Journal of Applied Bacteriology Symposium Supplement 1992, 73, 115S126S

Ecosystems in vegetable foods Barbara M. Lund AFRC Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, UK

1. Introduction, 115s 2. Numbers and types of bacteria normally present on vegetables in the field and after harvest, 115s 3. Properties of vegetables that influence the growth of bacteria, 117s 4. Growth of bacteria on vegetables, 120s

1. INTRODUCTION

The ecosystem in vegetable foods consists of: (1) The micro-organisms present, with their different metabolic capabilities and responses to the external environment. (2) The vegetable tissue, with its own particular structure, the pH of the tissue, the presence of organic acids and of anti-microbial compounds. The state of the tissue will be altered by its treatment after harvest. (3) The extrinsic, environmental factors including the temperature, water activity and free water and gaseous environment. Because of the scope of the subject, this paper will be concerned only with bacteria in these ecosystems. Many of the bacteria present on freshly harvested vegetables are those that were present on the vegetable in the field. Bacteria present on leaves are liable to be killed by drying and by ultraviolet light and many survive best in positions that are moist and protected from light (Leben 1988). Seasonal and diurnal changes (Leben 1988) and large differences in numbers of bacteria on individual leaves in the canopy of crop plants have been reported (Hirano et al. 1982; Hirano & Upper 1983). Rain and insects are thought to be important in the transfer of bacteria on and among field plants, and high relative humidity favours the survival and spread of bacteria on the plant surface (Leben 1988). While the plant is growing nutrients can be leached from within the leaves by the action of rain, dew, mist and fog, and in guttation liquid and this probably takes place largely through the hydathodes (Tukey 1971). The substances leached include inorganic nutrients (minerals), sugars, pectic substances, sugar alcohols, hormones, vitamins, alkaloids and phenolic substances. At the leaf surface there is Correspondence to: Dr B.M. Lund, AFRC Institute of Food Research, Normich Laboratory, Norwich Research Park, Colney, Normich NR4 7UA, UK.

4.1 Decay of vegetables by bacteria, 1 2 0 s 4.2 Extrinsic environmental factors that influence t h e growth of bacteria on vegetables, 120s 4.3 Microenvironments in vegetable foods and growth of Clostridium botulinum, 121S

5. References, 1 2 2 s

said to be a so-called 'boundary layer', i.e. a thin layer of air influenced by the leaf surface. Within this layer, which may be a maximum of 10 mm and a minimum o f less than 1 mm thick, the leaf influences the temperature, moisture and speed of air movement (Burrage 1971). Broad leaves are likely to have a thicker boundary layer than thin leaves, and hairy or ridged leaves a thicker layer than smooth leaves. The humidity at the leaf surface depends on the rate at which water vapour is transferred through the boundary layer. Leben (1988) reported that the distribution of bacteria on leaf surfaces is related to the wettability of the surface, and that bacteria are found in areas that best retain water. The leaves of some vegetables, such as cabbage, have extensive deposits of small particles of wax which may be expected to influence the distribution of bacteria, whereas 1eaves"of other vegetables such as lettuce are without such deposits. There is evidence that some bacteria adhere to leaves and cannot be removed easily by gentle washing (Preece & Wong 1981). The percentage o f plant pathogenic bacteria that adhere to leaves of their host plant was reported to be higher than that adhering to leaves of non-host plants and higher than that of saprophytic bacteria. 2. N U M B E R S AND TYPES OF BACTERIA NORMALLY PRESENT ON VEGETABLES I N THE FIELD AND AFTER HARVEST

The most numerous bacteria found on the surface of leafy vegetables are usually Gram-negative bacteria (Pederson & Fisher 1944). An examination of 37 heads of cabbage in the USA over a period of 2 years showed that the average number of bacteria on the outside leaves of unwashed heads (from which the outer leaves were removed) was 2 x 106/g while on the inside leaves it was 4 x 103/g (Keipper & Fred 1930). Washing reduced the number of the bacteria

