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Immuno-assay techniques for detecting yeasts in foods
International Journal of Food Microbiology, 19 (1993) 53-62 © 1993 Elsevier Science Publishers B.V. All rights reserved 0168-1605/93/$06.00
53
FOOD 00599
Immuno-assay techniques for detecting yeasts in foods W o u t e r J. M i d d e l h o v e n a a n d Serv6 N o t e r m a n s b Laboratory of Microbiology, Wageningen Agricultural University, Wageningen, The Netherlands and b National Institute of Public Health and Environmental Hygiene, Bilthoven, The Netherlands
A brief literature review on immuno-assay of yeast cell wall antigens is given. Special attention is paid to extracellular, thermostable yeast antigens (EPS), which are released to the growth medium by many yeast species. The EPS of Saccharomyces cerevisiae and of Stephanoascus ciferrii (syn. Candida ciferrii) could be specifically and sensitively detected by a sandwich ELISA, using an IgG raised in rabbits immunized with the EPS of these yeasts. The EPS ELISA of three basidiomycetous yeasts tested was not specific, that of Geotrichum candidam was genus-specific but was not sensitive. The EPS of Zygosaccharomyces bailii could be detected in a highly specific competitive ELISA but not in a sandwich ELISA or in a latex agglutination test. Key words: Candida sp.; Geotrichum candidum; Saccharomyces cerevisiae; Stephanoascus ciferrii; Zygosaccharomyces bailii; Extracellular yeast antigen; Immuno-assay
Introduction Y e a s t s a r e k n o w n to p r o d u c e a n t i g e n s in t h e i r cell wall. A t t e m p t s have b e e n m a d e to use a n t i b o d i e s for m i s c e l l a n e o u s p u r p o s e s , e.g., r a p i d d e t e c t i o n of infectious wild y e a s t s in b r e w e r ' s a n d b a k e r ' s y e a s t ( C a m p b e l l , 1971a). S e r o l o g i c a l classification o f f e r e d results sufficiently close to t h o s e o b t a i n e d by m o r p h o l o g i c a l a n d physiological tests to p r o v i d e a r a p i d a n d r e l i a b l e m e t h o d o f i d e n t i f i c a t i o n o f S a c c h a r o m y c e s a n d Z y g o s a c c h a r o m y c e s s p e c i e s ( C a m p b e l l , 1968, 1971a). W i t h the e x c e p t i o n o f c a p s u l a t e d strains, in which cell wall a n t i g e n s a r e m a s k e d , cells w e r e r e a d i l y a g g l u t i n a t e d by specific a n t i s e r a ( C a m p b e l l , 1971b). S e r o l o g y has b e e n u s e d as a tool in y e a s t classification ( C a m p b e l l , 1972a, 1972b). In t h e s e studies w h o l e y e a s t ceils w e r e used. P r o t e i n a c e o u s a n d h e a t - l a b i l e a n t i g e n s p r o b a b l y have p l a y e d a part. H e a t - s t a b l e a n t i g e n s can be e x t r a c t e d with p o t a s s i u m h y d r o x y d e f r o m t h e cell walls o f m a n y a s c o m y c e t o u s y e a s t s (Tsuchiya a n d T a g u c h i , 1980). U p to n i n e d i f f e r e n t a n t i g e n s c o u l d be i s o l a t e d f r o m o n e species. It a p p e a r s t h a t s o m e a n t i g e n s a r e p r e s e n t in m a n y a s c o m y c e t o u s yeasts a n d in s o m e b a s i d i o m y c e t o u s y e a s t s s t u d i e d ( T s u c h i y a a n d T a g u c h i , 1980). O t h e r a n t i g e n s display m o r e speci-
Correspondence address: W.J. Middelhoven, Laboratory of Microbiology, Wageningen Agricultural University, Wageningen, The Netherlands.
54 ficity (Tsuchiya et al., 1974). In Japan a Candida Check has been commercially available since 1971 (Iatron Laboratory, Tokyo) for rapid identification of Candida albicans (Robin) Berkhout and some other medically important yeasts (Fukazawa et al., 1968). It consists of factor antibodies (IgG) raised to ten different heat-stable cell wall antigens. It is claimed to be superior to usual methods of yeast identification in its speed and accuracy. Immuno-assay techniques are, however, not generally applied by yeast taxonomists. The chemical structures of immunodominant groups of some yeast mannans were elucidated by Suzuki et al. (1968), Ballou (1970), Raschke and Ballou (1971, 1972) and Ballou and Raschke (1974). These epitopes are side chains of the cell wall mannan. They consist of mannose and galactose residues. Other sugars have also been detected in yeast and fungal EPS. More data on the immuno-chemical determinants of several antigenic factors of yeasts are given by Fukazawa et al. (1980). Some other papers on the immunology of heat-stable yeast antigens and its application in yeast classification and identification are Tsuchiya et al. (1965a,b). Filamentous fungi excrete heat-stable antigens (Sakaguchi et al., 1969). It was proven by an enzyme-linked immuno-sorbent assay (ELISA) that genus-specific heat-stable antigens were excreted by each of the species belonging to the genera Cladosporium, Fusarium and Geotrichum. Cross-reactions between these genera were not observed (Notermans and Soentoro, 1986). The thermostable antigens of the related genera Penicillium and Aspergillus, however, were immunologically related, as were those of the genera Mucor and Rhizopus (Notermans and Soentoro, 1986). Antibodies directed against extracellular mould antigens already find practical application in the detection of moulds in food (Notermans and Heuvelman, 1985; Notermans et al., 1986, L i n e t al., 1986). Thermostable antigens, in contrast to heat-labile ones, permit the detection of fungal growth in the raw materials of canned food, pasteurized soft drinks and irradiated foods (Kamphuis et al., 1992). From these products the fungi can no longer be isolated. Foods in which fungi have grown are suspect for the presence of mycotoxins such as aflatoxin. The heat stable antigens are extracellular polysaccharides (EPS). They are cell wall constituents excreted into the growth medium (Notermans et al., 1987). In some cases the chemical structure of the immuno-dominant residues have been elucidated (Veeneman et al., 1987, 1989; Notermans et al., 1988). The yeast Candida albicans (Robin) Berkhout produces a mannan that can be detected by immuno-assay in the serum of candidiasis patients (Segal et al., 1979; Meckstroth et al., 1981; Lew et al., 1982; Fujita et al., 1986). Other yeasts also excrete thermostable antigens (Middelhoven and Notermans, 1988) which are recovered from the medium. This review deals with the results of this and of later studies, and with possible applications of EPS immuno-assay in food microbiology.
Methods and general properties of extracellular heat-stable yeast antigens
Methods The isolation of the extracellular polysaccharides (EPS) from the culture liquids was described by Notermans et al. (1987). Antibodies were raised in rabbits against
55 EPS released by seven ascomycetous and four basidiomycetous yeasts. The IgG fraction of the rabbit sera was isolated and freeze-dried (Middelhoven and Notermans, 1988; Steinbuch and Audran, 1969). The specificity of the antibodies was studied by reaction with culture filtrates of 63 ascomycetous and 21 basidiomycetous yeast species isolated from various habitats (Middelhoven and Notermans, 1988). This yeast collection represents about 15% of all yeast species known at present. Two enzyme-linked immuno-sorbent assay (ELISA) techniques were used: a competitive ELISA and a sandwich ELISA. In the competitive ELISA microtiter plates were coated with purified EPS. To the wells was added IgG directed against the EPS used for coating and culture liquid of the yeast under investigation, diluted 1/10 and 1/100. Blanks without culture filtrate were also run. After removing excess of IgG and EPS by rinsing, sheep anti-rabbit immunoglobulin conjugated to horse radish peroxidase was added. Again, excess of the conjugate was removed. The amount of peroxidase in the wells was determined at 450 nm after adding hydrogen peroxide and 5-aminosalicylic acid. The blank wells and the wells supplied with non-competitive EPS stained dark brown. Culture filtrates containing EPS that cross-reacted with the IgG prevented binding of the peroxidase conjugate to the trays. In such cases the wells stained light brown or not at all. In the sandwich ELISA technique wells of a microtiter tray were coated with rabbit lgG directed against an EPS. The wells were then supplied with culture filtrates of the yeasts under investigation, diluted 1/50, 1/250, 1/1250 and so on. Blanks were also run. After incubation and rinsing, a conjugate of horse radish peroxidase and rabbit IgG directed against the EPS used for coating was added. After incubation and rinsing, the presence of adsorbed peroxidase was demonstrated as described above. A brown pigment was indicative of the presence of EPS or of cross-reacting material in the culture filtrates. For experimental details, see Middelhoven and Notermans (1988).
