Food Microbiology, 1988, 5, 33-42

Immunof luorescence detection of mould - An aid to the Howard mould counting technique

Alastair Robertson .1, Naresh Patell and James G. Sargeantt 2

1 C a m p d e n F o o d a n d D r i n k R e s e a r c h A s s o c i a t i o n , C h i p p i n g C a m p d e n , G louces t e r sh i r e G L 5 5 6 L D , U K a n d 2 R H M R e s e a r c h Centre , H i g h W y c o m b e , B u c k i n g h a m s h i r e , U K

R e c e i v e d 8 J a n u a r y 1 9 8 8

Antibodies have been raised in rabbits to the soluble and insoluble fractions of a mixture of five moulds representing the major spoilage organisms in tomatoes. Further antibodies were raised to the same moulds after heat treaatment at 100°C for 6 min. Immunofluorescence microscopy has shown that there is little difference between the titres of the antisera from heated and native fungal mixtures, although those raised to the soluble mould fractions produced by far the highest titre. The antibody appears to cross react with both mould species of the same genera, and with moulds of different genera not present in the original cocktail. Little cross reaction was found to occur between the antibody and tomato tissue. The immunofluorescent detection of mould can be used to simplify and reduce errors associated with the Howard Mould counting technique by enabling easy identification and enumeration of mould filaments within the tomato paste matrix.

Introduction The Howard Mould Count (HMC), devel- oped some 70 years ago (Howard 1911) is the officially recognised method of assessing, and thus controlling, the amount of decayed fruit that has been incorporated into comminuted tomato products such as juices and 'pastes'. The method relies on a correlation between the proportion of decayed fruit in the product and the occurrence of mould filaments, as measured by microscopy (Howard and Stephenson 1917). At present there are different HMC legisla- tion levels amongst the various coun- tries, ranging from 40% H M C - w h i c h approximates to 2-2% decayed fruit in the final product, to 60% H M C - 4 . 0 % decayed fruit.

The method is labour-intensive, t ime

t Present address: Beeches Biotech, Buck- land St. Mary, Chard, Somerset, UK

0740-0020/88/010033 + 10 $02.00/0

consuming and is subject to operator variation both within and between labor- atories. The technique depends on the operators ability to distinguish mould filaments from tomato tissues and in many cases there are numerous doubts during mould identification which inevi- tably leads to errors in the final result. Finally although a high HMC count always indicates high decayed fruit con- tent a low count does not necessarily imply decay free raw material (Howard and Stephenson 1917). The use of stains for mould identification has been attempted, but mould specific stains have not been found. Alternative methods have been proposed (Jarvis 1977, Bishop 1982), but for various reasons, none have been considered acceptable by regulatory bodies or industry.

The evolution and development of immunological techniques (Notermans

© 1988 Academic Press Limited

34 A. Robertson et al.

and Heuvelman 1985, Lin et al. 1986), seems to offer the approach that immu- no fluorescent detection of mould may aid the HMC technique by overcoming the problems of distinguishing mould amongst tomato tissue, would reduce the time taken to carry out an analysis, remove constrictions posed by the shor- tage of highly skilled staff and provide accurate, reproducible and quant i ta t ive analysis of samples.

The success of such a method is totally dependent upon the immunogenicity of moulds and hence their ability to stimu- late an immt~nogenic response in a host animal in recognition to hyphal com- ponent(s). For the method to be reliable, the antibody must be mould specific and not cross react with the growth sub- strate. Furthermore, it should react with fungal components which are not subject to variations as a result of growth, substrate or age. It would be ideal, although not essential, if this component was common to all moulds and present at similar levels. However, lack of cross reactivity between moulds, could be over- come by the use of mixed antibody derived from mould 'cocktails'. Finally an antibody preparation must bind to fungal hyphae which may change in composition and structure with heat t rea tment during the processing of tomatoes into paste and juice.

To satisfy the above requirements the following five moulds which collectively represent the major fungal spoilage in tomatoes world-wide, were chosen for the preparation of an immunogen cocktail: Alternaria alternata, Botrytis cinerea, Fusarium solani, Rhizophus stolonifer and Mucor piriformis (Grogan et al. 1975, Ferrer and Owen 1959, Griffin and Savage 1983, Rowe and Farely 1981).

This paper reports the preparation and comparison of a series of antisera from the soluble and insoluble fractions of heated and unheated immunogen cock- tails. These antibodies have been used to

visualise mould mycelia in commercial tomato pastes by immunofluorescence microscopy.

Materials and Methods

Mould cultures Tomato moulds. Freeze dried samples of Alternaria alternata, Botrytis cinerea, Fusarium solani, Mucor piriformis and Rhi- zophus stolonifer (AFRC, Norwich) were revived by rehydration in sterile distilled water for 15 min and then cultured on tomato juice agar slopes (Oxoid, Hants) at 25°C for 7 days. The resulting culture was stored at 4°C until required.

