APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1990, p. 3851-3854 Vol. 56, No. 12 0099-2240/90/123851-04$02.00/0 Copyright © 1990, American Society for Microbiology Microflora of Partially Processed Lettuce J. A. MAGNUSON, A. D. KING, JR.,* AND T. TOROK Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street, Albany, California 94710 Received 13 July 1990/Accepted 2 October 1990

Bacteria, yeasts, and molds isolated from partially processed iceberg lettuce were taxonomically classified. The majority of bacterial isolates were gram-negative rods. Pseudomonas, Erwinia, and Serratia species were commonly found. Yeasts most frequently isolated from lettuce included members of the genera Candida, Cryptococcus, Pichia, Torulaspora, and Trichosporon. Comparatively few molds were isolated; members of the genera Rhizopus, Cladosporium, Phoma, AspergiUus, and Penicillium were identified.

Grey mold and soft rot have been the major decays of head ing primarily oflettuce with some added red cabbage or carrot lettuce shipped to the New York and Chicago markets. pieces. Depending upon the experiment, samples were stored These diseases were attributed to Botrytis cinerea, Pseudo- either in 1.5-mil (38-p.m-thick) polyethylene produce bags or monas spp., and Erwinia carotovora (2, 3). in commercial plastic bags supplied with the cut lettuce. The maximum shelf life of cut lettuce prepared in proc- Samples (50 g) were diluted 1:10 in water and stomached essing plants for salad or sandwich use and sold in plastic (Colworth Stomacher 400) for 2 min. Serial dilutions in 0.1% bags is 2 to 3 weeks, depending upon the processing, peptone (Difco) water were made from the wash water and packaging, and storage conditions. The shelf life of head plated on plate count agar (Difco) and oxytetracycline- lettuce is about the same. The shelf life is related to the glucose-yeast extract agar (OGY; Oxoid). OGY medium, physiology of lettuce (respiration rate) and to the microbial which has the antibiotic oxytetracycline added to restrict flora (10). bacterial growth, was used to measure growth of yeast and The microbial flora of partially processed fruits and vege- molds. Plates were incubated at 25°C for 3 to 5 days. tables, including lettuce, is a topic of great concern, since The study of the microflora was divided into two parts. In both food spoilage and safety are involved. The presence or the first part, a survey was made to determine which species absence, as well as the fate, of both human pathogens and are present on fresh lettuce obtained from lettuce proces- plant tissue spoilage organisms is important. sors, grocery stores, or the fresh vegetable wholesale mar- Human pathogens, such as Salmonella (6) and Shigella (5) ket. The second part of the study investigated the taxonomic spp., have been found in fresh lettuce. Listeria monocyto- composition of the microflora of several stored samples. genes, which has been implicated in food-borne disease Two sets of sealed or unsealed packages of lettuce stored outbreaks, has been isolated from cabbage (4), fresh produce two weeks at 1.1 to 2.2°C were sampled for the microflora. (8), and lettuce (24), while Petran et al. (18) failed to isolate This lettuce was obtained from processing plants, where the any from head lettuce or other vegetables. Pseudomonas packages were sealed by factory methods or left unsealed. marginalis has been implicated in lettuce deterioration (17). Every different-appearing colony was microscopically The microbial population of lettuce has been shown to checked and, if necessary, Gram stained. Morphologically increase under storage conditions (A. D. King, Jr., J. A. distinct colonies were isolated, and taxonomic identifica- Magnuson, T. Torok, and N. Goodman, J. Food Sci., in tions were performed by standard methods. The isolations press). Additional reasons for concern involve the use of were made over a period of 1 year. modified-atmosphere packaging and its possible influence on Bergey's Manual of Systematic Bacteriology (13, 22) was