Psychrophilic and Mesophilic Fungi in Frozen Food Products

Transcription

1 Psychrophilic and Mesophilic Fungi in Frozen Food Products HAROLD H. KUEHN' AND MILLARD F. GUNDERSON Department of Microbiological Research, ampbell Soup ompany, amden, New Jersey Received for publication February 96 ABSTRAT KUEHN, HAROLD H. (ampbell Soup o., amden, N.J.) AND MILLARD F. GUNDERSON. Psychrophilic and mesophilic fungi in frozen food products. Appl. Microbiol. : The mold flora of certain frozen pastries and chicken pies was investigated. Molds were determined qualitatively or quantitatively, or both, by preparing pour plates of the blended product and incubating the plates at various temperatures. The mesophilic fungal flora developed on plates incubated at 0 and 0, whereas psychrophilic fungi were obtained on plates incubated at 0 and. About,000 cultures of fungi, representing about 00 different species, were isolated from various products. Four different brands of blueberry, two brands of cherry pastries, two brands of apple, and one brand of raspberry pastries were examined. In addition, two brands of chicken pies were studied. Blueberry pastries had a much higher total fungal population than the other products, although different brands of blueberry pastries varied considerably. Blueberry pastries had from 47 to,86 psychrophilic fungi per g. herry pastries had about 70 to 0 psychrophiles per g, and apple pastries had 9 to 9 psychrophiles per g. hicken pies contained very few psychrophilic fungi, about per g. Aureobasidium pullulans was recovered most frequently. About 90 % of the psychrophilic fungi found in blueberry products was A. pullulans. Depending upon the brand of cherry pastry, either Phoma spp. or A. pullulans was the most common fungus present. Apple pastries also displayed brand variation, but were unique in having many mesophilic aspergilli. This genus was generally absent from other products. The Penicillium content of apple pastries was also rather high; 0 % of the psychrophilic flora was represented by this genus. The psychrophilic fungal flora of chicken pies was composed primarily of penicillia (0 %) and hrysosporium pannorum (46 %). Because yeasts and molds are ubiquitous, their existence in frozen food products is neither surprising nor unusual. However, until recently, little attention has been given to their occurrence in low-temperature environments (Gunderson, 96). The fungi most commonly isolated from frozen foods are the pathogens of fruits and vegetables, especially those molds associated with storage diseases. Since the pathology of a given fruit or vegetable often is ' Present address: Rohm and Haas o., Bristol, Pa. not macroscopically evident, infested and sound food frequently are combined. This results in the deterioration of the total finished frozen product, providing that environmental conditions, such as temperature, are suitable for fungal development. Enumeration and identification of fungi encountered in frozen food products is important in understanding the sources and kind of mold damage encountered. However, it is the enzymes elaborated by such fungi which cause deterioration of the product. The presence of the microorganism itself is not required once it has produced ani extracellular enzyme which initiates and carries out the spoilage of the product. An investigation of the enzymes produced and the conditions requisite for their elaboration represents a future area of research. A previous report (Kuehn and Gunderson, 96) reviewed the literature of fungi present in frozen food products and presented a preliminary survey of the mold flora in frozen blueberry and cherry pastries. ontinued investigations of several different brands of cherry and blueberry pastries have provided additional information regarding the fungal flora of such foods. In addition, certain other types of frozen food products have been studied., MATERIALS AND METHODS The products investigated included frozen blueberry, cherry, apple, and raspberry pastries, as well as frozeni chicken pies. The crust and filling portions were sampled as a unit in all studies, using the procedures explained in detail in a previous report (Kuehn and Gunderson, 96). Briefly, a weighed sample of product was blended, and pour plates were prepared. The petri plates were incubated at various temperatures including 0, 0,, and 0 for up to 0 days. Those fungi which grew at 0 were considered to be psychrophiles, those growing at as facultative psychrophiles, and those growing at 0 and 0 as mesophiles. An endeavor was made to study the mold flora of each product, both qualitatively and quantitatively, although this goal was not realized in all instances. To do this, all fungal colonies were subcultured and identified, if possible. This permitted a calculation of the quantity of each species per g of product. Several individual pastries or pies of each product and brand, from different retail outlets, were sampled at various times to secure a more accurate representation of each product.