116s B A R B A R A M . L U N D

on the outer leaves by a factor of 2.5. A much larger proportion of catalase-negative, small pin-point colonies, presumed to be lactic acid bacteria, was obtained from the interior of heads than from the outside. On the outer, aesthetically acceptable leaves of lettuce ca 6.3 x lo4 mesophilic, aerobic bacteria/g were detected while 32/g were found on the innermost leaves (Maxcy 1978). The numbers of these bacteria on whole heads and on lettuce prepared for institutional use were 1.6 x lo5 and 4.2 x 105/g respectively. On freshly harvested, hydroponically grown lettuce (34 samples) the count of aerobic bacteria on the entire head was between 3.8 x lo4 and 2.3 x 108/g with a mean of 2.2 x 107/g (Riser et al. 1984). Most of the Gramnegative rods isolated from the lettuce were Enterobacter cloacae, Citrobacter freundii and E. agglomerans. On freshly harvested celery irrigated with mains water in the L K numbers of mesophilic aerobic bacteria were between 8 x lo4 and 2 x 106/g of which 1 x lo3 - 5 x 103/g were pectate-degrading bacteria and 8@1 x 103/g were fluorescent pseudomonads (Robinson & Adams 1978). Studies of the bacteria on the curd of over 39 freshly harvested cauliflowers in the UK showed > lo5 mesophilic, aerobic bacteriatg which included large numbers of fluorescent pseudomonads (Lund 1981). The majority of the bacteria on the surfaces of freshly harvested plants are saprophytes, two of the major groups present are often pseudomonads and Enterobacteriaceae. The numbers and types of lactic acid bacteria on vegetable plants in a subtropical, moist region of the USA have been studied by Mundt et al. (1967) and Mundt & Hamer (1968). None of these bacteria was recovered from 14.7% of 156 samples of raw vegetables. Most of the failures to isolate these occurred during conditions of marked drought, and the total numbers of micro-organisms present were likely to be low. Leuconostoc mesenteroides and yellow enterococci were the organisms most frequently isolated. When rainfall was more abundant the average number Leuc. mesenteroides on vegetables including greens, summer squash, green beans, okra, southern peas and butter beans was 2.5 x 104/g while the average number of enterococci was 10z/g. On a range of vegetables from commercial processing lines the highest counts of aerobic mesophilic bacteria were on shredded cabbage where the numbers were up to 107/g (Garg e: al. 1990). These vegetables had all been washed and dipped in chlorinated water. Shredders used to prepare shredded lettuce and cabbage were major sources of contamination in the factory. T h e predominant microorganisms on the vegetables were Gram-negative rods, mostly pseudomonads. T h e numbers of lactic acid bacteria were between < 10 and 1.4 x 103/g; over 80% of the lactic acid bacteria were leuconostocs, the others were equal

ECOSYSTEMS I N VEGETABLE FOODS 117s

numbers of lactobacilli (about half of which were heterofermentative) and streptococci. When vegetables are cut and shredded release of cell contents will encourage growth of micro-organisms. Bolin et a/. (1977) reported that thorough washing to remove these released contents was important in prolonging the shelf-life of cut lettuce. All fruits and vegetables are permeated by air spaces that are connected in some way with the external atmosphere. In tomatoes gas exchange occurs through the region of attachment of the stem to the fruit (the stem scar). Air spaces constitute approximately 1% of the volume of potatoes and gaseous exchange occurs primarily through the lenticels. During the washing of tomatoes and potatoes, immersion in water that is cooler than the produce (by 6 1 4 ° C in the case of tomatoes) or the hydrostatic pressure on the produce immersed in the water can lead to a slight increase in weight due to the influx of water through the stem scar or the lenticels, respectively, and to an increase in decay caused by the intake of both water and soft-rot bacteria (Bartz & Showalter 1981; Bartz & Kelman 1985). Contamination of vegetables with food-borne pathogenic bacteria can result from the survival of such bacteria in soil, for example in the case of Bacillus cereus, CI. perfringens, CI. botulinum and perhaps to a lesser extent Listeria monocytogenes. T h e sources of contamination with other pathogens including Salmonella, Escherichia coli, and L . monocytogenes are manure, sewage sludge, polluted irrigation water, from animals and birds and from unhygienic practices after harvest. A survey of lettuce and fennel in Italy in 1973-1975 showed that the average number of faecal coliform bacteria on lettuce was 61/g (Ercolani 1976), about half of which were E. coli. Salmonella spp. were isolated from 60% of lettuce, the largest number being 30,’ 100 g. In a survey in The Netherlands in 1976 of vegetables obtained from retail outlets or from importers, 11% of samples were contaminated with > 100 E. colz/g, at least 8.8% carried Salmonella spp. and Salm. typhi was found on one sample (Tamminga et al. 1978a). A study of 100 samples of vegetables, mainly of the salad type, purchased from retail outlets in the UK showed that the numbers of mesophilic, aerobic bacteria present were between < 102/g and >109/g (Roberts et al. 1982). T h e largest numbers were found on bean shoots, cress, spring onions and water cress. Only 14 samples contained detectable numbers of E. coli, the largest number being 460/g on bean shoots. Salmonellas were isolated from only one sample, a lettuce. Bacillus cereus and CE. perfingens were isolated by enrichment from one third of samples but Staphylococcus aureus, Campylobacter, Shigella, Vibrio spp. and Yersinia enterocolztica were not isolated from any of the samples. In the case of salad vegetables prepared for storage under refrigeration, it is particularly important to consider the incidence of psychrotrophic, food-borne pathogens able to