Sodium chloride free tomato paste was steam pasteurised for 1 h and diluted with distilled water (100 g/l). The diluted paste was filtered (Whatman 4) and the resultant filtrate (tomato broth) aliquoted into 500 ml glass conical flasks. These flasks were plugged with a foam rubber stopper, steamed for a further 15 min and then inoculated with the individual mould cultures. The flask contents were stirred for 7 days at room temperature and the mycelia harvested by vacuum filtration. The mould mycelia were washed thoroughly with sterile water and were stored in sterile, air-tight containers at 4°C.

Penicillium chrysogenum. Cross-reactivity of the tomato mould polyclonal antibody was assessed with PeniciUium chrysogenum. Freeze dried P. chrysogenum was rehydrated as described above but was cultured on potato dextrose agar (Oxoid, Hants) slopes at 25°C for 7 days. A sample of the resultant culture was inoculated into malt extract broth and cultured for 6 days with continuous stirring. Harvesting the mycelia was as described for the tomato moulds.

Preparation of antigenic material 2 g of mycelia from each of the 5 moulds was weighed in duplicate into two 20 ml glass containers to give a total 10 g of mixed mycelia. To each sample was added 10 ml of sterile distilled water. One sample was steamed for 6 min to simulate the pasteurisa- tion occurring during commercial tomato paste manufacture. Both viable and heat treated mixed fungal samples were washed by resuspending the mycelia in distilled water, centrifuging (MSE) at 3000g for 5 min and decanting off the supernatant.

Immunofluorescence detection of mould 35

Moulds (Grown on Tomato broth for 7 days)

I

Mix 2 g of eoch mould (Total lOg) add IOml H20

l Freeze dry <

l Cryogrind (liquid N 2)

Mix 2 g of each mould (Total lOg) odd IOml HzO

Heat IO0°C- 6 min

Dissolve I00 mg powder into 5ml PBSt

Sonicate and centrifuge

f Soluble fraction Pellet (solid)

Dialyse (H~O) Resuspend m PBSt

Freeze dry Freeze

Fig. 1. Flow diagram showing the stages in immunogen preparation.

The two samples of washed mycelia were freeze dried (Edwards, Modulyo) and the lyophilised material ground under liquid nitrogen. The resulting mycelia powders were stored in glass vials in a vacuum desiccator at room temperature.

Preparation of soluble and insoluble immunogens 100 mg of each mixed mycelia powder was resuspended into 5 ml of phosphate buffered saline (Dulbecco and Vogt 1954) containing 0.05% v/v Tween 20 (PBSt). The suspensions were sonicated for 5 min and then centrifuged at 3000 g for 5 min. The supernatant, which represented the fungal soluble fraction, was dialysed against two changes of deionised water for 24 h at 4°C. Protein analysis (Lowry et al. 1951} on the dialysates of the native and heat-treated fungi were carried out. Both

samples were freeze dried and stored in a vacuum desiccator at -20°C.

The mycelial pellets, representing the fun- gal insoluble fraction from the centrifuged suspension, were repeatedly washed (× 10) as described above using 5 ml aliquots of PBSt. The final pellets were resuspended in phos- phate buffered saline (20 mg/ml) to provide stock solutions which were stored at -20°C until required.

Figure 1 shows a summary of the steps involved during the preparation of the four immunogens.

Immunisation The lyophilised soluble fractions of the native and heat-treated mould mixtures were recon- stituted in PBSt (1 mg/ml) anddiluted 1:1 with Freunds adjuvant. Samples of the insol-

36 A. Rober t son et el.

Table 1. Injection of soluble and insoluble immunogens

Injection Protein/solids Rabbit No. Preparation vol (ml) concentration (mg)

1 Heat treated soluble 1-0 0-50 2 Heat treated soluble 0.5 0.25 3 Unheated soluble 1-0 0.50 4 Unheated soluble 0.5 0.25 5 Heat treated insoluble 1.0 10 6 Heat treated insoluble 0-5 5 7 Unheated insoluble 1.0 10 8 Unheated insoluble 1.5 5

Table 2. Immunisat ion and bleeding schedule, o

Time (weeks)

0 Bleed (control) Immunise 5 Immunise 7 Bleed 8 Reimmunise 10 Bleed 24 Reimmunise 26 Bleed

uble fraction stock solutions were diluted 1 : 1 with Freunds adjuvant.

White New Zealand rabbits (2.5-3 kg) were injected subcutaneously with the insoluble and soluble preparations at five sites along the back of each side of the backbone as shown in Table 1. Details of booster injections and bleeding schedules are shown in Table 2. Blood was sampled via the marginal ear vein keeping all sample volumes below 10 ml for the general welfare of the animal.

