Psychrophilic and Mesophilic Fungi in Fruit-Filled Pastries

Transcription

1 Psychrophilic and Mesophilic Fungi in Fruit-Filled Pastries HAROLD H. KUEHN' AND MAILLARD F. GUNDERSON Department of Bacteriological Research, Campbell Soup Conmpany, Canmden, New Jersey Ileceived for publication February 23, 1962 ABSTRACT KLVEHN, HAROLD H. (Campbell Soup Co., Camden, N.J.) AND AMILLARD F. GUNDERSON. Psychrophilic and mesophilic fungi in fruit-filled pastries. Appl. AMicrobiol. 10: Surveys of the mold flora of frozen blueberry and cherry pastries were undertaken. Molds were enumerated by preparing pour plates of the blended product and incubating the plates at 0, 5, 10, and 20 C. In this manner, the total fungal content of the product could be ascertained from the 10 and 20 C plates, and the psychrophilic fungal population was represented by those fungi which grew at 0 and 5 C. The pastry portion, or crust, of the blueberry material was sampled separately from the filling portion. Certain differences in fungal flora w-ere apparent. A ureobasidium pullulans was the dominant fungus in crust at all temperatures of isolation. However, Penicillium thomii proved to be the most common mesophilic funguts in the filling portion, and A. pullulans was the most common psychrophile in the filling. Aspergilli were (quite common in the crust, but, in general, were absent from the fruit filling. Cherry pastries had a much smaller total fungal flora than did the blueberry product. However, A. pullulans again was the most prevalent fungus in cherry pastries at all temperatures of isolation. Certain differences in fungal flora were apparent in the two fruit products. Phomha spp. were almost completely absent in blueberries, but represented the second most common fungus in cherry pastries. Blueberry filling had 440 psychrophilic fungi per gram of sample (at 0 C), blueberry crust had 65 per gram, and cherry pastries had 77 per gram. Fungi have always been associated with spoilage and destruction of foodstuffs. In the spoilage of certain produicts, such as bread and fresh and preserved fruits, mold growth is evident to the unaided eye. However, this is not true with respect to the majority of food products, especially those which are consumed at a rapid rate so that their storage time is short. Standard textbooks dealing with food microbiology make frequent reference to the presence of molds in spoiling product. Recently, Frazier (1958) reviewed the entire aspect of food spoilage during storage. He pointed ouit that a few molds may naturally be associated with a given frtuit or vegetable as it grows in the 1 Present ad(ldiess: Holly Drive, Oakwood Lakes, Medford, N.J. field. However, other molds from soil, water, or air become lodged on or attached to the foods as they are picked, cleaned, processed, and stored. This is true for fruits, vegetables, cereals, meats, sea foods, eggs, poultry, milk and milk products, and many other types of food products. A convenient definition of food spoilage is that a food is spoiled when a consumer refuses it as food. This may vary from society to society, person to person, or even from one type of food to another. Spoilage of food by fungi renders the food unacceptable to the consumer and, consequently, it represents a monetary loss. The fungal and bacterial storage diseases of fruits and vegetables have been discussed and illustrated (USDA Bulletins, No. 98, et seq.). However, we have found little information concerning the fungi which occur in frozen food products; the species of such fungi have not been enumerated. Jensen (1954) made the following statements: "Great difficulty was experienced in identifying some of the psychrophilic molds. This field is an open one.... The problem of yeasts and molds in meat products is a promising field for research." Great progress has been made in the study of the bacteriological aspects of food spoilage, but the role of molds and yeasts has not been studied with equal thoroughness. The psychrophilic fungi and their role in the deterioration of frozen foods have not been a field of endeavor. Mold is definitely a problem in frozen food products. Moldy products result when frozen foods are allowed to attain temperatures of -10 C, or above. We know but little concerning the exact temperature which permits the development of mold and how much lower the temperature should be to prevent the appearance of mold. We do not know how long a period of time is required to produce moldiness at a given temperature. Little is known regarding fluctuating temperature in a freezer cabinet and its relation to the development of mold. Products become inoculated with the mold spores and accompany the ingredients into the processing plant, and there is a certain amount of airborne contamination within the plant. We do not know the relationship between the spore load of ingredient and the proneness to become moldy. All of these unknown factors would be interesting areas for study. Technologists have long been familar with the mold problem on meats held in refrigerated storage. Jensen (1954) reviewed the literature with respect to the subject of meats, and Frazier (1958) explored the role of molds in various food products. Wolf and Wolf (1950) enumerated 354