multiply on these foods during refrigerated storage. In a survey of pre-packed salads in the UK L . monocytogenes was detected in four of 60 samples (70%) (Sizmur & Walker 1988). A further survey of pre-packed, prepared salads and the individual ingredients from which these were made was reported by Velani & Roberts (1991). Listeria monocytogenes was found in eight (190/,) of 42 pre-packed mixed salads and in two (1.8%) individual salad ingredients. The higher rate of contamination of the prepared salads is likely to be due to contamination and spread of the organism during chopping, mixing and packaging of salads. The numbers of listerias were very low. Of 25 samples of fresh cut vegetables in T h e Netherlands L. monocytogenes was detected in 11 (44%); the numbers present were < 1OO/g (Beckers et al. 1989). Other workers have failed to isolate L. monocytogenes from a range of vegetables (Farber et al. 1989; Heisick et al. 1989) but have found it associated with 14% of radishes and 21% of potatoes (Heisick et a/. 1989). Contamination of cabbage in the field with this bacterium was the suspected cause of the outbreak of listeriosis associated with coleslaw in Canada in 1981 (Schlech et al. 1983). Strains of Aeromonas hydrophila and Aero. cavaae are found frequently on raw produce (Callister & Agger 1987; Berrang et al. 1989b). These bacteria can multiply at temperatures of 1 4 ° C and are associated with diarrhoea1 illness but their real status as human pathogens remains to be established (Archer & Kvenberg 1988). Yersinia enterocolitica has been detected, probably in very low numbers, in raw vegetables in France (Delmas & Vidon 1985) and The Netherlands (de Boer et al. 1986) and in prepared salad vegetables in the UK (Brocklehurst et al. 1987). T h e great majority of the strains isolated from these are considered to be non-pathogenic. This bacterium will also multiply at temperatures down to 1°C. 3. PROPERTIES OF VEGETABLES THAT INFLUENCE THE GROWTH OF BACTERIA

The properties of vegetables that influence growth of bacteria include their p H content of organic acids and of nutrients and the presence of anti-bacterial compounds. T h e p H of vegetables is usually in the range that will support the growth of bacteria (Table 1). Thus vegetables are subject to attack by soft-rot bacteria (Section 4.1) and will support the growth of a wide range of saprophytic bacteria as well as those pathogenic to humans. Outbreaks of typhoid, paratyphoid, cholera, food-poisoning due to Salmonella and B . cereus (Roberts et al. 1982), shigellosis (Anon. 1987) and botulism (Section 4.3) have been associated with vegetables and fruits. In a few cases the bacteria may not have multiplied on the produce.

118s B A R B A R A M . LUND

Table 1 Approximate pH values of some fresh vegetables and fruits

Product Vegetables Asparagus (buds and stalks) Beans (string) Beans (Lima) Beets Beets (sugar) Broccoli Brussels sprouts Cabbage (green) Carrots Cauliflower Celery

Corn (sweet) Eggplant Lettuce Olives Onions (red) Parsley Parsnip Peppers (green cherry) Peppers (red cherry) Peppers (several types) Pimentos Potato tubers Pumpkin Rhubarb

Spinach Squash Sweet potatoes Tomatoes (whole) Tomatoes, under ripe

Tomatoes, ripe Tomatoes, over ripe Turnips Fruits Apples Apricots Bananas Cherries Figs Grapefruit (juice) Grapes Limes Melons (cantaloupe) Melons (honeydew) Oranges (juice) Pears Plums Watermelons