Purification of the antisera The blood sample was allowed to coagulate for 2 h at room temperature. The clot was then released from the wall of the tube and allowed to retract overnight. The clear plasma (antiserum) was withdrawn and stored at -20°C.

The immunoglobulin fraction (IgG) was isolated from the antiserum using DEAE Affigel Blue (Biorad). Antiserum (1 mb was dialysed for 16 h against 20 mM Tris-HCL buffer containing 28 mM sodium chloride, pH 8.0. A column (1 x 10 cm) packed with Affigel Blue was equilibriated with 5 bed volumes of the above Tris buffer. The dialysed antisera

was applied to the column and eluted with 3 bed volumes of Tris buffer. The eluate was monitored at 280 nm and the unbound protein, comprising mainly of IgG, collected. The fractions containing the IgG were pooled and dialysed against deionised water for approximately 7 h and then freeze dried. The lyophilised IgG was reconstituted in PBSt to the original volume of 1 ml.

Immunofluorescence microscopy Previously washed mould material was sus- pended in PBSt (0.25 mg/ml) and, after gentle mixing, centrifuged for 1 min at 3000 g. The supernatant was discarded and the above washing procedure repeated. To the washed mould was added 750 .ul PBSt and 5 ~L1 of the antibody cocktail. The suspension was mixed gently and incubated for 30 min at 37°C. The mixture was centrifuged at 3000 g for 5 min, the supernatant discarded and the pellet washed twice using PBSt. The washed pellet was resuspended into 750 ~tl of PBSt and 10 ~tl ofFITC ant irabbi t goat IgG (Sigma) added. The suspension was mixed gently and incubated for 30 min at 37°C. The mixture was centrifuged and the supernatant dis- carded. Excess second antibody in the pellet was washed twice using PBSt. The final pellet was resuspended into 1 ml of PBSt and a drop of this suspension was placed onto a microscope slide, covered with a cover-slip and viewed under a Reichart Polyvar micro- scope fitted with an epi-illumination fluores- cence module. Photographs were taken using 400 ASA Kodak Kodacolor VR film.

The immunofluorescence detection of mould in tomato paste was carried out as follows: 200 ~l tomato paste sample, pre- diluted to a refractometer value of 8.5°Brix, was mixed with 4 ml of PBSt and centrifuged at 3000 g for 10 min. The supernatant was

Immunofluorescence detection of mould 37

Fig. 2. Immunofluorescence microscopy showing each of the five moulds used in the mixed antigen. (a) Alterneria alternata, (bl Botritis cinerea, (cl Fusarium solani, (dl Mucor piriformis, lel Rhizopus stolonifer, Magnification × 200.

QO

ii n

O

~D

O

D~

Immunofluorescence detection of mould 39

discarded and the pellet washed once more with 4 ml of PBSt to remove all soluble material. The resultant tomato paste pellet was resuspended in 750 ul PBSt and the antibody reaction carried out as described for the mould.

Results and Discussion Figure 2 shows immunofluorescence micrographs of the individual moulds used to prepare the mixed immunogen after reaction with the prepared antisera and a second FITC labelled antirabbit IgG raised in goat (see methods}. The results demonstrate that antibodies have been successfully raised in rabbits, which recognize the five composite moulds of the immunogen.

Visual comparisons of the fluorescence level between the ant iserum obtained from soluble and insoluble fractions, as well as native and heat treated materials, showed all to be active. However it was apparent from the brightness of the fluorescence that the titres of the antibody produced from the soluble fraction were considerably

higher than those from the insoluble mycelial wall fragments. The micro- graphs in Fig. 2 show that fluorescence and hence antibody binding is localised at the hyphal wall surface. As higher antibody titres were achieved from the immunogens prepared from the soluble mould fractions, the surface wall antigens are either solubilised by Tween 20 or also present in the hyphal proto- plasm.

No difference could be observed between the antibody titres of native and heat treated mould preparations. The practical implications of this finding are that future preparations of antisera will be carried out on native unheated mould, thus improving the simplicity of the operation.

Although the antisera clearly reacts with five moulds of the immunogen, PeniciUium chrysogenum was cultivated to assess its cross reactivity with other mould genera. Immunofluorescence mi- croscopy demonstrated that this fungus cross-reacted fully with the antiserum at

40 A. Robertson et al.

Fig. 3. Immunofluorescence microscopy showing mould in tomato paste with a Howard Mould Count of 70% positive fields. (a) mould hyphae typically from rotten fruit; (hi Geotric/~um candidum, machinery mould.

a f luorescence in tens i ty s imi la r to tha t of the t a rge t moulds. Thus, the p repara t ion of the immunogen has been carr ied out in such a way as to include an ant igenic mould component common to o ther fun-

gal genera. This observa t ion is in con- t ras t to previous workers (Note rmans and H e u v e l m a n 1985, Lin et al. 1986) who have shown high specificit ies of ant ibodies for the i r t a rge t moulds.