2 VOL. 96 FUNGI IN FROZEN FOOD PRODUTS TABLE. AlIternaria humicola. Alternaria spp... Aspergillus awamori. A. atinstelodami. A. candidus.. A. flavipes... A. flavus... A. versicolor... Au reobasidium pullulans... Botrytis cinerea... andida sp... ephalosporiumspp.. ladosporium cladosporioides.... epiphyllum.... herbarum... Haplographium sp... Horttiokrypsis sp.. Humitticola sp.. Mlucor spp... Myxrotrichum spinosll... Peyronellaea sp... Phialophora sp... Phomia spp... Pyrenochaeta sp.. Sphaeropsis sp.. Penicillium adamietzi P. brevi-compactum. P. capsulatum... P. ch.ermesinium... P. citrinum... P. corylophilum... P. cyaneo-fulvum... P. cyclopiutm.. P. decumbens... P. fellittanum... P. frequentans... P. funiculosum... P. granulosum... P. implicatum... P. janthinellum P. lapidosum. P. lividium series... P. martensii... P. muldticolor.. P. palitans... P. piscarium.. P. roqueforti... P. rulbrum... P. spinulosum... P. steekii... P. sublateritium... P. ierrestre... P. thoinii... P. thomii series... P. urticae... P. iviridicatum... Fungi isolated at various temperatures from blueberry pastries Brand A Brand B Brand Brand D ecies- 0 oc 0 O 0 0 it,00, , Species- P. wortmanii... TABLE. (ont.) Brand Brand A Brand B Brand D 0 0 c Total penicillia Unknown sp. K Unknown sp. K Unknown spp Total fungi per g,064,86, * Quantitative data unavailable. t Results expressed as no. per g. RESULTS AND DISUSSION A total of over,000 cultures of fungi representing 00 species has been isolated from the frozen food products analyzed in the present investigation. Four different brands of frozen blueberry pastries were sampled and incubated at various isolation temperatures, as shown in Table. 4 The analyses showed certain differences as well as certain similarities among the brands. Brand A had a total of,86 psychrophiles (growing at 0 ) per g, while brand B had,8 per g. However, brand had only 47 psy- 9 chrophiles (at ) per g, or about one-fifth as many as the other two brands. The psychrophilic flora of brand D was not studied, but its mesophilic flora compared in numbers with that of brand. About 90 % of the total psychrophilic population in brands A, B, and was attributed to Aureobasidium pullulans (brand A,,480/g; B,,9/g;, 96/g). In brand A, the second most prevalent psychrophile was Botrytis cinerea, with a frequency of 80 per g. Penicillia comprised the third most prevalent group, 6 showing 0 colonies per g of sample. In brand B, Botrytis was not isolated. The second most common fungi were Phoma species (7./g) and penicillia (7/g). Penicillia (/g) were the second most common fungi in brand, followed by an unidentified pycnidial species (4/g) designated as K-887. Two of the molds isolated from blueberry pastries are worthy of further comment. Penicillium lapidosum originally had been isolated from canned blueberries in 98 (Williams, ameron, and Williams, 94). Sclerotia formed by this species are sufficiently heat-resistant to allow them to survive the heat of the canning process. In fact, routine isolation of P. lapidosum is accomplished by heating soil samples to 80 F for min. The association of P. lapidosum with blueberries was borne out in the present study, in which this species was not isolated from any other source. One of the most interesting fungi to be encountered was Myxotrichum s'pinosum, an ascomycete of the family 4 Gymnoascaceae. This fungus originally was described in 90 (Massee and Salmon, 90) growing on dead wood