PH

Reference

5.7-6.1 ; 54-6.1 4.6 6.5; 5 . 6 6 . 5 ; 6.2 4.9-5.8 4.2-44 6.5 6.3; 6.3-6.6 54-6.0; 5.2-6.3; 5.8 4'9-5.2, 6.0; 4 . g 6 . 3 6.0-6.7 5 7 4 . 0 ; 5.6 7.3; 5.9-6.5; 7.1 4.5 6.0; 6 . g 6 . 4 3.6-3.8 5.3-5.8; 5.0 5.7-6.0; 5.8 53 5.6-7.0 5.3-5.8 54-5.6 4'3-5'2 5.6-6.2; 6.0 4.8-5.2 3.1-3'4 5.5-6.0; 5.1-6.8 54&5.4 5.3-5.6 4 , 2 4 3 ; 3.74.9 3.245 3.4-4.7 4.M.8 5.2-5.5; 5 2 - 5 6

1; 3 1 1; 3; 6 3 1 1 1; 3 1; 3; 6 1; 3

2'9-3.3 3.51.0 4.5-4.7; 4.5-5.2 3.247 4.6 3.0 3.4-4.5 1.8-2.0 6.2-6.5 6'3-6'7 3.w.3 3.M.7 2w.6 5.2-5.6

1 3 1; 3 3

1, Jaj 1986; 2, Burton 1966; 3, Banwart 1989; 4, 1990; 5, Wolf et al. 1079; 6, Sapers et al. 1984.

3 1 ; 3; 6 1; 3; 6 I 1; 3 1 1; 3; 6 1; 6 1

4 4 6 3 2; 6 1 1 1; 3 1 3 1; 3

5 5 5 1; 3

1 1 1 1

3 1 1 3 1; 3 1

Daeschel et al.

Some fruits have a p H level that is sufficiently high to allow the growth of bacteria. Tomatoes and peppers, with a pH range of 3-4-4.7 and 5.G7.0 respectively, are subject to bacterial decay. In the USA and Israel, for example, soft rot due to Erwinia carotovora has been a major cause of spoilage of tomatoes (McColloch et al. 1968). I n the USA tomatoes are washed after harvest and before shipment. If the fruits are immersed in water that is cooler than the fruit a decrease occurs in the internal gas pressure. This results in an intake of water and bacteria through the stem scar (Bartz & Showalter 1981). Immersion of tomatoes in dumptanks during washing has probably been a factor leading to infiltration and spoilage by Erw. carotovora ; this wastage should be avoided by using water that is slightly warmer than the incoming fruit. In the USA and Italy bacterial soft rot caused by Erw. carotovora has been reported as the most destructive market disease of peppers (Coplin 1980; Risse & Chun 1987). T h e ability of food-borne pathogenic bacteria to grow on the cut surfaces of tomatoes and some other fruits has been of recent concern. Strains of Salm. enteritidis, Salm. infantis and Salm. typhimurium multiplied, producing large numbers in 24 h at 22°C after inoculation on the cut surface of tomatoes with a p H between 3.99 and 4.37 (Asplund & Nurmi 1991). T h e major acidulants in tomatoes are citric (80%) and malic acids (10%). Salmonellas have been shown to multiply on the cut surface of watermelon (Gayler et al. 1955). After inoculation on cubes of papaya and jacima fruits, with a surface p H of 5.67 and 5.97 respectively, and watermelon Shigella spp., Salm. derby and Salm. typhi multiplied during storage at 25-27°C for up to 6 h (Escartin et al. 1989). I n 1990 an outbreak of infection with Salm. Chester associated with cantaloupe melons affected at least 245 persons in 30 states in the USA and an outbreak due to Salm. javiana associated with tomatoes affected 174 persons in four states. I n 1991 more than 400 cases of infection with Salm. poona occurred in 23 states in Canada and were associated with cantaloupes (Centers for Disease Control 1991). I n none of these outbreaks was the organism recovered from the incriminated food but in 1990 and 1991 workers at the Food and Drugs Administration (FDA) isolated many Salmonella serotypes from approximately 10/0 of the rinds of cantaloupes and watermelons. Many crop plants produce anti-microbial compounds (Skinner 1955; Davidson et al. 1983; Beuchat & Golden 1989). T h e anti-bacterial activity of herbs and spices in particular has been the subject of many studies. Vegetables have often been shown to have anti-bacterial properties. The most notable effects have been associated with horseradish, garlic, onion and leek. Horseradish vapour was reported to have an anti-bacterial effect (Foter & Gollick 1938). T h e vapour from garlic was shown to be inhibitory

