The microbial biota of land-grown vegetables may be expected to reflect that of the soils in which they are grown, although exceptions occur. The actinomycetes (Gram-positive branching forms) are the most abundant bacteria in stable soils, yet they are rarely reported on vegetable products. On the other hand, the lactic acid bacteria are rarely found in soil per se, but they are significant parts of the bacterial biota of plants and plant products. The overall exposure of plant products to the environment provides many opportunities for contamination by microorganisms. The protective cover of fruits and vegetables and the possession by some of pH values below which many organisms cannot grow are important factors in the microbiology of these products.
An attempt is made in this chapter to treat fruits and vegetables separately even though this is difficult. In common usage, products such as tomatoes and cucumbers are referred to as vegetables and yet from the botanical standpoint they are fruits. Lemons, oranges, and limes are fruits botanically as well as in common usage. By and large, the distinctions between fruits and vegetables are based on pH, irrespective of the lack of scientific merit.
FRESH AND FROZEN VEGETABLES
The incidence of microorganisms in vegetables may be expected to reflect the sanitary quality of the processing steps and the microbiological condition of the raw product at the time of processing.
In a study of green beans before blanching, Splittstoesser et al.50showed that the total counts ranged from 5.60 log10to over 6.00 log10in two production plants. After blanching, the total numbers were reduced to 3.00–3.60 log10/g. After passing through the various processing stages and packaging, the counts ranged from 4.72 to 5.94 log10/g. In the case of French-style beans, one of the greatest buildups in numbers of organisms occurred immediately after slicing. This same general pattern was shown for peas and corn. Preblanched green peas from three factories showed total counts per gram between 4.94 and 5.95 log10. These numbers were reduced by blanching but again increased suc-cessively with each processing step. In the case of whole-kernel corn, the postblanch counts rose both after cutting and at the end of the conveyor belt to the washer. Whereas the immediate post-blanch count was about 3.48 log10, the product had total counts of about 5.94 log10/g after packaging.
Between 40% and 75% of the bacterial biota of peas, snap beans, and corn was shown to consist of leuconostocs and “streptococci,” whereas many of the Gram-positive, catalase-positive rods resembled corynebacteria.48,49
125
126 Modern Food Microbiology
Table 6–1 Microbial Numbers in Some Fresh Vegetables
Products Log10cfu/g Reference
Red and green chicory and carrot mixture APC, 7.94 57
Coliforms, 7.03 57
Fecal coliforms, 6.74 57
Lactic acid bacteria, 6.18 57
Red chicory, endive, and carrot mixture APC, 6.14 57
Coliforms, 4.68 57
Fecal coliforms, 4.51 57
Lactic acid bacteria, 5.86 57
Bean sprouts APC, 7.26, 7.99 23
Coliforms, 7.49, 6.99
Broccoli APC, 3.97 37
Carrots APC, 4.20 37
Cauliflower APC, 6.97 37
Celery APC, 10.0 37
Coleslaw APC, 7.00 37
Radish APC, 6.04 37
Sprouts APC 8.7; Coliforms 7.2 55
Lettuce APC 8.6; Coliforms 5.6 55
Celery APC 7.5; Coliforms 55
Cauliflower APC 7.4; Coliforms 2.9 55
Broccoli APC 6.3; Coliforms 4.8 55
Fresh retail lettucea APC 6.94; Coliforms 3.25 30
Note: APC= aerobic plate count; cfu = colony-forming unit.
aMean of 10 samples, which also contained 1.64 E. coli and 5.62 fungi.
Numbers of bacteria that have been reported for a number of different fresh vegetables are summa-rized in Table 6–1. It may be noted that APCs of around 6 to 7 log10cfu/g are common among the vegetables listed, and that coliform numbers of around 5–6 log10are not uncommon.