2 FUNGI IN FRUIT-FILLED PASTRIES 350- the species of fungi which they found on meats, and also listed species reported by other inv,estigators. Sulzbacher (19350) stated that there were not as many yeasts and molds isolated from frozen meats as were isolated from frozen fruits and vegetables. I'erhaps the length of the incubation time, or the temperature, is the reason, but Hucker (1954) failed to mention the occurrence of any fungi in frozen vegetables, although he did study the microbial population of such products. Wallace and Tanner (1933) listed three species of yeasts and five of molds from fruit products. Lochhead and Jones (1936) presented figures varying from 95 to 19,100 yeasts and molds per gram of frozen fruits and Avegetables under certain storage conditions. However, these writers failed to identify the species involved. In a series of reports, Smart (1934, 1935, 1937, 1939) listed some genera of yeasts and molds isolated from frozen fruits and vegetables, butt there was no identification of the species present. Appreciation of the fact that certain fungi can grow at low temperatures was not held by too many. Wolf and Wolf (1947), in speaking of molds, made the statement that "at zero degrees Centigrade their growth is completely checked. " However in a recent review, Hawker et al. (1960) acknowledged that some ftungi grow below 0 C and that some even grow at temperatures "approaching -20 C." The importance of this problem increases with the growth of the frozen-food industry and with consumer acceptance of such products. Other workers have been interested in low-temperatture microbiology. For example, Ingraham and Stokes (1959) recently reviewed the literature of psychrophilic bacteria. The growth and survival of fungi at and below 0 C was reviewed about three decades ago by Berry and Magoon (1934). There have been a few reports concerning psychrophilic yeasts, including those by Scott (1957), Lawrence, Wilson, and Pederson (1959), and Hagen and Rose (1961). The latter workers provided a definition of terms. The purpose of the present paper is to provide an initial enutimeration of those fungi isolated from certain frozen frtuit-filled pastries. However, our interest is directed toward those fungi which grow at temperatures below 5 C. Fungi which were unable to grow at refrigerator temperatures were given only cursory treatment. MIATERIALS AND METHODS The products investigated included frozen blueberry pastries and frozen cherry pastries. The pastry portion, or crust, was sampled separately from the fruit filling in all experiments involving blueberry pastries. No effort was made to sample the crust and filling portions separately in cherry pastries. An 1 -g sample was removed aseptically from the produict, placed into a sterile blendor containing 99 ml of sterile water, and blended for 3 min. Then pour plates were prepared, using a 1-ml sample of the blended sample per petri plate. Therefore, each plate contained 0.1 g of the product. Petri plates were incubated at 0, 5, 10, or 20 C for enough time for the fungal flora to develop. This usually was 7 days at 20 C, and from 15 to 25 days at the lower temperatures. Those fungi which appeared on the 0 C plates were considered to be psychrophiles, and the fungi which grew at 5 C were regarded as facultative psychrophiles. The plates incubated at 20 C were controls and were examined to ascertain the total fungal content of the product. It was discovered that the fungal flora which developed at 10 C was approximately the same as that growing at 20 C. The media which were utilized during these studies included: Czapek Solution Agar (Difco); Potato Dextrose Agar (PDA, Difco); and malt extract agar (ph 5.0), which contained 2 % malt extract, 1.5 % agar, 2 %/o dextrose, and 0.1 % peptone. MIalt extract agar and PDA were used as the media for the pour-plate isolation procedure. An endeavor was made to survey the molds quantitatively and qualitatively. To do this accurately, each fungus colony had to be subcultured and identified. Although this procedure was followed at 0 and 5 C, where relatively few colonies appeared, it was not feasible at 10 and 20 C, at which temperatures the plates had many colonies. Therefore, at the higher temperatures, only an approximation was made of the nuimber of colonies of each species present. However, since we were primarily interested in the psychrophilic fungi developing at 0 and ) C, all fungal colonies on such plates were subcultured and identified, if possible. Numerous plate cultures were required to accomplish this, but no other method could be used to achieve the desired results. No attempt was made to identify the species of Penicillium which grew at temperatures of 10 and 20 C, because of the time factor involved and also because plates at these temperatures merely served to give an indication of the total fungal flora present. Penicillia and aspergilli, at times, were quite TABLE 1. Aspergillus candidus A. chevalieri A. flavus A. niger A. pseudoglaucus A. repens A. sydowi A. versicolor Aureobasidiumn (Pullularia) pullulans Aureobasidium sp. K-356 Cephalosporium acremnoniuxm Cladosporium herbarumn (ladosporium sp. Fusarium sp. Hornmodendrum hordei Mesophilic fuingi isolated front blueberry and cherry pastries Hormodendrum sp. Mucor ramnannianus Mucor sp. Penicilliuntii adainetzi P. funiculosum P. janthinellurm P. rubrunt P. thomii Penicilliamiii spp. (31 spp.) Phialophora spp. (6 spp.) Phoma glomerata P. hibernica Phomra spp. (? spp.) Rhinocladiella atrovirens Torula lucifuga Unknown spp. (16 spp.)