pathogen growth (9). It has been suggested that modified- used to make definitive identifications of bacterial isolates. atmosphere packaging might enhance or mask growth of Pectinolytic and proteolytic activities of the isolates were dangerous organisms. checked with MP7 agar (23) and gelatin liquefaction, respec- The microflora of partially processed foods, including tively. lettuce, is not well defined. The relative importance of The yeast population of lettuce was determined by isolat- physiological breakdown and microbial decay of tissue can- ing morphologically distinct colonies from the OGY or plate not be assessed until both of these spoilage causes are well count agar plates. For cultivation and maintenance of the described. The objective of this study was to identify bacte- pure cultures, YPDA (1% yeast extract, 1% Bacto-Peptone, ria, yeasts, and molds present on partially processed iceberg 1% dextrose, and 2% agar) was used. Identification of the lettuce. yeast isolates was carried out by standard methods (1, 12). Mold colonies were isolated from the OGY or plate count MATERIALS AND METHODS agar plates. Taxonomic identification was performed by using the identification media and methods described by Pitt A series of iceberg lettuce (Lactuca sativa) samples was and Hocking (20), Pitt (19), and Samson et al. (21). obtained from local grocers, wholesale markets, and process- ing plants. Lettuce examined included intact heads, cut leaves RESULTS or unpackaged), and samples stored for up to 79 (packaged Of 38 bacterial isolates from fresh lettuce, 95% were days at 1.1 to 2.8°C. Some samples were a salad mix consist- gram-negative rods, 5% were gram-positive rods, and none were gram-positive cocci. * Corresponding author. Numbers of isolates and their taxonomic classifications 3851 3852 MAGNUSON ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 1. Bacterial isolates from fresh lettuce TABLE 2. Taxonomic composition of bacterial populations on lettuce stored in sealed and unsealed packagesa Type and No. of species isolatesa Type of package No. of and species isolatesb Gram-positive rods Bacillus sp. 1 Sealed Curtobacterium flaccumfaciens 1 Erwinia herbicola 1 Pseudomonas fluorescens bv. I 2 Gram-negative rods Pseudomonasfluorescens bv. II 2 Alcaligenes faecalis 1 Pseudomonas fluorescens bv. III 1 Enterobacter agglomerans 1 Pseudomonas fluorescens bv. V 5 Erwinia carotovora 2 Pseudomonas putida bv. B 4 Erwinia herbicola 1 Flavobacterium breve 1 Unsealed Janthinobacterium lividum 1 Bacillus sp. 1 Pseudomonas cichorii 1 Enterobacter agglomerans 1 Pseudomonas fluorescens bv. I 1 Erwinia carotovora 3 Pseudomonas fluorescens bv. II 3 Lactobacillus plantarum 1 Pseudomonas fluorescens bv. V 14 Pseudomonas caryophylli 2 Pseudomonas putida bv. B 1 Pseudomonas fluorescens bv. I 1 Serratia liquefaciens 2 Pseudomonasfluorescens bv. II 1 Serratia marcescens 1 Pseudomonas putida bv. A 1 Xanthomonas campestris 2 Pseudomonas putida bv. B 1 Unidentified 4 a Polyethylene bags either sealed at processing plant (partial vacuum) or a Morphologically different colonies were isolated. unsealed. b Every different-appearing colony was isolated from each set of duplicate plates. appear in Table 1. Since the purpose of the taxonomic survey was to identify the types of organisnns nresent the number of isolates is a relative indication of tlhehefrequency of yielded 15 morphologically different bacterial types and the occurrence in the samples. sealed samples yielded 12. The results are shown in Table 2. On the basis of colony appearance, Pseudo)monas species, Analysis of the isolates showed that the cultures from Erwinia herbicola, Erwinia carotovora, the,a Enterobacter sealed packages consisted of 1 Erwinia and 14 Pseudomonas agglomerans group, and Serratia species w(ere determined isolates. The unsealed package cultures were slightly more to be commonly present in the lettuce sampl[es. diverse, with one Bacillus, four Erwinia, one Enterobacter, Pectinolytic and proteolytic activities ofF the bacterial