Lactic acid cocci are associated with many raw and processed vegetables.29These cocci have been shown to constitute from 41% to 75% of the aerobic plate count (APC) biota of frozen peas, snap beans, and corn.46 It has been shown that fresh peas, green beans, and corn all contained coagulase-positive staphylococci after processing.48 Peas were found to have the highest count (0.86 log10/g), whereas 64% of corn samples contained this organism. These authors found that a general buildup of staphylococci occurred as the vegetables underwent successive stages of processing, with the main source of organisms coming from the hands of employees. Although staphylococci may be found on vegetables during processing, they are generally unable to proliferate in the presence of the more normal lactic biota. Both coliforms (but not Escherichia coli) and enterococci have been found at most stages during raw vegetable processing, but they appear to present no public health hazard.47
In a study of the incidence of Clostridium botulinum in 100 commercially available frozen vacuum pouch-pack vegetables, the organism was not found in 50 samples of string beans, but types A and B spores were found in 6 of 50 samples of spinach.20
The total numbers of bacteria on frozen vegetables tend to be lower than on comparable nonfrozen products. This is due primarily to, (1) blanching of products prior to freezing, (2) selection of higher quality products for freezing, and (3) the die-off of some bacteria while in the frozen state (see
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Table 6–2 General Microbiological Quality of Frozen Vegetables
% Samples
No. of Meeting
Products Samples Microbial Group/Target Target Reference
Cauliflower 1,556 APC at 35◦C: 105/g or less 75 5
1,556 MPN coliforms:<20/g 79 5
1,556 MPN E. coliforms:<3/g 98 5
Corn 1,542 APC at 35◦C: 105/g or less 94 5
1,542 MPN coliforms:<20/g 71 5
1,542 MPN E. coli:<3/g 99 5
Peas 1,564 APC at 35◦C: 105/g or less 95 5
1,564 MPN coliforms:<20/g 78 5
1,564 MPN E. coli:<3/g 99 5
Blanched vegetables
(17 different) 575 Absence of fecal coliforms 63 49
575 n= 5, c = 3, m = 10, M = 103 33 49 575 n= 5, c = 3, m = 50, M = 103 70 49 Cut green beans,
leaf spinach, peas 144 Mean APC range for group: – 47
4.73–4.93 log10/g Lima beans, corn, broccoli
spears, brussels sprouts 170 Mean APC range for group: – 47 5.30–5.36 log10/g
French-style green beans,
chopped greens, squash 135 Mean APC range: – 47
5.48–5.51 log10/g Chopped spinach, cauliflower 80 Mean APC range:
5.54–5.65 log10/g – 47
Chopped broccoli 45 Mean APC: 6.26 log10/g – 47
Note: APC= Aerobic plate count; MPN = most probable number.
Chapter 16). It can be seen from Table 6–2 that APCs of the frozen green vegetables listed is around 5.0 log10cfu/g in contrast to fresh green vegetables in Table 6–1, which are higher. A summary of the log10APC for some frozen processed potato products is presented in Table 6–3. Since the inside of a fresh and undamaged potato is free of bacteria, the numbers summarized in this table may be taken to reflect post-cooking contamination for those that received heat treatment.
Table 6–3 Summary of log10APC for Some Frozen Processed Potato Products12
Products APC Range
Potato salad 2.6–5.1
Dried potatoes 2.5–5.5
Baked potatoes 4.0–6.4
French fries 2.0–6.7
Hash browns 3.2–8.7
128 Modern Food Microbiology
Table 6–4 General Chemical Composition of Higher Plant Materials Carbohydrates and related compounds
1. Polysaccharides—pentosan (araban), hexosans (cellulose, starch, xylans, fructans, mannans, galactans, levans)
2. Oligosaccharides—tetrasaccharide (stachyose), trisaccharides (robinose, mannotriose, raffinose), disaccharides (maltose, sucrose, cellobiose, melibiose, trehalose)
3. Monosaccharides—hexoses (mannose, glucose, galactose, fructose, sorbose), pentoses (arabinose, xylose, ribose,L-rhamnose,L-fucose)
4. Sugar alcohols—glycerol, ribitol, mannitol, sorbitol, inositols 5. Sugar acids—uronic acids, ascorbic acid
6. Esters—tannins
7. Organic acids—citric, shikimic,D-tartaric, oxalic, lactic, glycolic, malonic, etc.
Proteins—albumins, globulins, glutelins, prolamines, peptides, and amino acids Lipids—fatty acids, fatty acid esters, phospholipids, glycolipids, etc.