3 4 in England. It was not reported again until 97 when it was encountered in Iowa on oak bark and also on pineapple pericarp (Gilman, Tiffany, and Lichtwardt, 97). In neither instance did the investigators concern themselves with attempts to culture the fungus. During the present study, two strains of M. spinosum were isolated from blueberry pastries at, marking the first time this fungus has ever been obtained in pure culture. Further studies of these isolates will be reported elsewhere. Two different brands of frozen cherry pastries were sampled and investigated, using isolation temperatures of and 0. Results confirmed the preliminary findings TABLE. Fungi isolated at various temperatures from cherry pastries* Species Alternaria sp... Aposphaeria sp... Aspergillus candidus... A. versicolor... Aureobasidium pullulans. Botrytis cinerea... ephalosporium spp... ladosporium herbarum.. Geotricum candidum... Haplographium sp... Humicola sp... Monilia cinerea... Mucor spp... Phialophora sp... Phoma spp... Plenozythia sp... Sphaeropsis Sp... Penicillium adametzi... P. commune... P. cyclopium... P. decumbens... P. expansum... P. fellutanum... P. frequentans... P. implicatum... P. janthinellum series... P. lanoso-coerulum... P. martensii... P. palitans... P. puberulum... P. roqueforti... P. spinulosum... P. steckii... P. thomii... P. urticae... P. viridicatum... Total penicillia Unknown sp. K-.. Unknown sp. K-... Unknown spp... Total fungi per g Brand A Brand B 0 S 0.7 * Results expressed as no. per g KUEHN AND GUNDERSONA (Kuehn and Gunderson, 96) in establishing the far smaller fungal content of cherry pastries as compared with blueberry products. The two cherry products differed somewhat. In brand A (Table ), Phoma spp. comprised about 4 % (0 colonies/g) of the total of 7 mesophilic fungi per g of sample, whereas A. pullulans represented 0 % (/g), and penicillia represented 0% (.7/g). Brand B, however, had mesophilic fungal colonies per TABLE. Fungi isolated at various temperatures from apple pastries* Species Aspergillus amstelodami.. A. candidus... A. chevalieri... A. flavipes... A. flavus... A. niger... A. repens... A. versicolor... Total aspergilli.6.6 Aureobasidium pullulans. 0.4 ephalosporium spp hrysosporium pannorum. ladosporium herbarum...margarinomyces sp. 0.4 Peyronellaea sp Phialophora sp. Phoma spp Syncephalastrum racemo sum.. 8p Tritirachium sp Unknown sp. K-887. Unknown sp. K-. Unknown spp. Penicillium capsulatum., P. corylophilum. P. chrysogenum. 0.4 P. cyaneum. 0.4 P. cyclopium.. P. expansum. 0.4 P. fellutanum. P. frequentans series. P. janthinellum series....7 P. jenseni. 0.4 P. kapuscinskiii. 0.4 P. martensii. P. palitans. P. puberulum. p. resticulosum. p. roqueforti. P. spinulosu s...m...7. P.. P snom thomii.... P. urticae P. viridicatum. 0.8 P. waksmanik Total penicillia Total fungi per g Brand A Brand B 0 O * Results expressed as no. per g.. APPL. MIROBIOL

4 VOL. ) 96 FUNGI IN FROZEN FOOD PRODUTS g, of which A. pullulans formed % (7/g), penicillia ' (/g), Phoma spp. 6 % (9/g), and Geotrichum candidutm % (4/g). The latter species was not isolated from brand A, but was found to be a component of both the mesophilic and psychrophilic flora of brand B. The flora which developed on the isolation plates at was similar to that at 0, except for the smaller number of colonies. In brand A, Phoma spp. were still the most prevalent (9/g), followed by A. pullulans (7.6/g) and penicillia (./g). In brand B, A. pullulans was the most common fungus (77.8/g), followed by penicillia (/g), Phoma spp. (7.4/g), and G. candidum (7/g). Two brands of apple pastries were studied (Table ). These pastries had a high percentage of mesophilic and psychrophilic penicillia, but aspergilli were present only in the mesophilic flora. The mesophilic fungi in brand A included 8 % A. pullulans (4/g) with 44 % penicillia and 48 % penicillia and aspergilli combined. Tritirachium spp. were present in surprisingly large numbers, comprising 6.6 % of the total mesophilic fungal population. By comparison, mesophiles in brand B included 8 % aspergilli and 4 % penicillia. A. pullulans was completely absent. In the psychrophilic flora of brand A, there were 9 % penicillia and 6 % A. pullulans. In brand B, penicillia represented 79 % of the total psychrophilic flora, whereas A. pullulans was not present. No aspergilli were found in the psychrophilic flora of the apple pastries. The seeming inability of the aspergilli to grow at reduced temperatures was shown strikingly in this product. Although many aspergilli were isolated in this study, no strains were found TABLE 4. Fungi isolated at various tenmperatures from raspberry and miscellaneous pastries Raspberry Miscellaneous Species pastries pastries 0 (0 ) Alternaria sp Asperygilluts candidus A. flavus.. A. terres.. Aureobasidium pullulansl Botrytis cinerea andida sp.. hrysosporium pannorum ladosporium berbarum Geotrichum candidum Phialophora sp Phomna spp. Rhizopus arrhizus... Syncephalastrum racemosum Penicillium chrysogenum P. cyclopium P. lapidosum P. roqueforti P. rutbrum... Penicillium sp Unknown ascomycete K-97 growing at 0 or. Recently, in this laboratory, two isolates capable of growing at have been found. One raspberry pastry product was investigated, but only qualitative determinations of the fungal population were made. Table 4 lists the fungi isolated at 0 and. One of the more unusual fungi was a simple ascomycete, K-97, which appears to be similar to Anixiopsis or Pseudeurotium. This culture and several of the other unidentified fungi are being examined further in an endeavor to determine their exact taxonomic placement. During the course of bacteriological investigations of frozen pastry products in this laboratory, a number of fungi were encountered on plates incubated at 0. These fungi were identified and are listed in Table 4. Kuehn and Gunderson (96) reviewed the literature concerning fungal populations of frozen food products and included citations of fungi found on meats stored at refrigeration temperatures. Two of the most prevalent fungi known to occur on meats in cold storage are hryosporium pannorum (formerly known as Aleurisma carnis) and ladosporium herbarum (Jensen, 94). Our studies of two brands of frozen chicken pies revealed that. pannorum was indeed a very common organism in this product. Brand A (Table ) was sampled quantitatively and showed that. pannorum represented 46 % (6./g) of the total fungal population. Penicillia were slightly more numerous and represented about 0 % of all fungi present.. herbarum (6 %) was the third most common fungus. Although sampling of brand B was not quantitative, it revealed the same pattern except for the absence of. herbarum. Jensen (94) stated that penicillia have not TABLE. Psychrophilic fungi isolated from chicken pies Species Brand A Brand B Au reobasidium pullulans... O. * hrysosporium pannorum 6. t ladosporium herbarum 0.9 Mucor sp... Phoma sp Penicillium brevi-compactum P. chrysogenum P. cyclopium P. decumbens series P. expansum P. frequentans.. P. martensii... P. nigricans P. palitans... P. resticulosum... P.roqueforti... P. spinulosum P. sublateritium..... P. urticae P. viridicatum Total penicillia.4 Total fungi. * Results expressed as no. per g. t Quantitative data unavailable.

5 6 KUEHN AND GUNDERSON APPL. MIROBIOL. been found on frozen meats in the United States, although they were encountered in studies conducted in France. Because penicillia were so common in chicken pies, it would be interesting to study their occurrence in other frozen meat products. LITERATURE ITED GILMAN, J.., L. H. TIFFANY, AND R. W. LIHTWARDT. 97. Fungi new to Iowa. Proc. Iowa Acad. Sci. 64:8-9. GUNDERSON, M. F. 96. Mold problem in frozen foods, p Proc. Low Temperature Mlicrobiol. Symp., ampbell Soup o., amden, N.J. JENSEN, L. B. 94. Microbiology of meats. Garrard Press, hampaign, Ill. KUEHN, H. H., AND M. F. GUNDERSON. 96. Psychrophilic and mesophilic fungi in fruit-filled pastries. Appl. Microbiol. 0:4-8. MASSEE, G., AND E. S. SALMON. 90. Researches on coprophilous fungi. II. Ann. Botany 6:7-9. WILLIAMS,.., E. J. AMERON, AND 0. B. WILLIAMS. 94. A facultatively anaerobic mold of unusual heat resistance. Food Res. 6:69-7.