Heat-stable yeast antigens Of the eleven yeast species studied, nine produced extracellular antigens. EPS of Candida milleri Yarrow CBS 6897 and Rhodotorula mucilaginosa (J6rgensen) Harrison St.41 failed to raise antibodies in a rabbit. The antibodies directed against ascomycetous EPS generally were very specific. Cross-reactions were seen with a few related species (see below). With basidiomycetous yeasts quite different results were obtained: the antibodies reacted with many culture filtrates including those of many ascomycetes (Middelhoven and Notermans, 1988). Immuno-assay of the extracellular antigens can be used for the quantitative determination of yeast cells in foods only if the excreted amounts of EPS under all conditions are proportional to the cell density. Attempts were made to check this for some yeast species (Middelhoven et al., 1988) by growing these in different growth media and in chemostat cultures, and by following EPS excretion in growing cultures. It appeared that yeasts behaved differently. Stephanoascus ciferrii M.Th. Smith et al. CBS4856 (syn. Candida ciferrii Kreger-van Rij) and the soil yeast Hansenula wickerhamii Capriotti CBS4307 were grown on different carbon sources. In the stationary growth phase the amounts of excreted EPS were
56 proportional to the dry weight of the cultures. This was also true of glucose-limited and ammonia-limited chemostat cultures of St. ciferrii CBS4856 at various growth rates. Results with Saccharomyces cereuisiae Reess ex Hansen K1502 were different. Under anaerobic conditions much less antigen was present in the medium than in well-aerated cultures. In the latter most of the EPS was excreted during the late exponential and stationary growth phases. On solid growth medium no EPS was excreted, but it could be extracted from the cells with citrate buffer at 120°C (Koch and Rademacher, 1980) or by sonic disruption. Release of the antigen by treatment with snail enzymes proved that the EPS of S. cerevisiae is a cell wall constituent. Only small amounts were detected in lysed protoplasts (Middelhoven et al., 1988). The chemical structures of the immuno-dominant side chain of the cell wall mannan of S. cerevisiae and two other yeasts has been elucidated (Ballou, 1970; Ballou and Raschke, 1974).
Heat-stable antigens of some food-borne yeasts
Basidiomycetes EPSs isolated from cultures of Rhodosporidium toruloides Banno CBS14, Sporidiobolus salmonicolor Fell et Talman CBS5937 and Trichosporon cutaneum (De Beurmann et al.) Ota CBS8111 were immunogenic but those of Rhodotorula mucilaginosa (J6rgensen) Harrison St.41 failed to raise antibodies in a rabbit. The antibodies of the above-mentioned yeasts were, however, not specific. Cross-reactions with many other basidiomycetes were observed. Antibodies directed against EPS of Rh. toruloides and of Tr. cutaneum alsoreacted with many culture filtrates of ascomycetes in a competitive ELISA (Middelhoven and Notermans, 1988). It is concluded that EPS ELISA of basidiomycetous yeasts is unsuitable for specific detection of these yeasts in foods. Candida milleri Yarrow The type strain of this species, CBS6897, was isolated from San Francisco sour dough (Yarrow, 1978). The species is common in ensiled maize and other ensiled vegetable crops (Middelhoven and Franzen, 1986; Middelhoven et al., 1990) in which it takes part in aerobic deterioration (Middelhoven and Van Baalen, 1988). The EPS of C milleri CBS6897 failed to raise antibodies in a rabbit but culture filtrates of other strains cross-reacted with antibodies directed against EPS of Saccharomyces exiguus Reess ex Hansen CBS379 (see below). Geotrichum candidum Link This yeast-like fungus is common in foods that passed a lactic acid fermentation. Its EPS behaved like that of filamentous fungi in being genus-specific. Cross-reactions were seen with other Geotrichum species (Notermans and Soentoro, 1986), with its teleomorph Galactornyces reessii (Van der Walt) Redhead et Mulloch
57 CBS179.60 and with some other, related yeast-like fungi, e.g., Trichosporiella sp., Hyphozyma sp., Rhinocladiella atrovirens Nannf. CBS291.65 and Hortaea werneckii (Horta) Nishimura et Miyaji CBSl11.31, and with some true yeasts, viz. Candida blankii Buckley et Van Uden CBS1898, Candida hydrocarbofumarica Yamada et al. ex Ramirez CBS6734 and Zygoascus hellenicus M.Th. Smith et Batenburg-van de Vegte CBS5839 (syn. Candida hellenica (Verona et Picci) King et Jong) (Middelhoven et al., 1989). The latter true yeasts are known to be related to yeast-like fungi (M.Th. Smith, personal communication). The sandwich ELISA of G. candidum CBS144.88 EPS was not very sensitive (titers upto 1/1250) and was not reproducible (Middelhoven and Notermans, 1988). Saccharomyces cerevisiae Meyen ex Hansen All strains of S. cerevisiae tested in the sandwich ELISA showed positive reactions (titers 1/250-1/1250). Cross reactions in the EPS sandwich ELISA of this yeast occurred with culture liquids of Kluyveromyces marxianus (Hansen) Van der Walt CBS712 and of Zygosaccharomyces florentinus Castelli ex Kudriavzev CBS746 and CBS748. These yeast species are easily distinguished from S. cerevisiae by their ability to utilize ethylamine, cadaverine and lysine as sole nitrogen sources (Barnett et al., 1990). Within the genus Saccharomyces cross-reactions were observed with other strains of S. cerevisiae and with S. bayanus Saccardo CBS380, S. chevalieri Guillermond CBS400, S. globosus Osterwalder CBS424 and S. italicus Castelli CBS459. These species are classed under S. cerevisiae (Yarrow and Nakase, 1975; Yarrow, 1984) because they are interfertile and have the same DNA base composition. The EPS ELISA of S. cerevisiae confirmed modern taxonomic insights. As mentioned above, the EPS of S. cerevisiae K1502, and possibly that of other strains, was released from aerated cultures only in the late exponential and stationary growth phases, and was excreted only in small amounts in anaerobic cultures. The latter phenomenon is not shown by all strains as high EPS titers could be demonstrated in specimens of beer and wine up to ten years old (unpublished). Attempts were made to set up a latex agglutination test in order to replace the sandwich ELISA of S. cerevisiae EPS by a less time-consuming immuno-assay technique. Latex agglutination has been successfully applied in the determination of mould EPS (Kamphuis et al., 1989). The latex globules were coated with IgG directed against S. cerevisiae EPS and subsequently supplied with diluted culture filtrates of several yeasts. It was demonstrated (unpublished results) that S. cerevisiae EPS could be detected as sensitively as in the sandwich ELISA but the latex agglutination was less specific: many yeast species cross-reacted. This lack of specificity is prohibitive to application of this immuno-assay in food microbiology. Saccharomyces exiguus Reess ex Hansen This yeast has frequently been isolated from fermented foods like sauerkraut and cucumber brine, and from maize silage (Middelhoven and Franzen, 1986; Middelhoven et al., 1990). Besides Candida milleri Yarrow and Candida lambica