Immunofluorescence detection of mould 41

However a mould antibody of low speci- ficity has part icular advantages in this application, allowing moulds not included in the immunogen to be de- tected.

Figure 3 shows immunofluorescence micrographs of mould filaments in com- mercial tomato pastes, est imated to have 70% positive fields by the Howard Mould Count technique. Figure 3a shows mould in paste typically from inclusion of rot- ten fruits whereas Fig. 3b demonstrates strongly fluorescing Geotrichum candi- dum (machinery mould) providing further evidence that the antibody binds to mould genera not included in the original immunogen cocktail. There appears to be some background fluores- cence by the tomato cells, but it is not yet clear whether this is due to non-specific binding by the FITC labelled second antibody, cross reaction by the mould specific antibody or merely reflection of fluorescent light from the mycelia by the tomato cells. The former presents no serious problem and can be overcome by first flooding the non-specific binding sites with an unlabelled antirabbit IgG. Some cross reaction may be occurring since the moulds used in this study were cultured in a tomato based medium, and contamination of the immunogen by tomato proteins is possible. However, this growth medium was chosen with no available information on the effects of substrate on mould metabolism and immunogenicity and it was therefore considered necessary to maintain the organisms in a metabolic and morpho- logical state as similar as possible to that

found in tomato paste. In the light of the data presented in this paper, future batches of antibody will be raised to moulds grown on a synthetic medium.

The results of this study suggest that an immunochemical technique could be used for the unambiguous visual count- ing of mould fi laments and would over- come a major problem of the Howard Mould counting technique. However, work is now progressing on providing objective reproducible quanti tat ive tech- niques based on enzyme linked, or flu- orescence measurement methods. Before such a technique can be developed, pre- l iminary work is necessary to investi- gate and quantify the level of antibody binding to each mould, as well as the effects of hyphal matur i ty on the level of fluorescence. This work is currently in progress.

Acknowledgements T h e authors would like to thank Dr S. Alcock and Mr K. Brown of the Micro- biology Depar tment at CFDRA, for growing and heat t reat ing the moulds. We are also grateful to Dr C. Dennis (CFDRA) for helping with the initial selection of the appropriate tomato spoil- age organism and to the AFRC Insti tute of Food Research, Norwich, for providing the freeze dried samples. Thanks are due to Mr M. Hall for performing the Howard Mould Count and Dr D. Bennet t (RHM) and Miss M. Howlet t (CFDRA) for the fluorescence microscopy. The authors gratefully acknowledge the Ministry of Agriculture, Fisheries and Food for fund- ing this work.

References Bishop, R. H., Duncan, C. L. and Evancho, G. M. (1982) Estimation of fungal contamination in

tomato products by chemical assay for chitin. J. Food Sci. 47, 437. Dulbecco, R. and Vogt, M. (1954) Plaque formation and isolation of pure lines with

poliomyelitis viruses. J. Exp. Med. 99, 167-199.

42 A. Robertson et al.

Ferrer, J. B. and Owen, J. H. (1959) Botrytis cinerea the cause of ghost spot disease of tomato. Phytopathology 49, 411-417.

Griffin, M. J. and Savage, M. J. (eds) (1983) Control of Pests and Diseases of Protected Crops 1983. Tomatoes. ADAS Booklet 2243 MAFF Publications, Alnwick, UK.

Grogan, R. G., Kimble, K. A. and Misagti, I. (1975) A stem canker disease of tomato caused by Alternaria alternata f. sp. lycopersici. Phytopathology GS, 880-886.

Howard, B. J. (1911) Tomato Ketchup under the microscope with practical suggestions to ensure a clean product. US Dept of Agriculture, Bureau of Chemistry, Circular No. 68, Washington DC.

Howard, B. J. and Stephenson, C. H. (1917) Microscopical studies on tomato products. US Dept of Agriculture Bulletin No. 581.

Jarvis, B. (1977) A chemical method for the estimation of mould in tomato products. J. Food Technol. 12, 581.

Lin, H. H., Lister, R. M. and Cousin, M. A. (1986) Enzyme-linked immunosorbent assay for the detection of mould in tomato puree. J. Food Sci. 51,180-183.

Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.

Rowe, R. C. a~d Farely, J. D. (1981) Strategies for controlling Fusarium crown and root rot in greenhouse tomatoes. Pl. Dis. Rep. 65, 107-117.

Notermans, S. and Heuvelman, C. J. (1985) Immunological detection of moulds in foods by using the enzyme-linked immunosorbent assay (ELISA); preparation of antigens. Int. J. Food Micro. 2, 247-258.