ECOSYSTEMS IN VEGETABLE FOODS

to Mycobacterium butyricum and M . smegmatis and to a lesser extent to E. coli, Serratia marcescens and B . subtilis (Walton et al. 1936). Crushed garlic tissue was inhibitory to a wide range of Gram-positive and Gram-negative bacteria (Al-Delaimy & Ali 1970; Karaioannoglou et al. 1977; Mantis et al. 1978; Saleem & Al-Delaimy 1982; see also Fenwick & Hanley (1985)). The anti-bacterial activity of garlic was destroyed by heating at 80-90°C (Saleem & Al-Delaimy 1982). In order to prepare garlic powders with the maximum anti-bacterial activity the conditions advised were (i) drying the garlic at temperatures below 60°C; (ii) restricting the load on trays; (iii) reduction of loss of anti-bacterial activity in the powder by storage under nitrogen and at &2"C (Pruthi et al. 1959a,b,c,d,e, cited by Marth (1955)). The fact that some commercially prepared dehydrated garlic (and onion) retained its anti-bacterial activity was shown by Johnson & Vaughn (1969), who reported that freshly reconstituted, dehydrated garlic and onion killed Salm. typhimurium and E. coli. Fresh onion juice and homogenates also have an antimicrobial effect (Love11 1937; Fuller & Higgins 1940; Vaughn 1951; Virtanen & Mattikala 1959; Al-Delaimy & Ali 1970) which can be sufficiently strong to interfere with pour-plate counts of bacteria in/on onion (Wei et al. 1967). The interference with counts of B. subtilis could be overcome by the addition of potassium sulphite (0-5-1.6%) to the liquid used to homogenize the onion. Dehydrated onion prepared commercially or in the laboratory had an antibacterial effect (Wei et al. 1967). The main anti-bacterial compound in crushed garlic is considered to be allicin (di-(2 propenyl) thiosulphinate) (Cavallito et al. 1944; Stoll & Seebeck, 1948, 1951), which

is formed from a precursor compound alliin (S-allyl-Lcysteine-sulphoxide) by enzyme activity when fresh garlic is crushed (Fig. 1) (Stoll & Seebeck 1949, 1951). Onion contains S-methylcysteine sulphoxide and S-n-propyl cysteine sulphoxide. When onion is crushed these cysteine sulphoxides are converted enzymically to thiosulphinates, which have a somewhat weaker anti-microbial effect than the allythiosulphinate formed in crushed garlic (Virtannen & Matikkala 1959). The effect of allkin is generally attributed to its interaction with thiol-containing compounds (Fenwick & Hanley 1985). Reduction by such compounds as cysteine and dithiothreitol to di-(propenyl) disulphide decreases the anti-microbial effect, but this can be restored by oxidation with hydrogen peroxide. During the preparation of sauerkraut Gram-negative bacteria were the predominant organisms present initially on cabbage leaves but the numbers declined rapidly, before the numbers of Gram-positive bacteria increased (Pederson & Fisher 1944). Substances in macerated cabbage had a bactericidal effect on the Gram-negative bacteria and much less effect on Gram-positive bacteria. Many other vegetables were tested but showed little or no anti-bacterial effect. T h e bactericidal effect of the juice of some varieties of cabbage was as active as the juice of onions. Despite the formation of these compounds, garlic, onion and other plant tissues are susceptible to bacterial spoilage. Even growth of food-poisoning bacteria is not necessarily prevented by the presence of these anti-bacterial agents. T h e addition of fresh garlic or onion or their oils or extracts to foods has in some cases reduced the growth of microorganisms (Brocklehurst et al. 1983). The addition of garlic or onion oils to a pork slurry was not sufficient to inhibit growth of C1. botulinum types B or E (De Wit et al. 1979).

I

Allylsulphenic acid

I

+

2 s--0-

CHZ HzN-CH

I I

COOH

Al Iicin (Cavallito)

CHZ

2 HZN-C

CH 3

II

$ 2 HZO

Fig. 1 The enzymic decomposition of alliin to allicin (Stoll & Seedbeck 1951)

COOH

a- Aminoacrylic

Pyruvic acid

acid Scheme

I

2C=O

I

I

COOH Alliin

119s

II:

+2NH3

120s B A R B A R A M. LUND

An anti-bacterial effect of a different type has been reported recently, resulting in the inhibition of L. monocytogenes by carrot tissue. The numbers of viable L. monocytogenes decreased upon contact with whole, shredded or sliced carrots (Beuchat & Brackett 1990; Nguyen-the & Lund 1991). The anti-listerial effect was very unstable; it was destroyed by some vigorous methods of maceration of carrot tissue. It diminished rapidly at temperatures between 4 and 20°C and was destroyed by boiling. The anti-listerial effect was inhibited in anaerobic conditions but not by catalase, peroxidase or superoxide dismutase, all of which could be expected to protect against the toxic effects of peroxide or active oxygen species (Nguyen-the & Lund 1992).

4. GROWTH OF B A C T E R I A ON VEGETABLES

4.1 Decay of vegetables by bacteria

Bacteria are a major cause of disease and decay of vegetables in the field as well as after harvest. The ability of bacteria to degrade plant tissue is attributed to their ability to degrade pectic substances, which are important components of the middle lamella and the primary cell walls of the parenchymatous tissue of dicotyledonous and monocotyledonous plants (Fig. 2) (Selvendren 1985). The major constituents of pectic substances are rhamnogalacturonans. Those in which a proportion of the galacturonic acid residues are present as methyl esters are designated pectinic acids or pectins ; those without methyl ester groups are called pectic acids.

t

0-5-2 p n

1

-Primary -Middle

cell wall lamello

-

According to Selvendren (1985) there is still debate about the localization of pectins within the walls of parenchymatous tissues. T h e middle lamella of parenchymatous tissue is thought to consist principally of the calcium salts of pectins. T h e properties of pectic substances in plant cell walls have been reviewed by Selvendren (1985). T h e ability of bacteria to cause rotting of plant tissue results mainly from their production of an array of enzymes that attack the rhamnogalacturonan polymer by hydrolysis (hydrolases) or by elimination (lyases) (Collmer & Keen 1986; Kotoujansky 1987). T h e bacteria that are mainly responsible for rotting of vegetables after harvest are Erw. carotovora and certain fluorescent pseudomonads (Pseudomonas marginalis) (Lund 1983; Liao & Wells 1987a, b). They also contribute to the spoilage of prepared, ready-touse, fresh vegetables (Nguyen-the & Prunier 1989). The formation of pectic enzymes by a wide range of bacteria was reviewed by Rombouts & Pilnik (1980). These enzymes are produced by many plant pathogenic bacteria including species of Erwinia, Pseudomonas and Xanthomonas. Other bacteria that form pectic enzymes and cause softening of plant tissue include Aero. liquefaciens, associated with the softening of ripe olives (Vaughn et al. 1969; Hsu & Vaughn 1969), Cytophaga spp. (Liao & Wells 1987c), Bacillus spp. and Clostridium spp. (Lund 1983), Leuc. mesenteroides (Juven et al. 1985) and Agrobacterium tumefaciens biovar 3 (McGuire et al. 1991). Bacteria reported to form pectic enzymes but not shown to cause marked softening of plant tissue include Flavobacterium spp. (Lund 1969), Klebsiella pneumaniae and Y . enterocolitica (Starr et al. 1977; Chatterjee et al. 1979; Bagley & Starr 1979), K l . oxytoca, Rhizobium spp., Arthrobacter spp. and Bacteroides spp. The type and amount of pectic enzymes formed by softrot bacteria enable them to degrade the tissue of vegetables. T o what extent the formation of more limited types and quantities of pectic enzymes by other bacteria facilitates their growth in association with plant tissue is not known.

"

Branched peptic polymer

Cellulose microfibrils Xyl og lucan

-

60 nrn-

Fig. 2 Pectic polymers in the primary wall of dicotyledonous and

monocotyledonous plants (Selvendren, R. R . personal communication)

4.2 Extrinsic environmentalfactors that influence the growth of bacteria on vegetables

Many human pathogens including Salmonella, Shigella and Vibrio cholerae, are able to survive and multiply on plant tissue. After a recent outbreak of shigellosis attributed to contamination of commercially distributed, shredded lettuce it was reported that a strain of Shigella sonnei multiplied rapidly on shredded lettuce at 22°C (Davis et al. 1988). The ability of bacteria to multiply on vegetables is influenced strongly by environmental factors including the storage temperature, the presence of free water, the relative humidity, and the gaseous environment. There has been

ECOSYSTEMS I N VEGETABLE FOODS 121s

considerable interest in the possible use of modified atmospheres or controlled atmospheres to extend the storage life of vegetables (Lioutas 1988; Zagory & Kader 1988; Huxsoll & Bolin 1989; Myers 1989). Controlled atmospheres usually contain a lower concentration of oxygen and a higher concentration of carbon dioxide than is found in air. This tends to slow the respiration of the vegetable, retard ripening and maintain quality for a longer time than during storage in air. The minimum oxygen concentration that will avoid injury to produce is ca 2% in most cases. Below this level anaerobic respiration may occur (Myers 1989). Controlled or modified atmospheres are combined with refrigeration for greatest effectiveness. A study of the effect of controlled atmospheres on the growth of L. monocytogenes on asparagus, broccoli and cauliflower and on the shelf life of these vegetables was conducted by Berrang et al. (1989a). A high relative humidity was maintained during the experiments. The controlled atmospheres extended the storage time at 4 or 15°C for which the vegetables remained acceptable for consumption, but did not affect the growth of L. monocytogenes. Similar results were obtained with Aero. hydrophila (Berrang et al. 1989b). Controlled atmosphere storage had similar effects on the sensory qualities and the natural microflora of broccoli (Brackett 1989) and bell peppers (Brackett 1988). Modified but not controlled atmospheres may be used for packaging vegetables but respiration of the produce can result in high concentrations of carbon dioxide and depletion of oxygen to concentrations below 1-2%. Spoilage of such products is associated with high counts of facultatively anaerobic bacteria, and usually occurs before any significant multiplication of Clostridium spp. If severe depletion of oxygen occurred in packs then it would be expected that a decrease in quality of the product would make it unsaleable before growth of Clostridium spp. had occurred. In some cases, however, microenvironments have become established in vegetable products other than canned foods and have resulted in growth of C1. botulinum and outbreaks of botulism.

4.3 Microenvironments in vegetable foods and growth of Clostridium botulinum

Some outbreaks of botulism have resulted from the use of partially processed vegetable products in which the risk had not been foreseen but in which the microenvironments allowed the growth of Cl. botulinum. Potato salad has been implicated in three outbreaks of type A botulism in the USA. One in Colorado in 1969 involved six people (Ryan et al. 1971; Cherrington 1974), one in New Mexico in 1978 affected 34 people (Centers for Disease Control 1978) and one in Colorado in 1978 affected

eight people (Seals et al. 1981). In at least two of these incidents the salad was prepared from potatoes that had been baked in aluminium foil, and kept for several days at room temperature. Potatoes that were surface or stabinoculated with spores of type A Cl. botulinum, wrapped in aluminium foil, baked, then left at 22°C became toxic in 6-7 d when they were not always overtly spoiled (Sugiyama et al. 1981). Boiled potatoes inoculated with spores, used immediately to prepare potato salad with a p H of 5.2 and incubated anaerobically at room temperature for 9 d failed to become toxic (Seals et al. 1981). Plain potatoes treated similarly were toxic in 24 h. Both proteolytic and nonproteolytic strains of CI. botulinum have been reported to survive commercial cooking processes for vacuum-packed potatoes and to produce toxin during storage, sometimes before obvious spoilage (Tamminga et al. 1978b; Notermans et al. 1981; Lund et al. 1988). In 1983 an outbreak of botulism occurred in Peoria, Illinois, in which 28 people were hospitalized and one died. Type A toxin was involved and sautked onions were identified as the probable food vehicle (Solomon & Kautter 1986). It was claimed that the sautked onions were prepared freshly each morning and that any leftovers were discarded at the end of the day. Strains of CI. botulinum type A were isolated from patients and the skins of onions, and spores that were inoculated into onions that had been sautCed in margarine and cooled, which were then incubated at 35°C to simulate the holding temperature in the restaurant (Soloman & Kautter 1986). As few as two spores/g of these strains in sautked onions resulted in growth and toxin formation within 48 h at 35°C. When the appearance of the food was normal. I t was concluded that the margarine probably created an anaerobic environment that allowed growth of the organism. An outbreak of type A botulism occurred in Japan in 1984 and involved 36 people of whom 11 died (Hayashi et al. 1986). T h e causative food was vacuum-packed, deepfried, mustard-stuffed lotus root. After the stuffing had been placed in the lotus roots and the food was refrigerated the p H and the water activity of the stuffing increased to levels that allowed growth of Cl. botulinum. Growth of this organism and toxin formation occurred, however, only if the product was subjected to a heat treatment which was part of the manufacturing process. Whether the heat treatment killed competing bacteria or altered the food so as to provide more favourable conditions for growth was not determined. The authors considered that vacuum packaging did not enhance growth of Cl. botulinum in the product. It may have contributed to the outbreak in that people assumed that the vacuum-packaged food had a longer shelf life than products not so packed. Between July and September 1985 36 cases of type B botulism were caused by food served in a restaurant in

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Vancouver, British Columbia (Centers for Disease Control 1985; St Louis et ul. 1988). Commercially prepared bottled, chopped garlic in soybean oil was implicated epidemiologically. Although the product was labelled ‘Keep refrigerated’ in very small print, the jar of garlic in the restaurant had been kept at room temperature (25-32°C). The chopped garlic in soybean oil was produced by rehydration of dry chopped garlic to which soybean oil was added. The product was filled into 8 oz glass jars. Each jar contained 125 g of garlic and 90 ml of water-in-oil emulsion, pH 5.85.9. Inoculation studies showed that type -4 and proteolytic type B strains multiplied and formed toxin in the product during incubation at 35°C (Solomon & Kautter 1988). The product remained organoleptically acceptable even when highly toxic. Toxin was also formed when the product was incubated a t room temperature. (It is of interest that a type B strain from a patient involved in the outbreak produced a relatively high level of toxin in the product.) One or two non-proteolytic type B strains grew and formed toxin in the chopped garlic in oil incubated at room temperature. Thus, both onions and garlic in an oil menstruum supported the outgrowth of and toxin formation by Cl. botulinum types A and B from a minimal inoculum of 1-5 spores/g while remaining organoleptically acceptable. In 1989 three cases of type A botulism were attributed to the consumption of a chopped garlic-in-oil product (Morse et a/. 1990). The remains of the product contained high concentrations of the organism and the toxin. After this second outbreak the US FDA ordered companies to stop making any garlic-inoil mixes that are protected only by refrigeration. Such products must now contain specific levels of microbial inhibitors or the acidifying agents, such as phosphoric or citric acid. In 1987 four circus performers in Sarogota, Florida, suffered symptoms of botulism after eating coleslaw prepared from packaged, shredded cabbage mixed with coleslaw dressing (Solomon et a!. 1990). Clostridium botulinum type A toxin and spores were found in the coleslaw dressing which contained many pieces of cabbage and carrot. .4s the p H of the coleslaw dressing was reported to be 3.5 it was likely that CI. botulinum had not grown in the coleslaw but in the shredded cabbage which it was suspected had been packed in a modified atmosphere. An investigation of the incidence of Cl. botulinunr in fresh cabbages from supermarkets in Washington DC showed that type A strains were isolated from 12 of 88 cabbages (13.6%). Shredded cabbage was inoculated with a mixture of spores (about 100-200/g) and 250 g samples of shredded cabbage were packaged in oxygen-impermeable bags under an atmosphere of CO, , 70%, N,, 30Oh, and stored at 22-2.5-C. Toxin was formed from type ‘1 spores, but not tqpe B spores, after 4-6 d, while the cabbagc was still organoleptically acceptable, as determined by appearance, odour and texture.

Although mushrooms are not strictly a vegetable it is worth noting in this context that they are liable to be contaminated with spores of Cl. botulinum. The decrease of oxygen concentration that can occur in film-wrapped packs, due to the respiration of the mushrooms, can result in conditions that allow growth of CI. botulinum and formation of toxin before spoilage is apparent (Hauschild 1989). For this reason, it has been advised that holes are made in the overwrap so that the oxygen concentration does not drop below about 6%. This was reported to prevent the production of botulinum toxin without reducing significantly the advantage of the packaging method. Malizio & Johnson (1991) evaluated the risk of toxin formation in enoki mushrooms inoculated with C1. botulinzlm spores and incubated at 6-27°C in vacuum packs. With levels of up to 1000 spores/ pack toxin was formed by proteolytic strains at 15-27”C, but the mushrooms were visibly spoiled at the time of toxin formation. Detailed results were not given for nonproteolytic strains. 5. REFERENCES

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Ecosystems in vegetable foods.

Journal of Applied Bacteriology Symposium Supplement 1992, 73, 115S126S Ecosystems in vegetable foods Barbara M. Lund AFRC Institute of Food Research...
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