58 (Lindner et Genoud) Van Uden et Buckley it is the major agent causing the aerobic deterioration of maize silage (Middelhoven and Van Baalen, 1988). Nonsporulating strains are classed as the anamorph Candida holmii (J6rgensen) Meyer et Yarrow which can be distinguished from Candida milleri only by its DNA base composition and vitamin requirement (Yarrow, 1978; Middelhoven and Van Baalen, 1988). An easier way to distinguish these important food-borne yeasts would be desirable. IgG directed against EPS of S. exiguus CBS379 reacted in a competitive ELISA but not in a sandwich ELISA (Middelhoven and Notermans, 1988). This may be indicative of the presence of only one immunogenic residue per EPS molecule. If this is true, formation of a network of IgG and EPS molecules, necessary in a successful sandwich ELISA, is impossible. In the competitive ELISA the antibodies were not as specific as those directed to EPS of other ascomycetous yeasts: cross-reactions were observed with eight other yeast species occurring in foods, C. milleri (except for its type strain, CBS6897) included. It is concluded that the EPS ELISA of S. exiguus can not distinguish C. holmii and C. milleri and hence is of little value in microbiological food analysis. Stephanoascus ciferrii Smith, Van der Walt et Johannsen This yeast, which is also known as Candida ciferrii Kreger-van Rij, is associated with animals (Kreger-van Rij, 1965), is frequently isolated from meat products (Dalton et al., 1984), from soil (Middelhoven et al., 1985) and from some ensiled vegetable crops (Middelhoven et al., 1990). IgG directed against the EPS of St. ciferrii CBS4856 was very specific. Cross-reactions were only observed with culture filtrates of the very rare soil yeast Arxula terrestris (Van der Walt et Johannsen) Van der Walt et al. CBS6697 (syn. Trichosporon terrestre Van der Walt et Johannsen, but not of all other yeasts (Middelhoven and Notermans, 1988) and yeast-like fungi (Middelhoven et al., 1989) tested. In a sandwich ELISA St. ciferrii EPS could be detected in culture filtrates diluted 1/31,250 (Middelhoven and Notermans, 1988; Middelhoven et al., 1988). The amount of EPS excreted by St. ciferrii CBS4856 was proportional to the cell density under a wide variety of growth conditions (see above). These properties make the EPS ELISA of St. ciferrii an ideal immuno-assay for sensitive and specific detection of this yeast. Zygosaccharomyces bailii (Lindner) Guillermond This yeast causes much annoyance in the food industry. Its ascospores are thermoresistant (Put et al., 1976) and survive mild pasteurization. This yeast is osmotolerant and very resistant to preservatives like benzoic and acetic acids (Sand, 1973; Pitt, 1974). Spoilage of soft drinks and fruit juice concentrates often is caused by Z. bailii or the closely related Z. bisporus Naganishi. IgG directed against the EPS of Z. bailii Na was very specific. In the competitive ELISA it reacted only with culture filtrates of Z. bailii and Z. bisporus (Middelhoven and Notermans, 1988). However, it gave no response in a sandwich ELISA or in a latex agglutination test. For these immuno-assay techniques it is necessary that a network of IgG and EPS molecules is formed. Failure of the IgG
59 to react in these immuno-assays possibly indicates availability of only one immunogenic residue (epitope) per EPS molecule. Nevertheless, attempts were made to set up a latex agglutination test. The procedure was like that described for S. exiguus (see above), but agglutination was aimed at by addition of concanavalin A which is known to link mannans. In this way complexes of EPS molecules may be obtained containing several epitopes. Under certain conditions agglutination was seen, but the test was not sensitive and not specific. Many yeast culture filtrates caused agglutination (unpublished results).
Discussion
Most yeasts studied produced heat-stable extracellular antigens (EPS). For lack of specificity of the raised antibodies, growth of basidiomycetous yeasts could not be demonstrated by immuno-assay of the excreted EPS. Ascomycetous EPS, however, in most cases was species- or genus-specific and antibodies directed against these could be used to detect yeast growth even in foods and drinks in which the yeasts had died, e.g., Saccharomyces cerevisiae in beer and wine. Proportionality of EPS excretion and cell density was not shown by all yeast strains tested and was not shown under all growth conditions. Thus detection of yeast EPS in food samples only provided a qualitative indication and did not tell how many yeasts were present. Some yeasts, e.g., S. cerecisiae K1502, did excrete only small amounts of EPS in young exponentially growing cultures. Infections of foods and drinks with yeasts behaving like this strain of S. cerevisiae can not be detected in an early stage. The sensitivity of the EPS immuno-assay can be increased by treatment with citrate buffer at 120°C which is known to dissolve the cell wall mannans (Koch and Rademacher, 1980). Another way to detect yeast infections in an early stage could be detection of the EPS by immuno-assay in crude enrichment cultures inoculated with the food sample under investigation. In this way, identification of the yeast infection, which normally takes 2-3 weeks, might be speeded up considerably. The EPS of most ascomycetous yeasts could be detected in a sandwich ELISA but EPS antibodies of some yeasts, e.g., Saccharornyces exiguus CBS379 and Zyaosaccharomyces bailii Na, identified EPS only in a competitive ELISA, which is less sensitive. A sandwich ELISA takes about 24 h. Attempts to replace this immuno-assay by a more rapid one which requires less skill, e.g., a latex agglutination test, failed. The test for S. cerecisiae EPS was as sensitive as the sandwich ELISA but was not as specific. The studies carried out so far have demonstrated that a general application of immunological detection of yeasts based on detection of EPS is not yet feasible. Possibly, the somatic heat-stable yeast antigens will be more useful for this purpose (Tsuchiya and Taguchi, 1980). The EPS of some yeasts (e.g., most ascomycetes tested) is almost species-specific while the EPS of other yeasts (e.g., basidiomycetes) is not. Nevertheless, the studies have shown that in specific cases
60
immunological detection of yeast EPS may be an attractive method. This is especially the case if a fermentation process has to be controlled or if a certain type of yeast causes a special problem in food, like spoilage. This overview provides some basis information in this area.
Acknowledgement The skillful but unsuccessful attempts made by Ms Jos6 M. Eijkelkamp to set up a latex agglutination test for Saccharomyces cerevisiae and Zygosaccharomyces bailii are gratefully acknowledged.
References Ballou, C. (1970) A study of three yeast mannans. J. Biol. Chem. 245, 1197-1203. Ballou, C. and Raschke, W. (1974) Polymorphism of the somatic antigen of yeast. Science 184, 127-134. Barnett, J.A., Payne, R.W. and Yarrow, D. (1990) Yeasts: Characterization and Identification. Cambridge University Press, Cambridge, UK. Campbell, I. (1968) Serological identification scheme for the genus Saccharomyces. J. Appl. Bacteriol. 31,515-524. Campbell, I. (1971a) Comparison of serological ans physiological classification of the genus Saccharomyces. J. Gen. Microbiol. 63, 189-198. Campbell, I. (1971b) Antigenic properties of yeasts of various genera. J. Appl. Bacteriol. 34, 237-242. Campbell, I. (1972a) Numerical analysis of the genera Saccharornyces and Kluyveromyces. J. Gen. Microbiol. 73, 279-301. Campbell, I. (1972b) Simplified identification of yeasts by a serological technique. J. Inst. Brewing 78, 225-229. Dalton, H.K., Board, R.G. and Davenport, R.R. (1984) The yeasts of British fresh sausage and minced beef. Antonie van Leeuwenhoek 50, 227-248. Fujita, S., Matsubara, F. and Matsuda, T. (1986) Enzyme-linked immunosorbent assay measurements of fluctuations in antibody titer and antigenemia in cancer patients with and without candidiasis. J. Clin. Microbiol. 23, 568-575. Fukazawa, Y., Shinoda, T. and Tsuchiya, T. (1968) Response and specificity of antibodies for Candida albicans. J. Bacteriol. 95,754-763. Fukazawa, Y., Nishikawa, A., Suzuki, M. and Shinoda, T. (1980) Immunochemical base of the serologic specificity of the yeast: Immunochemical determinants of several antigenic factors of yeasts. In: H. Preusser (Ed.) Medical Mycology, Zbl. Bakt. Suppl. 8, Gustav Fischer Verlag, Stuttgart, New York, pp. 127-136. Kamphuis, H.J., Notermans, S., Veeneman, G.H., van Boom, J.H. and Rombouts, F.M. (1989) A rapid and reliable detection method of molds in food using a latex agglutination assay. J. Food Prot. 52, 244-247. Kamphuis, H.J., van der Horst, M.I., Samson, R.A., Rombouts, F.M. and Notermans, S. (1992) Mycological conditions of maize products. Int. J. Food Microbiol. 16, 237-245. Koch, Y. and Rademacher, K.H. (1980) Chemical and enzymatic changes in the cell walls of Candida albicans and Saccharomyces cerevisiae by scanning microscopy. Can. J. Microbiol. 26, 965-970. Kreger-van Rij, N.J.W. (1965) Candida ciferrii, a new yeast species. Mycopathol. Mycol. Appl. 26, 49-52. Lew,, M.A., Siber, S.R., Donahue, D.M. and Majorca, F. (1982) Enhanced detection with an enzymelinked immunosorbent assay of Candida mannan in antibody-containing serum after heat extraction. J. Infect. Dis. 145, 45-56.