one Lactobacillus, and six Pseudomonas isolates. It is isolates are shown in Fig. 1. Proteolytic actiivity was much evident that the genus Pseudomonas figures prominently in more common than pectinolytic activity. the microfloral population of fresh and stored lettuce under Some isolates were found which belonged tio genera whose both sealed and unsealed conditions. members have human pathogenic capabilities (13, 22). These A wide variety of yeast species (a total of 72 isolates; 24 isolates included members of the genera Alc6zligenes, Bacil- species belonging to 11 genera) were isolated from fresh lus, Enterobacter, Erwinia, Flavobacterium, Pseudomonas, lettuce. Yeast species most frequently isolated from lettuce and Serratia. The species were Alcaligenesfazecalis, Entero' included members of the genera Candida, Cryptococcus, bacter agglomerans, Erwinia herbicola, Serratia lique- Pichia, Torulaspora, and Trichosporon (Table 3). faciens, and Serratia marcescens. From unsealed stored samples of cut lettuce, a wide In the tests of the composition of the bacte-rial population variety of yeast species could be isolated. In the sealed bags, of stored cut lettuce samples, the two unssealed samples only a few mainly facultative anaerobe species prevailed, Pichia fermentans and Torulaspora delbrueckii being the most frequent isolates. This was true for both short- and long-term storage. 100 Mr PEOMNYMACTWVWIY Comparatively few molds were isolated because of the infrequent appearance of mold colonies on the plates from - PROTE1CACTVM1 lettuce or salad mix. Members of the genera Aspergillus, 0) Cladosporium, Penicillium, Phoma, and Rhizopus were identified on the petri plates. Individual species most fre- 00 quently identified were Aspergillus niger, Botrytis allii, 0~ Penicillium oxalicum, Penicillium canescens, Penicillium variabile, and Scopulariopsis brevicaulis. 0- 0 0 CL DISCUSSION Bacterial, yeast, and mold counts on fresh and processed 20 lettuce are reported elsewhere (King et al., in press). Bac- terial counts ranged from 105 to 107 bacteria per g, while GRAM+ GRAM- UNSEAkLED SEALED yeast populations were in the range of 103 to 106. Molds were RODS RODS SAMPI'LES SAMPLES noted infrequently. FIG. 1. Proteolytic and pectinolytic activities of the bacterial In our experiments, the majority of bacteria identified isolates. from fresh lettuce were gram-negative rods. A total of 36 VOL. 56, 1990 MICROFLORA OF PARTIALLY PROCESSED LETTUCE 3853

TABLE 3. Yeasts isolated from fresh lettuce, salad mix, and The Pseudomonas species identified in this study belong stored samples to the same rRNA and DNA homology groups, with the Type of sample No. of exception of Pseudomonas caryophylli (13). This indicates and species isolates that the environment in lettuce culture and processing is a was not Fresh lettuce strong selection factor. Pseudomonas aeruginosa Arthroascus javanensis 1 identified in this study but is reported to have been isolated Candida musae 2 from lettuce (11). However, Green et al. (7) could not find it Candida sake 1 in any of 200 lettuce samples. In a related study, Wright et al. Cryptococcus albidus 5 (26) identified several members of the family Enterobac- Cryptococcus curvatus 1 teriaceae, including members of the genera Klebsiella, En- Cryptococcus flavus 1 terobacter, and Serratia, as well as Pseudomonas aerugi- Cryptococcus laurentii 3 Debaryomyces hansenii 1 nosa, from salads containing several vegetables including Filobasidium capsuligenum 2 lettuce. Filobasidiella neoformans 1 In our experiments, many of the bacterial isolates were Pichia carsonii 1 proteolytic but surprisingly few were pectinolytic. While in Pichia fermentans 7 earlier studies proteolytic activity seemed very common in Rhodosporidium infirmo-miniatum 1 it appears unlikely that this activity would Rhodotorula glutinis 2 lettuce isolates, Saccharomyces cerevisiae 1 be an important factor in substrate degradation. Pectinolytic Stephanoascus ciferrii 1 bacteria are known to produce a number of plant diseases, Torulaspora delbrueckii 1 e.g., soft rot. Our results are in contrast to those of Liao and Tremella foliacea 2 Wells (14), who isolated Erwinia carotovora and fluorescent Trichosporon cutaneum 4 Pseudomonas spp. from lettuce. Similarly, Nguyen-The and Trichosporonoides sp. 1 Prunier (17) found a clear relationship between the deterio- Salad mix ration of chicory (endive) salads and the count of fluorescent Candida castellii 1 pectinolytic Pseudomonas marginalis. Erwinia herbicola Candida musae 1 was the next most common soft rot-causing bacteria isolated Cryptococcus albidus 1 by them. Cryptococcus laurentii 1 A large group of yeasts was isolated and identified in this Debaryomyces hansenii 2 study. To our knowledge, this is the first information on the Pichia fermentans 3 Saccharomyces cerevisiae 2 yeast population of lettuce. We observed different yeast Torulaspora delbrueckii 2 species in fresh and stored samples and changes in the yeast population due to the modified atmosphere in sealed stored Stored lettuce products. However, the yeasts did not seem to be involved Candida parapsilosis 1 in spoilage of lettuce initially, because their growth rate is Candida sake 2 lower than that of bacteria (King et al., in press), but they Cryptococcus humicolus 1 Cryptococcus laurentii 1 can use the cell fluids on physically or biologically damaged Hanseniaspora valbyensis 1 lettuce. Kluyveromyces marxianus 1 Molds, especially B. cinerea, at one time were a major Pichia fermentans 7 problem of market lettuce, as noted by previous reports (2, Rhodotorula glutinis 1 3). In this study we found few molds on lettuce and no B. Torulaspora delbrueckii 5 cinerea. This may be due to the development of control measures, such as the use of fungicides in the field (16) and of modified atmosphere transportation of head lettuce (25) or modified atmosphere packaging of shredded lettuce. We isolates were identified from fresh lettuce. Of these, 20 conclude that the infrequent presence of molds means that isolates were Pseudomonas species and 14 were Pseudomo- those present were chance contaminants rather than a nor- nas fluorescens bv. V. mal part of the lettuce flora. Bacteria isolated and identified from fresh lettuce heads There was a change of flora during long-term storage, were different from those of processed lettuce. There were presumably due to less competition among yeast and bacte- no gram-positive bacteria identified from petri plates from rial species under conditions of changing atmosphere and processed cut lettuce, and there were only two identified lettuce metabolism. With unsealed bags that have more fresh lettuce. The number of gram-negative genera and from oxygen, greater varieties of both bacteria and yeasts were species identified from processed lettuce was less than that observed. There was much less of bacterial flora in from fresh lettuce. This result may mean that the washing diversity have a different enriched during processing had removed some soil-borne microorgan- the sealed bags, which atmosphere isms. Maxcy (15) also found a predominance of gram- in carbon dioxide that selects for the surviving microorgan- negative rods, many of them nonproteolytic. isms. Though fewer species were present, yeast growth was Of the bacterial isolates identified from processed cut more enhanced in the sealed samples than it was in the lettuce, 20 were from 6 Pseudomonas species. Sealed pack- unsealed samples. ages of lettuce had a buildup of carbon dioxide and a These experiments were conducted to identify the micro- reduction of oxygen (King et al., in press). This may explain flora of lettuce so an assessment of importance of microbial the different populations of bacteria isolated from sealed spoilage could be made. Lettuce has a limited shelf life, as do lettuce bags compared with those isolated from the unsealed other partially processed vegetables. This paper defines the bags. aerobic microflora. 3854 MAGNUSON ET AL. APPL. ENVIRON. MICROBIOL.

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