Nucleic acids and derivatives—purine and pyrimidine bases, nucleotides, etc.
Vitamins—fat soluble (A, D, E), water soluble (thiamine, niacin, riboflavin, etc.) Minerals—Na, K, Ca, Mg, Mn, Fe, etc.
Water
Others—alkaloids, porphyrins, aromatics, etc.
Spoilage
The general composition of higher plants is presented in Table 6–4, and the composition of 21 common vegetables is presented in Table 6–5. The average water content of vegetables is about 88%, with an average content of 8.6% carbohydrates, 1.9% proteins, 0.3% fat, and 0.84% ash. The total percentage composition of vitamins, nucleic acids, and other plant constituents is generally less than 1%. From the standpoint of nutrient content, vegetables are capable of supporting the growth of molds, yeasts, and bacteria and, consequently, of being spoiled by any or all of these organisms. The higher water content of vegetables favors the growth of spoilage bacteria, and the relatively low carbohydrate and fat contents suggest that much of this water is in available form. The pH range of most vegetables is within the growth range of a large number of bacteria, and it is not surprising, therefore, that bacteria are common agents of vegetable spoilage. The relatively high oxidation–reduction (O/R) potential of vegetables and their lack of high poising capacity suggest that the aerobic and facultative anaerobic types would be more important than the anaerobes. This is precisely the case; some of the most ubiquitous etiologic agents in the bacterial spoilage of vegetables are species of the genera Erwinia and Pectobacterium, and they are associated with plants and vegetables in their natural growth environment. The common spoilage pattern displayed by these organisms is referred to as bacterial soft rot.
Bacterial Agents
The genus Erwinia has been delimited by the transfer of ten species and 5 subspecies to two new genera, and they are listed in Table 6–6. It should be noted that some taxonomists are not in full accord with the transfer of some of the erwiniae to the genus Pectobacterium, suggesting that further changes are likely to occur (see reference 63). Many of the plant-associated pseudomonads have
Vegetable and Fruit Products 129
Table 6–5 Vegetable Foods: Approximate Percentage Chemical Composition
Vegetable Water Carbohydrates Proteins Fat Ash
Beans, green 89.9 7.7 2.4 0.2 0.8
Beets 87.6 9.6 1.6 0.1 1.1
Broccoli 89.9 5.5 3.3 0.2 1.1
Brussels sprouts 84.9 8.9 4.4 0.5 1.3
Cabbage 92.4 5.3 1.4 0.2 0.8
Cantaloupe 94.0 4.6 0.2 0.2 0.6
Cauliflower 91.7 4.9 2.4 0.2 0.8
Celery 93.7 3.7 1.3 0.2 1.1
Corn 73.9 20.5 3.7 1.2 0.7
Cucumbers 96.1 2.7 0.7 0.1 0.4
Lettuce 94.8 2.9 1.2 0.2 0.9
Onions 87.5 10.3 1.4 0.2 0.6
Peas 74.3 17.7 6.7 0.4 0.9
Potatoes 77.8 19.1 2.0 0.1 1.0
Pumpkin 90.5 7.3 1.2 0.2 0.8
Radish 93.6 4.2 1.2 0.1 1.0
Spinach 92.7 3.2 2.3 0.3 1.5
Squash, summer 95.0 3.9 0.6 0.1 0.4
Sweet potatoes 68.5 27.9 1.8 0.7 1.1
Tomatoes 94.1 4.0 1.0 0.3 0.6
Watermelon 92.1 6.9 0.5 0.2 0.3
Mean 88.3 8.6 2.0 0.3 0.8
Source: Watt and Merrill.60
Table 6–6 The Transfer of 10 Former Erwinia spp. and Five Subspecies to the New Genera Pectobacterium and Brenneria (Summarized from Reference 18)
Pectobacterium Formerly Erwinia spp.