61 Lin, H.H., Lister, R.M. and Cousin, M.A. (1986) Enzyme-linked immunosorbent assay for detection of molds in tomato puree. J. Food Sci. 51, 180-192. Meckstroth, K.L., Reiss, E., Keller, J.W. and Kaufman, L. (1981) Detection of antibodies and antigenemia in leukemic patients with candidiasis by enzyme-linked immunosorbent assay. J. Infect. Dis. 144, 24-32. Middelhoven, W.J., De Kievit, H. and'Biesbroek, A.L. (1985) Yeast species utilizing uric acid, adenine, n-alkylamines and diamines as sole source of carbon and energy. Antonie van Leeuwenhoek 51, 289-301. Middelhoven, W.J. and Franzen, M.M. (1986) The yeast flora of ensiled whole-crop maize. J. Sci. Food Agric. 37, 855-861. Middelhoven, W.J. and Notermans, S. (1988) Species-specific extracellular antigen production by ascomycetous yeasts, detected by ELISA. J. Gen. Appl. Microbiol. 34, 15-26. Middelhoven, W.J., Slingerland, R.J. and Notermans S. (1988) The effect of growth conditions on the excretion of extracellular antigens by three ascomycetous yeasts. Antonie van Leeuwenhoek 54, 235-244. Middelhoven, W.J. and Van Baalen, A.H.M. (1988) Development of the yeast flora of whole-crop maize during ensiling and during subsequent aerobiosis. J. Sci. Food Agric. 42, 199-207. Middelhoven, W.J., De Hoog, G.S. and Notermans, S. (1989) Carbon assimilation and extracellular antigens of some yeast-like fungi. Antonie van Leeuwenhoek 55, 165-175. Middelhoven, W.J., De Jong, I.M. and De Winter, M. (1990) Yeasts and fungi occurring in ensiled whole-crop maize and other ensiled vegetable crops. Antonie van Leeuwenhoek 57, 153-158. Notermans, S. and Heuvelman, C.J. (1985) Immunological detection of moulds in food by using enzyme-linked immunosorbent assay (ELISA); preparation of antigens. Int. J. Food Microbiol. 2, 247-258. Notermans, S., Heuvelman, C.J., Van Egmond, H.P., Paulsch, W.E. and Besling, J.R. (1986) Detection of mould in food by enzyme-linked immunosorbent assay. J. Food Prot. 49, 786-791. Notermans, S. and Soentoro, P.S.S. (1986) Immunological relationship of extracellular antigens produced by different mould species. Antonie van Leeuwenhoek 52, 393-401. Notermans, S., Veeneman, G.H., van Zuylen, C.W.E.M., Hoogerhout, P. and van Boom, J.H. (1988) (1,5)-linked /3-D-galactofuranosides are immunodominant in extracellular polysaccharides of Penicilliurn and Aspergillus species. Mol. Immunol. 25, 975-979. Notermans, S., Wieten, G., Engel, H.W.B., Rombouts, F.M., Hoogerhout, P. and Van Boom, J.H. (1987) Purification and properties of extracellular polysaccharide (EPS) antigens produced by different mould species. J. Appl. Bacteriol. 62, 157-166. Pitt, J.L. (1974) Resistance of some food spoilage yeasts to preservatives. Food Technol. Austr. 26, 238-241. Put, H.M.C., De Jong, J., Sand, F.E.M.J. and Van Grinsven, A.M. (1976) Heat resistance studies on yeasts causing spoilage of soft drinks. J. Appl. Bacteriol. 40, 135-152. Raschke, W.C. and Ballou, C.E. (1971) lmmunochemistry of the phosphomannan of the yeast Kloeckera brevis. Biochemistry 10, 4130-4135. Raschke, W.C. and Ballou, C.E. (1972) Characterization of a yeast mannan containing N-acetyl-D-glucosamine as an immunochemical determinant. Biochemistry 11, 3807-3816. Sakaguchi O., Yokota, K. and Suzuki, M. (1969) Immunochemical and biochemical studies of fungi. XIII. On the galactomannans isolated from mycelia and culture filtrates of several filamentous fungi. Jpn. J. Microbiol. 13, 1-7. Sand, F.E.M.J. (1973) Recent investigations on the microbiology of fruit juice concentrates. Proc. Int. Fruit Juice Union, Vienna, pp. 185-216. Segal, E., Berg, R.A., Pizzo, P.A. and Bennett, J.E. (1979) Detection of Candida antigens in sera of patients with candidiasis by an enzyme-linked immunosorbent assay inhibition technique. J. Clin. Microbiol. 10, 116-118. Steinbuch, M. and Audran, R. (1969) The isolation of IgG from mammalian sera with the aid of caprylic acid. Arch. Biochem. Biophys. 134, 279-284. Suzuki, S., Sunayama, H. and Saito, T. (1968) Studies on the antigenic activity of yeasts. I. Analysis of the determinant groups of the mannan of Saccharomyces cerevisiae. Jpn. J. Microbiol. 12, 19-24.
62 Tsuchiya, T., Fukuzawa, Y. and Kawakita, S. (1965a) Significance of serological studies on yeasts. Mycopath. Mycol. Appl. 26, 1-15. Tsuchiya, T., Fukuzawa, Y, Kawakita, S. lmai, M. and Shinoda, T. (1965b) Serological classification of the genus Saccharomyces. Jpn. J. Microbiol. 9, 149-159. Tsuchiya, T., Fukuzawa, Y., Taguchi, M. Nakase, T. and Shinoda, T. (1974) Serologic aspects on yeast classification. Mycopathol. Mycol. Applic. 53, 77-91. Tsuchiya, T. and Taguchi, M. (1980) Antigenic relationships among various yeasts, in: H. Preusser (Ed.), Medical Mycology. Zbl. Bakt. Suppl. 8, Gustav Fischer Verlag, Stuttgart, New York, pp. 137-146. Veeneman, G.H., Notermans, S., Liskamp, R.M.J., van der Marel, G.A. and van Boom, J.H. (1987) Solid-phase synthesis of a naturally occurring/3-(1,5)-linked D-galactofuranosyl heptamer containing the artificial arm L-homoserine. Tetrahedron Lett. 28, 6695-6698. Veeneman, G.H., Notermans, S., Hoogerhout, P. and van Boom, J.H. (1989) Synthesis of an immunologically active component of the extracellular polysaccharide produced by Aspergillus and Penicillium species. Rec. Trav. Chim. Pays Bas 106, 344-350. Yarrow, D. (1978) Candida milleri sp. nov. Int. J. Syst. Bacteriol. 28, 608-610. Yarrow, D. and Nakase, T. (1975) DNA base composition of species of the genus Saccharomyces. Antonie van Leeuwenhoek 41, 81-88. Yarrow, D. Saccharomyces Meyen ex Reess. In: N.J.W. Kreger-van Rij (Ed.), The Yeasts, a Taxonomic Study. 3rd Edn. Elsevier, Amsterdam, pp. 379-395.