3 356 KUEHN AND GUNDERSON [VOL. 10 numerous on plates incubated at 10 and 20 C, but, in general, were few in number on plates incubated at 0 C. RESULTS AND DISCUSSION The most frequently encountered fungus in our studies proved to be Aureobasidium (Pullularia) pullulans (de TABLE 2. Aureobasidium pullulans Botryotrichum sp. Cephalosporium acremonium Cephalosporium sp. Cladosporium cladosporioides C. herbarum Cladosporium sp. Mucor ramannianus M. fragilis section M. plutmbeus M. hiemalis Penicillium chrysogenum P. cyclopium P. frequentans P. janthinellum P. jenseni Psychrophilic fungi isolated from blueberry and cherry pastries P. martensii P. olivino-viride P. palitans P. puberulum P. purpurescens P. restrictum P. thomii P. velutinum P. waksmani P. urticae Penicillium spp. Phoma glomerata Phoma spp. Sphaeropsidaceae sp. K-351 Tuberculariaceae sp. K-212 Unknown spp. (5) TABLE 3. Numbers of psychrophilic fungi (at 0 C) from the filling portion of blueberry pastries A ureoba- Peni- Total sidium cillium Mucor colonies/ Petri plate no. pullulans spp. ramannianus plate Total Average/plate Avg/gram of sample TABLE 4. Numbers of psychrophilic fungi (at 0 C) from the crust portion of blueberry pastries Total Aureobasidium Penicillium Unknown colonies/ Petri plate no. puillulans spp. K-351 plate Total Average/plate Avg/gram of sample Bary) Arnaud, a so-called black yeast. An examination was made of the crust portion of blueberry pastries, and at each isolation temperature A. pullulans was the most TABLE 5. Molds isolated from crust and filling of cherry pastries on plates incubated at 5 C Total Average Average per Species colonies per plate gram of sample Aureobasidium pullulans Phoma spp Unknown sp. K Penicillium spp Cephalosporium spp Unknown sp. K Papularia sphaerosperma Alternaria sp Mucor plumbeus M. fragilis section Cladosporium herbarum Chrysosporium pannorumn TABLE 6. Psychrophilic fungi (grou'ing at 0 C) isolated from crust and filling of cherry pastries Cephalo- Papu- Aureoba- Peni- sporiuns laria Total sidium Pizoma cilliurn acre- Unknown sphaero- colonies/ Petri plate no. pullulans spp. spp. nionium K-351 sperma plate Total Avg/plate Avg/gram of sample *...~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.... FIG. 1. Isolation pour plate from fruit filling of blueberry pastries incubated at 20 C.

4 FUNGI IN FR'UIT-FILLED PASTRIES 35 7 prevalent species. Not only the total number of fungi, but also the types of fuingi, decreased with decrease in temperatuire from 20 (Table 1) to 0 C (Table 2). This was to be expected, since the growth optimum for most fungi has been reported to be above 15 C. Although 44 different species of fungi were isolated from the crust at temperatures of 10 and 20 C, only 8 were found at 5 C and 5 at O C. At 10 and 20 C, the most common species in the filling portion of blueberry pastry was Penicillium thomii, rather than A. pullulans as in the crust portion. However, at the lower temperatures of ; and 0 C, the psychrophile A. pullulans developed, but P. thomii was not found except for a single colony at, 5 C. The same trend of decreasing numbers of fungi with decrease in temperature was found in the filling as in the crust portion. At 10 and 20 C, 33 species of fungi were encountered; 12 were found at 5 C, and only 7 at 0 C. It is interesting to note that only one culture of P. thomii was isolated from the crust portion, although this species was so prevalent in filling. Tables 3 and 4 present a quantitative and qualitative summary of psychrophilic fungi isolated from separate samplings of blueberry filling and crust. Comparison of the two tables shows that the filling portion had a much greater fungal content than did the crust. The filling had 440 psychrophiles per gram of sample, while the crust had only 65 per gram. In each instance, A. pullulans was the most common fungus, comprising 90 % of the fungi present. Only three types of fungi were found at 0 C in each instance: A. pullulans, Penicillium spp., and an unknown Sphaeropsidaceous fungus, K-351, in the crust, and A. pullulans, Penicillium spp., and Mllucor ramannianus in the filling. An interesting difference between the fungi in crust and those in filling was the almost complete absence of species of Aspergillus in filling. Only one colony representing an Aspergillus, A. niger, was found in filling; this could very well have been an air contaminant, since this species was otherwise completely absent in all of our