P. carotovorum subsp. atrosepticum E. carotovora subsp. atroseptica P. carotovorum subsp. carotovorum E. carotovora subsp. carotovora P. carotovorum subsp. betavasculorum E. carotovora subsp. betavasculorum P. carotovorum subsp. odoriferum E. carotovora subsp. odorifera P. carotovorum subsp. wasabiae E. carotovorum sp. wasabiae
P. cacticidum E. cacticida
P. chrysanthemi (formerly Erwinia) P. cypripedii (formerly Erwinia) Brenneria (all former Erwinia spp.)
B. alni, B. nigrifluens, B. paradisiaca, B. quercina, B. rubrifaciens, and B. salicis
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Table 6–7 Some Bacteria that Cause Field and Storage Spoilage of Vegetables and Fruits (See Text for Others)
Organisms Spoilage Condition/Products
Corynebacterium michiganenese Vascular wilt, canker; leaf and fruit spot on tomatoes, others
C. nebraskense Leaf spot, leaf blight, and wilt of corn
C. sepedonicum Tuber rot of white potatoes
Curtobacterium flaccumfaciens Bacterial wilt of beans (formerly Corynebacterium)
Pseudomonas agarici and P. tolaasii Drippy gill of mushrooms
P. corrupata Tomato pith necrosis
Pseudomonas cichorii group Bacterial zonate spot of cabbage and lettuce Pseudomonas marginalis group Soft rot of vegetables, side slime of lettuce P. morsprunorum group (formerly P. phaseolicola) Halo blight of beans
P. syringae pv. syringae Bacterial canker of stone fruit trees
Formerly P. glycinea Disease of soybeans
Formerly P. lachrymans Angular leaf spot of cucumbers
Formerly P. pisi Bacterial blight of pears
P. tomato group Bacterial speck of tomatoes
Xanthomonas campestris
pv. campestris Black rot of cabbage and cauliflower
X. oryzae pv. oryzae Bacterial blight of rice
pv. oryzicola Bacterial leaf streak of rice
Rathyibacter spp. Gumming diseases of plants
Janthinobacterium agaricidamnosum Soft rot of mushrooms
Streptomyces spp. Potato scab
Xanthomonas axonopodis pv. citri Citrus canker
Xylella fastidiosa Pierce’s disease of grapes
Ralstonia spp. Wilt of tomatoes
Erwinia amylovora Fire blight of apple/pear trees
Acidovorax valerianellae Black water-soaked spots of lamb’s lettuce
been transferred to new genera including Acidovorax, Burkholderia, and Hydrogenophaga. Changes have been made to the genus Xanthomonas, and it appears that this process will continue with the emphasis more on molecular genetic methods than the traditional phenotypic approaches. The genus Pantoea is closely related to Erwinia, and it along with Citrobacter and Klebsiella are probably more important in the storage spoilage of vegetables than is now apparent. The bacterial genera most often associated with field and storage spoilage of vegetables are Pseudomonas, Pectobacterium, Erwinia, Xanthomonas, and some of the specific spoilage conditions are listed in Table 6–7.
Soft rots occur in plants of a number of species and those of carrots are well known. A “soft” rot refers to the mushy consistency of the plant or vegetable in contrast to some other spoilage conditions where the product remains firm. The bacteria most commonly associated with the soft rotting of carrots are Pectobacterium spp., especially P. carotovorum subsp. carotovorum and P. carotovorum subsp.
odoriferum. After the spoilage process is initiated, a number of soil-dwelling bacteria are involved,
Vegetable and Fruit Products 131
and they include Pseudomonas spp. as well as Bacillus, Paenibacillus, and Clostridium. For fruits and vegetables like tomatoes and potatoes, P. carotovorum subsp. carotovorum causes soft rot by entering surface wounds. For root vegetables such as carrots, the roots are protected for some. Plant roots are protected from invading microorganisms by their possession of hydrogen peroxide and superoxide, and invading microorganisms produce catalase and superoxide dismutase to overcome this defense.
The Pseudomonas syringae group as well as erwiniae produce these enzymes.
The cementing substance of the vegetable body induces the formation of pectinases, which act by hydrolyzing pectin, thereby producing the mushy consistency. In potatoes, tissue maceration has been shown to be caused by an endopolygalacturonate transeliminase of Pectobacterium origin.28 P.
chrysanthemi produces two pectin methyesterases, at least seven pectate lyases, a polygalacturonase, and a pectin lyase.19Once pectin is destroyed, oligogalacturonidases are formed and utilized by the varied microbiota. Because of the early and relatively rapid growth of bacteria, molds, which tend to be crowded out, are of less consequence in the spoilage of vegetables that are susceptible to bacterial agents.
Once the outer plant barrier has been destroyed by these pectinase producers, nonpectinase producers no doubt enter the plant tissues and help bring about fermentation of the simple carbohydrates that are present. The quantities of simple nitrogenous compounds present, the vitamins (especially the B-complex group), and minerals are adequate to sustain the growth of the invading organisms until the vegetables have been essentially consumed or destroyed. The malodors that are produced are the direct result of volatile compounds (such as NH3, volatile acids, and the like) produced by the biota. When growing in acid media, microorganisms tend to decarboxylate amino acids, leaving amines that cause an elevation of pH toward the neutral range and beyond. Complex carbohydrates such as cellulose are generally the last to be degraded, and a varied biota consisting of molds and other soil organisms is usually responsible, as cellulose degradation by Erwinia spp. is doubtful. Aromatic constituents and porphyrins are probably not attacked until late in the spoilage process, and again by a varied biota of soil types. The genus Brenneria causes diseases of trees such as bark cankers, necrosis on walnut trees, oozing of sap from acorns, etc.18
The genes of P. carotovorum subsp. carotovorum that are involved in potato tuber maceration have been cloned. Plasmids containing cloned DNA mediated the production of endopectate lyases, exopec-tate lyase, endopolygalacturonase, and cellulases.39The Escherichia coli strains that contained cloned plasmids showed that endopectate lyases with endopolygalacturonase or exopectate lyase caused mac-eration of potato tuber slices. These enzymes, along with phosphatidase C and phospholipase A, are involved in soft rot by this organism. Carrots infected with Agrobacterium tumefaciens undergo senes-cence at a faster rate because of increased ethylene synthesis. In normal uninfected plants, ethylene synthesis is regulated by auxins, but A. tumefaciens increases the synthesis of indoleacetic acid, which results in increased levels of ethylene.
The citrus canker pathogen, Xanthomonas anonopodis pv. (pathovar) citri wreaked havoc on the Florida citrus industry in the late 1990s. The canker consists of external scabs and cork-like lesions that occur on oranges, limes, grapefruits, and other citrus fruits. The organism enters fruit trees through leaf stomata. Once inside, they employ a type III secretion system (see Chapter 22) to induce increased cell division that leads to the brownish cankers. The infected fruits produce more ethylene, which leads to senescence, increased ripening, and premature falling of fruits from trees. This infection does not destroy trees. The bacterium is spread from tree to tree by wind, rain, insects, and other nonspecific means.
The genus Xanthomonas is undergoing reclassification, thus some of the species and pathovars noted in Table 6–6 are likely to be changed. Most form yellow mucoid and smooth colonies and
132 Modern Food Microbiology
Figure 6–1 Tomato diseases—(A and B) nailhead spot; (C and D) bacterial spot; (E) bacterial canker; (F) bacterial spot. Source: From Agriculture Handbook 28, USDA, 1968, “Fungus and Bacterial Diseases of Fresh Tomatoes.”
produce the yellow-pigmented xanthomonadins. The mucoid colonies are due to xanthans, which are typical of the genus.56Bacterial canker of stone fruits is caused by P. syringae pv. syringae, and this pathovar has been reported to cause disease in over 180 species of plants.25
Some of the more important bacteria that cause field and storage spoilage of vegetables are pre-sented in Table 6–7. Some genera and species listed are undergoing taxonomic changes. The plant
Vegetable and Fruit Products 133
Figure 6–2 Lima bean diseases—(A and B) pod blight; (C) seed spotting; (D) yeast spot. Pea diseases—(E) pod spot; (F) anthracnose; (G) scab. Source: From Agriculture Handbook 303, USDA, 1966, Chapter 5.
corynebacteria represent a diverse collection, many of which do not belong to this genus. Some have been transferred to the genus Curtobacter. The plant pathogenic and field spoilage pseudomonads and xanthomonads are also diverse.
The appearance of some market vegetables undergoing bacterial and fungal spoilage is shown in Figures 6–1 and 6–2.
134 Modern Food Microbiology
Fungal Agents
A synopsis of some of the common spoilage conditions of vegetables and fruits is presented in Table 6–8. Some of these spoilage conditions are initiated preharvest and others postharvest. Among the former, Botrytis invades the flower of strawberries to cause gray mold rot, Colletotrichum invades the epidermis of bananas to initiate banana anthracnose, and Gloeosporium invades the lenticels of apples to initiate lenticel rot.13The largest number of market fruit and vegetable spoilage conditions occur after harvesting, and although the fungi most often invade bruised and damaged products, some enter specific areas. For example, Thielaviopsis invades the fruit stem of pineapples to cause black rot of this fruit, and Colletotrichum invades the crown cushion of bananas to cause banana crown rot.13 Black rot of sweet potatoes is caused by Ceratocystis, neck rot of onions by Botrytis allii, and downey mildew of lettuce by Bremia spp.9Some of the spoilage conditions listed in Table 6–8 are discussed below.
Gray mold rot. This condition is caused by Botrytis cinerea, which produces a gray mycelium. This type of spoilage is favored by high humidity and warm temperatures. Among the vegetables affected are asparagus, onions, garlic, beans (green, lima, and wax), carrots, parsnips, celery, tomatoes, endives, globe artichokes, lettuce, rhubarb, cabbage, Brussels sprouts, cauliflower, broccoli, radish, rutabagas, turnips, cucumbers, pumpkin, squash, peppers, and sweet potatoes. In this disease, the causal fungus grows on decayed areas in the form of a prominent gray mold. It can enter fruits and vegetables through the unbroken skin or through cuts and cracks.
Sour rot (oospora rot, watery soft rot). This condition of vegetables is caused by Geotrichum candidum and other organisms. Among the vegetables affected are asparagus, onions, garlic, beans (green, lima, and wax), carrots, parsnips, parsley, endives, globe artichokes, lettuce, cabbage, Brussels sprouts, cauliflower, broccoli, radishes, rutabagas, turnips, and tomatoes. The causal fungus is widely distributed in soils and on decaying fruits and vegetables. Drosophila melanogaster (fruit fly) carries spores and mycelial fragments on its body from decaying fruits and vegetables to growth cracks and wounds in healthy fruits and vegetables. Because the fungus cannot enter through the unbroken skin, infections usually start in openings of one type or another.27
Rhizopus soft rot. This condition is caused by Rhizopus stolonifer and other species that make vegetables soft and mushy. Cottony growth of the mold with small black dots of sporangia often covers the vegetables. Among those affected are beans (green, lima, and wax), carrots, sweet potatoes, potatoes, cabbage, Brussels sprouts, cauliflower, broccoli, radish, rutabagas, turnips, cucumbers, can-taloupes, pumpkins, squash, watermelons, and tomatoes. This fungus is spread by D. melanogaster, which lays its eggs in the growth cracks on various fruits and vegetables. The fungus is widespread and is disseminated by other means also. Entry usually occurs through wounds and other skin breaks.
Phytophthora rot. This market condition, caused by Phytophthora spp., occurs largely in the field as a blight and fruit rot of market vegetables. It appears to be more variable than some other market
“diseases” and affects different plants in different ways. Among the vegetables affected are asparagus, onions, garlic, cantaloupes, watermelons, tomatoes, eggplants, and peppers.
Anthracnose. This plant disease is characterized by spotting of leaves, fruit, or seed pods. It is caused by Colletotrichum coccodes and other species. These fungi are considered weak plant pathogens. They live from season to season on plant debris in the soil and on the seed of various plants such as the tomato. Their spread is favored by warm, wet, weather. Among the vegetables affected are beans, cucumbers, watermelons, pumpkins, squash, tomatoes, and peppers.
Blue mold rot is a post-harvest disease of apples and pears that is caused by Penicillium expansum while gray mold rot is caused by Botrytis cinerea. In an attempt to control these fungi on apples and pears, two biocontrol agents have been tested—Candida sake and Pantoea agglomerans.32When