53
FOOD 00599
Immuno-assay techniques for detecting yeasts in foods W o u t e r J. M i d d e l h o v e n a a n d Serv6 N o t e r m a n s b Laboratory of Microbiology, Wageningen Agricultural University, Wageningen, The Netherlands and b National Institute of Public Health and Environmental Hygiene, Bilthoven, The Netherlands
A brief literature review on immuno-assay of yeast cell wall antigens is given. Special attention is paid to extracellular, thermostable yeast antigens (EPS), which are released to the growth medium by many yeast species. The EPS of Saccharomyces cerevisiae and of Stephanoascus ciferrii (syn. Candida ciferrii) could be specifically and sensitively detected by a sandwich ELISA, using an IgG raised in rabbits immunized with the EPS of these yeasts. The EPS ELISA of three basidiomycetous yeasts tested was not specific, that of Geotrichum candidam was genus-specific but was not sensitive. The EPS of Zygosaccharomyces bailii could be detected in a highly specific competitive ELISA but not in a sandwich ELISA or in a latex agglutination test. Key words: Candida sp.; Geotrichum candidum; Saccharomyces cerevisiae; Stephanoascus ciferrii; Zygosaccharomyces bailii; Extracellular yeast antigen; Immuno-assay
Introduction Y e a s t s a r e k n o w n to p r o d u c e a n t i g e n s in t h e i r cell wall. A t t e m p t s have b e e n m a d e to use a n t i b o d i e s for m i s c e l l a n e o u s p u r p o s e s , e.g., r a p i d d e t e c t i o n of infectious wild y e a s t s in b r e w e r ' s a n d b a k e r ' s y e a s t ( C a m p b e l l , 1971a). S e r o l o g i c a l classification o f f e r e d results sufficiently close to t h o s e o b t a i n e d by m o r p h o l o g i c a l a n d physiological tests to p r o v i d e a r a p i d a n d r e l i a b l e m e t h o d o f i d e n t i f i c a t i o n o f S a c c h a r o m y c e s a n d Z y g o s a c c h a r o m y c e s s p e c i e s ( C a m p b e l l , 1968, 1971a). W i t h the e x c e p t i o n o f c a p s u l a t e d strains, in which cell wall a n t i g e n s a r e m a s k e d , cells w e r e r e a d i l y a g g l u t i n a t e d by specific a n t i s e r a ( C a m p b e l l , 1971b). S e r o l o g y has b e e n u s e d as a tool in y e a s t classification ( C a m p b e l l , 1972a, 1972b). In t h e s e studies w h o l e y e a s t ceils w e r e used. P r o t e i n a c e o u s a n d h e a t - l a b i l e a n t i g e n s p r o b a b l y have p l a y e d a part. H e a t - s t a b l e a n t i g e n s can be e x t r a c t e d with p o t a s s i u m h y d r o x y d e f r o m t h e cell walls o f m a n y a s c o m y c e t o u s y e a s t s (Tsuchiya a n d T a g u c h i , 1980). U p to n i n e d i f f e r e n t a n t i g e n s c o u l d be i s o l a t e d f r o m o n e species. It a p p e a r s t h a t s o m e a n t i g e n s a r e p r e s e n t in m a n y a s c o m y c e t o u s yeasts a n d in s o m e b a s i d i o m y c e t o u s y e a s t s s t u d i e d ( T s u c h i y a a n d T a g u c h i , 1980). O t h e r a n t i g e n s display m o r e speci-
Correspondence address: W.J. Middelhoven, Laboratory of Microbiology, Wageningen Agricultural University, Wageningen, The Netherlands.
54 ficity (Tsuchiya et al., 1974). In Japan a Candida Check has been commercially available since 1971 (Iatron Laboratory, Tokyo) for rapid identification of Candida albicans (Robin) Berkhout and some other medically important yeasts (Fukazawa et al., 1968). It consists of factor antibodies (IgG) raised to ten different heat-stable cell wall antigens. It is claimed to be superior to usual methods of yeast identification in its speed and accuracy. Immuno-assay techniques are, however, not generally applied by yeast taxonomists. The chemical structures of immunodominant groups of some yeast mannans were elucidated by Suzuki et al. (1968), Ballou (1970), Raschke and Ballou (1971, 1972) and Ballou and Raschke (1974). These epitopes are side chains of the cell wall mannan. They consist of mannose and galactose residues. Other sugars have also been detected in yeast and fungal EPS. More data on the immuno-chemical determinants of several antigenic factors of yeasts are given by Fukazawa et al. (1980). Some other papers on the immunology of heat-stable yeast antigens and its application in yeast classification and identification are Tsuchiya et al. (1965a,b). Filamentous fungi excrete heat-stable antigens (Sakaguchi et al., 1969). It was proven by an enzyme-linked immuno-sorbent assay (ELISA) that genus-specific heat-stable antigens were excreted by each of the species belonging to the genera Cladosporium, Fusarium and Geotrichum. Cross-reactions between these genera were not observed (Notermans and Soentoro, 1986). The thermostable antigens of the related genera Penicillium and Aspergillus, however, were immunologically related, as were those of the genera Mucor and Rhizopus (Notermans and Soentoro, 1986). Antibodies directed against extracellular mould antigens already find practical application in the detection of moulds in food (Notermans and Heuvelman, 1985; Notermans et al., 1986, L i n e t al., 1986). Thermostable antigens, in contrast to heat-labile ones, permit the detection of fungal growth in the raw materials of canned food, pasteurized soft drinks and irradiated foods (Kamphuis et al., 1992). From these products the fungi can no longer be isolated. Foods in which fungi have grown are suspect for the presence of mycotoxins such as aflatoxin. The heat stable antigens are extracellular polysaccharides (EPS). They are cell wall constituents excreted into the growth medium (Notermans et al., 1987). In some cases the chemical structure of the immuno-dominant residues have been elucidated (Veeneman et al., 1987, 1989; Notermans et al., 1988). The yeast Candida albicans (Robin) Berkhout produces a mannan that can be detected by immuno-assay in the serum of candidiasis patients (Segal et al., 1979; Meckstroth et al., 1981; Lew et al., 1982; Fujita et al., 1986). Other yeasts also excrete thermostable antigens (Middelhoven and Notermans, 1988) which are recovered from the medium. This review deals with the results of this and of later studies, and with possible applications of EPS immuno-assay in food microbiology.
Methods and general properties of extracellular heat-stable yeast antigens
Methods The isolation of the extracellular polysaccharides (EPS) from the culture liquids was described by Notermans et al. (1987). Antibodies were raised in rabbits against
55 EPS released by seven ascomycetous and four basidiomycetous yeasts. The IgG fraction of the rabbit sera was isolated and freeze-dried (Middelhoven and Notermans, 1988; Steinbuch and Audran, 1969). The specificity of the antibodies was studied by reaction with culture filtrates of 63 ascomycetous and 21 basidiomycetous yeast species isolated from various habitats (Middelhoven and Notermans, 1988). This yeast collection represents about 15% of all yeast species known at present. Two enzyme-linked immuno-sorbent assay (ELISA) techniques were used: a competitive ELISA and a sandwich ELISA. In the competitive ELISA microtiter plates were coated with purified EPS. To the wells was added IgG directed against the EPS used for coating and culture liquid of the yeast under investigation, diluted 1/10 and 1/100. Blanks without culture filtrate were also run. After removing excess of IgG and EPS by rinsing, sheep anti-rabbit immunoglobulin conjugated to horse radish peroxidase was added. Again, excess of the conjugate was removed. The amount of peroxidase in the wells was determined at 450 nm after adding hydrogen peroxide and 5-aminosalicylic acid. The blank wells and the wells supplied with non-competitive EPS stained dark brown. Culture filtrates containing EPS that cross-reacted with the IgG prevented binding of the peroxidase conjugate to the trays. In such cases the wells stained light brown or not at all. In the sandwich ELISA technique wells of a microtiter tray were coated with rabbit lgG directed against an EPS. The wells were then supplied with culture filtrates of the yeasts under investigation, diluted 1/50, 1/250, 1/1250 and so on. Blanks were also run. After incubation and rinsing, a conjugate of horse radish peroxidase and rabbit IgG directed against the EPS used for coating was added. After incubation and rinsing, the presence of adsorbed peroxidase was demonstrated as described above. A brown pigment was indicative of the presence of EPS or of cross-reacting material in the culture filtrates. For experimental details, see Middelhoven and Notermans (1988).