isolation studies. There were many mesophilic isolates of aspergilli (Table 1). Most of these aspergilli are known to occur in constituents of the flour, and therefore it is not surprising to encounter them in the crust portion in these studies. The results of a survey of the mold flora of frozen cherry pastries showed that Aureobasidium pullulans was the most prevalent fungus encountered at each temperature. Seventeen different species of fungi were isolated from plates incubated at 20 C, twelve species appeared at 5 C (Table 5), and only six different fungi grew at 0 C. A. pullulans comprised 69 % of the fungi which grew at 5 C; Phoma spp. represented 15 %, and the unknown species, K-351, represented 8 % of the total. A. pullulans was found to comprise 56 c of all fungi which grew at 0 C, while Phoma spp. represented 24 % of the total. Comparison of psychrophilic fungi in blueberry filling (Table 4) with those in cherry pastries (Table 6) showed that the latter product had a much lower psychrophilic flora (by a factor of 10). \'Ioreover, although A. pullulans was the dominant fungus in each type of fruit filling, the fillings displayed certain differences in mold flora. Phoma spp. were entirely absent from blueberry filling at 0 C and formed only a minor percentage of species isolated at higher temperatures, although this genus was represented by a substantial portion of fungi isolated from cherry pastries. In addition, the unknown Sphaeropsidaceous fungus, K-351, was isolated only once from the blueberry product, but it was quite common in cherry pastries where it represented 8 % of all fungi isolated at 5 C. The unexpectedly heavy population of A. pullulans in frozen-fruit products is quite interesting and worthy of further study. Figure 1 shows a typical isolation plate of blueberry filling. This plate, incubated at 20 C, has many black colonies of A. pullulans. ACKNOW-LEDGMENTS We wish to thank the following persons for examining certain cultures and either identifying them or confirming our identifications: C. W. Hesseltine, Northern Regional Laboratory, U.S.D.A., Peoria, Ill. (Mucor spp.); W. B. Cooke, U.S. Public Health Service, Cincinnati, Ohio; E. G. Simmons, Quartermaster Depot, Natick, Mass.; G. F. Orr, UCLA, Los Angeles, Calif.; J. W. Carmichael (Chrysosporium pannorum), University of Alberta, Edmonton, Canada; and S. J. Hughes, Canadian Department of Agriculture, Ottawa, Canada. LITERATURE CITEI) BERRY, J. A., ANI) C. A. MAGOON Growth of fungi at and below 0 C. Phytopathology 24: FRAZIER, W. C Food microbiology. McGraw-Hill Book Co., New York. HAGEN, P. O., AND A. H. RoSE A psychrophilic Crypto- CoCCIUS. Can. J. Microbiol. 7: HAWKER, L. E., A. H. LINTON, B. F. FOLKES, AND M. J. CARLILE An introduction to the biology of microorganisms. Edward Arnold, London. HITCKER, G. J Low temperature organisms in frozen vegetables. Food Technol. 8: INGRAHAM, J. L., AND J. L. STOKES Psychr ophilic bacteria. Bacteriol. Rev. 23: JENSEN, L. B Microbiology of meats. Garrard Press, Champaign, Ill. LAWRENCE, N. L., D. C. WILSON, AND C. S. PEDERSON The growth of yeasts in grape juice stored at low temperatures. II. The types of yeast and their growth in pure culture. Appl. Microbiol. 7:7-11. LOCHHEAD, A. G., AND A. H. JONES Studies of numbers and types of microorganisms in frozen vegetables and fruits. Food Research 1: SCOTT, W. J Water relations of food spoilage microorganisms. Advances in Food Research 7: SMART. H. F Microorganisms surviving the storage period of frozen-pack fruits. Phytopathology 24: SMART, H. F Growth and survival of microorganisms at sub-freezing temperatures. Science 82:525. SMART, H. F Types and survival of some microorganisms in

5 358 [VOL. 10 KUEHN AND GUNDERSON frozen-pack peas, beans and sweet corn grown in the east. Food Research 2: SMART, H. F Microbiological studies on commercial packs of frozen fruits and vegetables. Food Research 4: SULZBACHER, W. L Survival of microorganisms in frozen meat. Food Technol. 4: U.S. DEPARTMENT OF AGRICULTIURE. Market disease of fruits and vegetables. Miscellaneous Publications No. 98, 121, 168, 228, 292, 340, 440, 498, and 541. WALLACE, G. I., AND F. W. TANNER Microbiology of frozen foods. III. Longevity of pure cultures of microorganisms frozen in various menstra. Fruit Products J. 14: WOLF, F. A., AND F. T. WOI,F The fungi, vol. 2. John Wiley & Sons, New York. WOLF, F. T., AND F. A. WOLF Chemical agents for the control of molds on meats. Miycologia 42: