in PBSGT) was added to each well and the plates incubated for 2h at 37°C. The plates were washed as before and 0.15 ml substrate solution (0.4 mg ml-' o-phenylene diarnine in 0 . 0 2 4 ~ citrate-0.05 M phosphate buffer [pH 5.01

592 M. B. Fuluhee et al.

containing 0.04% hydrogen peroxide) added. After 30 min at 37°C the reaction was stopped by the addition of 0.05 ml of 2.5 M HzS04 and absorbances read at 490 nm.

Preparation of cheese samples A number of commercial brands of processed cheese were used throughout. The method of preparation was essentially that described by Fowler et al. (1975).

Samples (2.5 t 0.1 g) were weighed out and dispersed evenly in 10 ml of 0 . 0 2 M HCI. The pH was adjusted to 2.0 k 0.1 and the sample heated at 98°C for 5 min. After cooling to 20"C, the volume was adjusted to 12.5 ml with 0.02M HCI and centrifuged for 20 min at 4000 x g and 4°C. The supernatant was removed after standing at 6 7 ° C for 30 min and filtered through a double layer of Whatman filter paper (no. 1) to give a clear extract.

An extract of cheese, labelled as preservative free and confirmed as nisin free using the ELISA, was prepared as above and used as analyte-free matrix.

Recovery was determined by spiking the initial extracts of nisin-free cheese with a range of known concentrations of nisin and processing as above. Samples were assayed in duplicate.

The intra-assay and interassay coefficients of variation were determined using three different samples of nisin-containing cheese. These were analysed on the same day and on different days. Four commercial brands (A, B, C and D) of nisin containing processed cheese were used for analysis using the ELISA and the bioassay. Samples A and B were individual portions of cheese spread; C and D were 200-g packs and D was a low-fat sprcad. Triplicate samples of each brand were used and each sample was assayed in duplicate. A nisin-free cheese spread was used as control.

Bioassay The bioassay method of Fowler and co-workers was used (Fowler et al., 1975).

Results

A standard curve for nisin is shown in Fig. 1. The limit of detection of the assay, defined as the mean zero value plus two standard deviations (n=6), is 1.9 x l o p 2 i.u. ml-'. To determine the presence of interfering material in the food matrix, standards were diluted in analyte-free matrix (AFM) rather than assay diluent (PBSGT). This gave a reduced response in the ELISA but had little effect on the overall shape of the standard curve.

Doubling dilutions in the range 150 to 1:1600 were prepared from an extract of commercial cheese containing nisin. An absorbance value for each dilution was obtained using the ELISA. The nisin content of the 1 :400 dilution was then determined from the standard curve produced using AFM. Using this value, the nisin contents of the other dilutions were calculated and these data, along with their measured absorbance values, were plotted on the standard curve (Fig. 2). The parallelism with the standard curve indicates that the same epitopes are being recognized in the sample as in the standard (Darley et al., 1988). Nisin inactivated using the procedure described by Fowler and co-workers (1975) gave no response in the ELISA. The recovery of nisin from cheese samples spiked with commercially used concentrations of nisin averaged 85% (Table 1).

Nisin ELISA 593

1 .oo

0.80

0.60

0.40

0.20

0.00 0.00 0.20 0.40 0.60 0.80 1.00

[NISIN] i.u.rn-'

Figure 1. Standard curve for nisin. Standards in PBSGT; values are the means of six determinations +1 a.d.

1.00 r

0.00 0.20 0.40 0.60 0.80 1.00

"ISIN] i.u.ml-'

Figure 2. Interpolated values for serial dilutions of a nisin-containing checsc extract. 0, interpolated values from commercial cheese: 0. standards in antigcn-free matrix, 1:400 dilution corresponds to 1.2 i.u. ml I nisin.

Table 1. Recovery of nisin from spiked cheese samples

Concentration of nisin recovered ( I u rnl ') Percentage rccovery in pdrentheses

Conccntration 01 nibin added (i u. mi I )

9.25 7.40 (80%")

27.8 22.2 (80%) 37.0 33.3 (90%)

18.5 16.7 (90%)

74.0 62.9 (85%)

594

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M . B. Falahee et al.

2

O F I L I I

0 2 4 6 8 10. 12 14 16

ELISA i.u.ml ' Figure 3. Corrclation between nisin ELISA and bioassay. Assays performed using siandards in analytc-free matrix.

The results of analysis of a number of processed cheese samples containing nisin as preservative are shown in Table 2. All samples were diluted 11200 prior to analysis in the ELISA. The intra- and interassay coefficients of variation for analysis were 5.3%) (n=6) and 10.9% (n=6) respectively.

The ELISA and bioassay were compared by regression analysis using standards prepared in analyte-free matrix (Fig. 3) and gave good correlation (r=0.995).

Discussion

The nisin ELISA correlates well with the bioassay both with standards in analyte-free matrix and with commercial cheese samples. I t is insensitive to inactivated nisin and analyte-free matrix, indicating the detection of epitopes associated with the bioactive molecule and the absence of interfering materials in processed cheese. The specificity of immunoassays in this regard is a particular advantage since, unlike a bioassay, it is insensitive to interference from other antimicrobials that may be present, such as polyphosphates. The ELISA is more sensitive, and more amenable to automation and analysis of multiple samples than the labour-intensive bioassay, although the result is obtained no sooner.

Application of the ELISA is likely to he limited at present since, though nisin is permitted in many countries, maximum permissible levels are not always prescribed.

Table 2. Analysis of cheese samples containing nisin as preservative by bioassay and ELISA

Concentration of nisin in cheese (i.u. g- ' ) * ? SD

Sample Bioassay ELISA

A B C D

478 k 43.9 210 k 26.5 316 k 33.6 449 2 38.6

45x 34.3 251 k 15.8 317 t 25.6 406 Itr 14.1

*Assuming 85% recovery.

Nisin E LISA 595

The need for a modern analytical technique is likely to increase, however. There is much current interest in the activity of other bacteriocins produced by lactic acid bacteria and their potential for improving the quality and safety of foods (Klaenham- mer, 1988). Nisin has been shown to possess activity against emerging food poisoning hazards such as Listeria nionocyfogenes (Benkerroum & Sandine, 1988) and the nisin-producing gene has been cloned and sequenced opening the way to its expression in sifu by other food-borne microorganisms (Buchman et al., 1988; Kaletta & Entian, 1989; Dodd et a l . , 1990). Such developments could well result in the expansion and more precise regulation of the use of nisin in foods for which an ELISA would be invaluable.

Acknowledgments

We would like to acknowledge the valued advice of Dr S. Hampton and the AFRC for financial support to MBF.

References

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(Received 17 November 1989, revised and accepted 30 May I990)