Heat-stable yeast antigens Of the eleven yeast species studied, nine produced extracellular antigens. EPS of Candida milleri Yarrow CBS 6897 and Rhodotorula mucilaginosa (J6rgensen) Harrison St.41 failed to raise antibodies in a rabbit. The antibodies directed against ascomycetous EPS generally were very specific. Cross-reactions were seen with a few related species (see below). With basidiomycetous yeasts quite different results were obtained: the antibodies reacted with many culture filtrates including those of many ascomycetes (Middelhoven and Notermans, 1988). Immuno-assay of the extracellular antigens can be used for the quantitative determination of yeast cells in foods only if the excreted amounts of EPS under all conditions are proportional to the cell density. Attempts were made to check this for some yeast species (Middelhoven et al., 1988) by growing these in different growth media and in chemostat cultures, and by following EPS excretion in growing cultures. It appeared that yeasts behaved differently. Stephanoascus ciferrii M.Th. Smith et al. CBS4856 (syn. Candida ciferrii Kreger-van Rij) and the soil yeast Hansenula wickerhamii Capriotti CBS4307 were grown on different carbon sources. In the stationary growth phase the amounts of excreted EPS were
56 proportional to the dry weight of the cultures. This was also true of glucose-limited and ammonia-limited chemostat cultures of St. ciferrii CBS4856 at various growth rates. Results with Saccharomyces cereuisiae Reess ex Hansen K1502 were different. Under anaerobic conditions much less antigen was present in the medium than in well-aerated cultures. In the latter most of the EPS was excreted during the late exponential and stationary growth phases. On solid growth medium no EPS was excreted, but it could be extracted from the cells with citrate buffer at 120°C (Koch and Rademacher, 1980) or by sonic disruption. Release of the antigen by treatment with snail enzymes proved that the EPS of S. cerevisiae is a cell wall constituent. Only small amounts were detected in lysed protoplasts (Middelhoven et al., 1988). The chemical structures of the immuno-dominant side chain of the cell wall mannan of S. cerevisiae and two other yeasts has been elucidated (Ballou, 1970; Ballou and Raschke, 1974).
Heat-stable antigens of some food-borne yeasts
Basidiomycetes EPSs isolated from cultures of Rhodosporidium toruloides Banno CBS14, Sporidiobolus salmonicolor Fell et Talman CBS5937 and Trichosporon cutaneum (De Beurmann et al.) Ota CBS8111 were immunogenic but those of Rhodotorula mucilaginosa (J6rgensen) Harrison St.41 failed to raise antibodies in a rabbit. The antibodies of the above-mentioned yeasts were, however, not specific. Cross-reactions with many other basidiomycetes were observed. Antibodies directed against EPS of Rh. toruloides and of Tr. cutaneum alsoreacted with many culture filtrates of ascomycetes in a competitive ELISA (Middelhoven and Notermans, 1988). It is concluded that EPS ELISA of basidiomycetous yeasts is unsuitable for specific detection of these yeasts in foods. Candida milleri Yarrow The type strain of this species, CBS6897, was isolated from San Francisco sour dough (Yarrow, 1978). The species is common in ensiled maize and other ensiled vegetable crops (Middelhoven and Franzen, 1986; Middelhoven et al., 1990) in which it takes part in aerobic deterioration (Middelhoven and Van Baalen, 1988). The EPS of C milleri CBS6897 failed to raise antibodies in a rabbit but culture filtrates of other strains cross-reacted with antibodies directed against EPS of Saccharomyces exiguus Reess ex Hansen CBS379 (see below). Geotrichum candidum Link This yeast-like fungus is common in foods that passed a lactic acid fermentation. Its EPS behaved like that of filamentous fungi in being genus-specific. Cross-reactions were seen with other Geotrichum species (Notermans and Soentoro, 1986), with its teleomorph Galactornyces reessii (Van der Walt) Redhead et Mulloch
57 CBS179.60 and with some other, related yeast-like fungi, e.g., Trichosporiella sp., Hyphozyma sp., Rhinocladiella atrovirens Nannf. CBS291.65 and Hortaea werneckii (Horta) Nishimura et Miyaji CBSl11.31, and with some true yeasts, viz. Candida blankii Buckley et Van Uden CBS1898, Candida hydrocarbofumarica Yamada et al. ex Ramirez CBS6734 and Zygoascus hellenicus M.Th. Smith et Batenburg-van de Vegte CBS5839 (syn. Candida hellenica (Verona et Picci) King et Jong) (Middelhoven et al., 1989). The latter true yeasts are known to be related to yeast-like fungi (M.Th. Smith, personal communication). The sandwich ELISA of G. candidum CBS144.88 EPS was not very sensitive (titers upto 1/1250) and was not reproducible (Middelhoven and Notermans, 1988). Saccharomyces cerevisiae Meyen ex Hansen All strains of S. cerevisiae tested in the sandwich ELISA showed positive reactions (titers 1/250-1/1250). Cross reactions in the EPS sandwich ELISA of this yeast occurred with culture liquids of Kluyveromyces marxianus (Hansen) Van der Walt CBS712 and of Zygosaccharomyces florentinus Castelli ex Kudriavzev CBS746 and CBS748. These yeast species are easily distinguished from S. cerevisiae by their ability to utilize ethylamine, cadaverine and lysine as sole nitrogen sources (Barnett et al., 1990). Within the genus Saccharomyces cross-reactions were observed with other strains of S. cerevisiae and with S. bayanus Saccardo CBS380, S. chevalieri Guillermond CBS400, S. globosus Osterwalder CBS424 and S. italicus Castelli CBS459. These species are classed under S. cerevisiae (Yarrow and Nakase, 1975; Yarrow, 1984) because they are interfertile and have the same DNA base composition. The EPS ELISA of S. cerevisiae confirmed modern taxonomic insights. As mentioned above, the EPS of S. cerevisiae K1502, and possibly that of other strains, was released from aerated cultures only in the late exponential and stationary growth phases, and was excreted only in small amounts in anaerobic cultures. The latter phenomenon is not shown by all strains as high EPS titers could be demonstrated in specimens of beer and wine up to ten years old (unpublished). Attempts were made to set up a latex agglutination test in order to replace the sandwich ELISA of S. cerevisiae EPS by a less time-consuming immuno-assay technique. Latex agglutination has been successfully applied in the determination of mould EPS (Kamphuis et al., 1989). The latex globules were coated with IgG directed against S. cerevisiae EPS and subsequently supplied with diluted culture filtrates of several yeasts. It was demonstrated (unpublished results) that S. cerevisiae EPS could be detected as sensitively as in the sandwich ELISA but the latex agglutination was less specific: many yeast species cross-reacted. This lack of specificity is prohibitive to application of this immuno-assay in food microbiology. Saccharomyces exiguus Reess ex Hansen This yeast has frequently been isolated from fermented foods like sauerkraut and cucumber brine, and from maize silage (Middelhoven and Franzen, 1986; Middelhoven et al., 1990). Besides Candida milleri Yarrow and Candida lambica
58 (Lindner et Genoud) Van Uden et Buckley it is the major agent causing the aerobic deterioration of maize silage (Middelhoven and Van Baalen, 1988). Nonsporulating strains are classed as the anamorph Candida holmii (J6rgensen) Meyer et Yarrow which can be distinguished from Candida milleri only by its DNA base composition and vitamin requirement (Yarrow, 1978; Middelhoven and Van Baalen, 1988). An easier way to distinguish these important food-borne yeasts would be desirable. IgG directed against EPS of S. exiguus CBS379 reacted in a competitive ELISA but not in a sandwich ELISA (Middelhoven and Notermans, 1988). This may be indicative of the presence of only one immunogenic residue per EPS molecule. If this is true, formation of a network of IgG and EPS molecules, necessary in a successful sandwich ELISA, is impossible. In the competitive ELISA the antibodies were not as specific as those directed to EPS of other ascomycetous yeasts: cross-reactions were observed with eight other yeast species occurring in foods, C. milleri (except for its type strain, CBS6897) included. It is concluded that the EPS ELISA of S. exiguus can not distinguish C. holmii and C. milleri and hence is of little value in microbiological food analysis. Stephanoascus ciferrii Smith, Van der Walt et Johannsen This yeast, which is also known as Candida ciferrii Kreger-van Rij, is associated with animals (Kreger-van Rij, 1965), is frequently isolated from meat products (Dalton et al., 1984), from soil (Middelhoven et al., 1985) and from some ensiled vegetable crops (Middelhoven et al., 1990). IgG directed against the EPS of St. ciferrii CBS4856 was very specific. Cross-reactions were only observed with culture filtrates of the very rare soil yeast Arxula terrestris (Van der Walt et Johannsen) Van der Walt et al. CBS6697 (syn. Trichosporon terrestre Van der Walt et Johannsen, but not of all other yeasts (Middelhoven and Notermans, 1988) and yeast-like fungi (Middelhoven et al., 1989) tested. In a sandwich ELISA St. ciferrii EPS could be detected in culture filtrates diluted 1/31,250 (Middelhoven and Notermans, 1988; Middelhoven et al., 1988). The amount of EPS excreted by St. ciferrii CBS4856 was proportional to the cell density under a wide variety of growth conditions (see above). These properties make the EPS ELISA of St. ciferrii an ideal immuno-assay for sensitive and specific detection of this yeast. Zygosaccharomyces bailii (Lindner) Guillermond This yeast causes much annoyance in the food industry. Its ascospores are thermoresistant (Put et al., 1976) and survive mild pasteurization. This yeast is osmotolerant and very resistant to preservatives like benzoic and acetic acids (Sand, 1973; Pitt, 1974). Spoilage of soft drinks and fruit juice concentrates often is caused by Z. bailii or the closely related Z. bisporus Naganishi. IgG directed against the EPS of Z. bailii Na was very specific. In the competitive ELISA it reacted only with culture filtrates of Z. bailii and Z. bisporus (Middelhoven and Notermans, 1988). However, it gave no response in a sandwich ELISA or in a latex agglutination test. For these immuno-assay techniques it is necessary that a network of IgG and EPS molecules is formed. Failure of the IgG
59 to react in these immuno-assays possibly indicates availability of only one immunogenic residue (epitope) per EPS molecule. Nevertheless, attempts were made to set up a latex agglutination test. The procedure was like that described for S. exiguus (see above), but agglutination was aimed at by addition of concanavalin A which is known to link mannans. In this way complexes of EPS molecules may be obtained containing several epitopes. Under certain conditions agglutination was seen, but the test was not sensitive and not specific. Many yeast culture filtrates caused agglutination (unpublished results).
Discussion
Most yeasts studied produced heat-stable extracellular antigens (EPS). For lack of specificity of the raised antibodies, growth of basidiomycetous yeasts could not be demonstrated by immuno-assay of the excreted EPS. Ascomycetous EPS, however, in most cases was species- or genus-specific and antibodies directed against these could be used to detect yeast growth even in foods and drinks in which the yeasts had died, e.g., Saccharomyces cerevisiae in beer and wine. Proportionality of EPS excretion and cell density was not shown by all yeast strains tested and was not shown under all growth conditions. Thus detection of yeast EPS in food samples only provided a qualitative indication and did not tell how many yeasts were present. Some yeasts, e.g., S. cerecisiae K1502, did excrete only small amounts of EPS in young exponentially growing cultures. Infections of foods and drinks with yeasts behaving like this strain of S. cerevisiae can not be detected in an early stage. The sensitivity of the EPS immuno-assay can be increased by treatment with citrate buffer at 120°C which is known to dissolve the cell wall mannans (Koch and Rademacher, 1980). Another way to detect yeast infections in an early stage could be detection of the EPS by immuno-assay in crude enrichment cultures inoculated with the food sample under investigation. In this way, identification of the yeast infection, which normally takes 2-3 weeks, might be speeded up considerably. The EPS of most ascomycetous yeasts could be detected in a sandwich ELISA but EPS antibodies of some yeasts, e.g., Saccharornyces exiguus CBS379 and Zyaosaccharomyces bailii Na, identified EPS only in a competitive ELISA, which is less sensitive. A sandwich ELISA takes about 24 h. Attempts to replace this immuno-assay by a more rapid one which requires less skill, e.g., a latex agglutination test, failed. The test for S. cerecisiae EPS was as sensitive as the sandwich ELISA but was not as specific. The studies carried out so far have demonstrated that a general application of immunological detection of yeasts based on detection of EPS is not yet feasible. Possibly, the somatic heat-stable yeast antigens will be more useful for this purpose (Tsuchiya and Taguchi, 1980). The EPS of some yeasts (e.g., most ascomycetes tested) is almost species-specific while the EPS of other yeasts (e.g., basidiomycetes) is not. Nevertheless, the studies have shown that in specific cases
60
immunological detection of yeast EPS may be an attractive method. This is especially the case if a fermentation process has to be controlled or if a certain type of yeast causes a special problem in food, like spoilage. This overview provides some basis information in this area.
Acknowledgement The skillful but unsuccessful attempts made by Ms Jos6 M. Eijkelkamp to set up a latex agglutination test for Saccharomyces cerevisiae and Zygosaccharomyces bailii are gratefully acknowledged.
References Ballou, C. (1970) A study of three yeast mannans. J. Biol. Chem. 245, 1197-1203. Ballou, C. and Raschke, W. (1974) Polymorphism of the somatic antigen of yeast. Science 184, 127-134. Barnett, J.A., Payne, R.W. and Yarrow, D. (1990) Yeasts: Characterization and Identification. Cambridge University Press, Cambridge, UK. Campbell, I. (1968) Serological identification scheme for the genus Saccharomyces. J. Appl. Bacteriol. 31,515-524. Campbell, I. (1971a) Comparison of serological ans physiological classification of the genus Saccharomyces. J. Gen. Microbiol. 63, 189-198. Campbell, I. (1971b) Antigenic properties of yeasts of various genera. J. Appl. Bacteriol. 34, 237-242. Campbell, I. (1972a) Numerical analysis of the genera Saccharornyces and Kluyveromyces. J. Gen. Microbiol. 73, 279-301. Campbell, I. (1972b) Simplified identification of yeasts by a serological technique. J. Inst. Brewing 78, 225-229. Dalton, H.K., Board, R.G. and Davenport, R.R. (1984) The yeasts of British fresh sausage and minced beef. Antonie van Leeuwenhoek 50, 227-248. Fujita, S., Matsubara, F. and Matsuda, T. (1986) Enzyme-linked immunosorbent assay measurements of fluctuations in antibody titer and antigenemia in cancer patients with and without candidiasis. J. Clin. Microbiol. 23, 568-575. Fukazawa, Y., Shinoda, T. and Tsuchiya, T. (1968) Response and specificity of antibodies for Candida albicans. J. Bacteriol. 95,754-763. Fukazawa, Y., Nishikawa, A., Suzuki, M. and Shinoda, T. (1980) Immunochemical base of the serologic specificity of the yeast: Immunochemical determinants of several antigenic factors of yeasts. In: H. Preusser (Ed.) Medical Mycology, Zbl. Bakt. Suppl. 8, Gustav Fischer Verlag, Stuttgart, New York, pp. 127-136. Kamphuis, H.J., Notermans, S., Veeneman, G.H., van Boom, J.H. and Rombouts, F.M. (1989) A rapid and reliable detection method of molds in food using a latex agglutination assay. J. Food Prot. 52, 244-247. Kamphuis, H.J., van der Horst, M.I., Samson, R.A., Rombouts, F.M. and Notermans, S. (1992) Mycological conditions of maize products. Int. J. Food Microbiol. 16, 237-245. Koch, Y. and Rademacher, K.H. (1980) Chemical and enzymatic changes in the cell walls of Candida albicans and Saccharomyces cerevisiae by scanning microscopy. Can. J. Microbiol. 26, 965-970. Kreger-van Rij, N.J.W. (1965) Candida ciferrii, a new yeast species. Mycopathol. Mycol. Appl. 26, 49-52. Lew,, M.A., Siber, S.R., Donahue, D.M. and Majorca, F. (1982) Enhanced detection with an enzymelinked immunosorbent assay of Candida mannan in antibody-containing serum after heat extraction. J. Infect. Dis. 145, 45-56.
61 Lin, H.H., Lister, R.M. and Cousin, M.A. (1986) Enzyme-linked immunosorbent assay for detection of molds in tomato puree. J. Food Sci. 51, 180-192. Meckstroth, K.L., Reiss, E., Keller, J.W. and Kaufman, L. (1981) Detection of antibodies and antigenemia in leukemic patients with candidiasis by enzyme-linked immunosorbent assay. J. Infect. Dis. 144, 24-32. Middelhoven, W.J., De Kievit, H. and'Biesbroek, A.L. (1985) Yeast species utilizing uric acid, adenine, n-alkylamines and diamines as sole source of carbon and energy. Antonie van Leeuwenhoek 51, 289-301. Middelhoven, W.J. and Franzen, M.M. (1986) The yeast flora of ensiled whole-crop maize. J. Sci. Food Agric. 37, 855-861. Middelhoven, W.J. and Notermans, S. (1988) Species-specific extracellular antigen production by ascomycetous yeasts, detected by ELISA. J. Gen. Appl. Microbiol. 34, 15-26. Middelhoven, W.J., Slingerland, R.J. and Notermans S. (1988) The effect of growth conditions on the excretion of extracellular antigens by three ascomycetous yeasts. Antonie van Leeuwenhoek 54, 235-244. Middelhoven, W.J. and Van Baalen, A.H.M. (1988) Development of the yeast flora of whole-crop maize during ensiling and during subsequent aerobiosis. J. Sci. Food Agric. 42, 199-207. Middelhoven, W.J., De Hoog, G.S. and Notermans, S. (1989) Carbon assimilation and extracellular antigens of some yeast-like fungi. Antonie van Leeuwenhoek 55, 165-175. Middelhoven, W.J., De Jong, I.M. and De Winter, M. (1990) Yeasts and fungi occurring in ensiled whole-crop maize and other ensiled vegetable crops. Antonie van Leeuwenhoek 57, 153-158. Notermans, S. and Heuvelman, C.J. (1985) Immunological detection of moulds in food by using enzyme-linked immunosorbent assay (ELISA); preparation of antigens. Int. J. Food Microbiol. 2, 247-258. Notermans, S., Heuvelman, C.J., Van Egmond, H.P., Paulsch, W.E. and Besling, J.R. (1986) Detection of mould in food by enzyme-linked immunosorbent assay. J. Food Prot. 49, 786-791. Notermans, S. and Soentoro, P.S.S. (1986) Immunological relationship of extracellular antigens produced by different mould species. Antonie van Leeuwenhoek 52, 393-401. Notermans, S., Veeneman, G.H., van Zuylen, C.W.E.M., Hoogerhout, P. and van Boom, J.H. (1988) (1,5)-linked /3-D-galactofuranosides are immunodominant in extracellular polysaccharides of Penicilliurn and Aspergillus species. Mol. Immunol. 25, 975-979. Notermans, S., Wieten, G., Engel, H.W.B., Rombouts, F.M., Hoogerhout, P. and Van Boom, J.H. (1987) Purification and properties of extracellular polysaccharide (EPS) antigens produced by different mould species. J. Appl. Bacteriol. 62, 157-166. Pitt, J.L. (1974) Resistance of some food spoilage yeasts to preservatives. Food Technol. Austr. 26, 238-241. Put, H.M.C., De Jong, J., Sand, F.E.M.J. and Van Grinsven, A.M. (1976) Heat resistance studies on yeasts causing spoilage of soft drinks. J. Appl. Bacteriol. 40, 135-152. Raschke, W.C. and Ballou, C.E. (1971) lmmunochemistry of the phosphomannan of the yeast Kloeckera brevis. Biochemistry 10, 4130-4135. Raschke, W.C. and Ballou, C.E. (1972) Characterization of a yeast mannan containing N-acetyl-D-glucosamine as an immunochemical determinant. Biochemistry 11, 3807-3816. Sakaguchi O., Yokota, K. and Suzuki, M. (1969) Immunochemical and biochemical studies of fungi. XIII. On the galactomannans isolated from mycelia and culture filtrates of several filamentous fungi. Jpn. J. Microbiol. 13, 1-7. Sand, F.E.M.J. (1973) Recent investigations on the microbiology of fruit juice concentrates. Proc. Int. Fruit Juice Union, Vienna, pp. 185-216. Segal, E., Berg, R.A., Pizzo, P.A. and Bennett, J.E. (1979) Detection of Candida antigens in sera of patients with candidiasis by an enzyme-linked immunosorbent assay inhibition technique. J. Clin. Microbiol. 10, 116-118. Steinbuch, M. and Audran, R. (1969) The isolation of IgG from mammalian sera with the aid of caprylic acid. Arch. Biochem. Biophys. 134, 279-284. Suzuki, S., Sunayama, H. and Saito, T. (1968) Studies on the antigenic activity of yeasts. I. Analysis of the determinant groups of the mannan of Saccharomyces cerevisiae. Jpn. J. Microbiol. 12, 19-24.
62 Tsuchiya, T., Fukuzawa, Y. and Kawakita, S. (1965a) Significance of serological studies on yeasts. Mycopath. Mycol. Appl. 26, 1-15. Tsuchiya, T., Fukuzawa, Y, Kawakita, S. lmai, M. and Shinoda, T. (1965b) Serological classification of the genus Saccharomyces. Jpn. J. Microbiol. 9, 149-159. Tsuchiya, T., Fukuzawa, Y., Taguchi, M. Nakase, T. and Shinoda, T. (1974) Serologic aspects on yeast classification. Mycopathol. Mycol. Applic. 53, 77-91. Tsuchiya, T. and Taguchi, M. (1980) Antigenic relationships among various yeasts, in: H. Preusser (Ed.), Medical Mycology. Zbl. Bakt. Suppl. 8, Gustav Fischer Verlag, Stuttgart, New York, pp. 137-146. Veeneman, G.H., Notermans, S., Liskamp, R.M.J., van der Marel, G.A. and van Boom, J.H. (1987) Solid-phase synthesis of a naturally occurring/3-(1,5)-linked D-galactofuranosyl heptamer containing the artificial arm L-homoserine. Tetrahedron Lett. 28, 6695-6698. Veeneman, G.H., Notermans, S., Hoogerhout, P. and van Boom, J.H. (1989) Synthesis of an immunologically active component of the extracellular polysaccharide produced by Aspergillus and Penicillium species. Rec. Trav. Chim. Pays Bas 106, 344-350. Yarrow, D. (1978) Candida milleri sp. nov. Int. J. Syst. Bacteriol. 28, 608-610. Yarrow, D. and Nakase, T. (1975) DNA base composition of species of the genus Saccharomyces. Antonie van Leeuwenhoek 41, 81-88. Yarrow, D. Saccharomyces Meyen ex Reess. In: N.J.W. Kreger-van Rij (Ed.), The Yeasts, a Taxonomic Study. 3rd Edn. Elsevier, Amsterdam, pp. 379-395.