Fate of Salmonella in Salpicon, a Mexican Cold Shredded Beef Salad

197

EDUARDO FERNANDEZ-ESCARTIN*1, JOSEFINA SALDANA-LOZANO, and OFELIA RODRIGUEZ-GARCIA

Laboratorio de Microbiologia Sanitaria, Facultad de Ciencias Quimicas, Universidad de Guadalajara, Guadalajara 44420, MexicoDownloaded from http://meridian.allenpress.com/jfp/article-pdf/56/3/197/1665152/0362-028x-56_3_197.pdf by guest on 24 September 2021

(Received for publication June 10, 1992)

ABSTRACT recognized as an important contributory factor in foodborne outbreaks of salmonellosis (4,17). The fate of Salmonella during the preparation and storage of Salmonella is highly sensitive to the inhibitory effect salpicon, a cold shredded beef salad commonly consumed in of acetic acid. Chung and Goepfert (7) reported that pH 5.4 Mexico, was evaluated. Salmonella contamination was introduced was the minimum pH at which salmonellae would initiate by a person shredding the cooked beef, who previously had growth in laboratory media, when acetic acid was used as handled raw pork containing several native serotypes of Salmo acidulant. A different behavior of salmonellae in a food nella at 9,000 salmonellae per g. The salad was prepared with 0, 2, or 4% vinegar and was held for 48 h (8 at 24-26°C and 40 at system is to be expected. Morever, survival time in a food 5-7°C). The optimal concentration of vinegar for acceptable salad as salpicon might be of concern, especially if we consider flavor was 4%. The initial number of salmonellae in salads the low infective dose, less than 100 cells that has been prepared with 0 or 4% vinegar was 20/g. Salmonella populations reported in some foodborne salmonellosis outbreaks (9,18). increased to 5,000 after 48 h. Salmonella did not grow in salad The objective of this study was to determine the fate of containing 4% vinegar (pH 5.3); however, the organism was Salmonella in salpicon prepared by a food handler who had isolated at levels of 7 cells per g at 48 h of incubation. Fourteen previously handled raw pork containing a known number of different serovars of Salmonella were isolated from the salads. Salmonella. Critical control points in the preparation and storage of salpicon are shredding the beef, the concentration of vinegar, and rapid MATERIALS AND METHODS cooling of salad before serving. Growth potential of Salmonella typhimurium in cooked and shred ded beef stored under conditions of temperature abuse Foodbome outbreaks in the United States are most Beef from the abdominal transverse muscle was obtained frequently associated with foods prepared in foodservice from a meat market, and 600 g was cut into 50- to 100-g portions. establishments and the home where mishandling occurs NaCl (15 g) and 1.5 L of water were added, and the beef was (5,6,14). Although data are not available to document the cooked at 110°C for 30 min. The meat was then shredded by an situation in Mexico, many traditional meals are prepared individual whose hands were previously sanitized with Iodophor and served in such a way that foods are frequently exposed solution containing 25 ppm free iodine. The meat was inoculated to contamination. Further, such foods are held and served with a 24-h culture of S. typhimurium (isolated from pork in our under conditions favorable for the survival and growth of laboratory) diluted in 0.1 % peptone water to provide approximately pathogens. 200 CFU/g of meat. The inoculum was incorporated into the meat by mixing for 3 min. The meat was divided into two portions and Salpicon is a popular cold beef salad of Mexico. To incubated at 20 or 30° + 1°C, respectively. These temperatures are prepare it, beef is cooked and then hand shredded. The beef common in kitchens of Guadalajara depending on season. Samples is mixed with minced tomato, sliced onion, minced avocado, were taken at 0, 4, and 8 h postinoculation, and Salmonella was cooked peas, salt, and vinegar is added. Once prepared, the enumerated. Salmonella counts were determined on each sample salad does not receive additional heat treatment and may be in duplicate by the direct pour plate method using bismuth sulfite kept at kitchen temperature for several hours before serv agar incubated at 35 °C for 48 h. All media used in this work were ing. Beef is sometimes mentioned as vehicle in outbreaks obtained from BIOXON, Mexico. At zero time, the count was of foodborne diseases (1). On the other hand, raw pork made in sextuplicate to calculate the coefficient of variation. There were no problems in counting Salmonella colonies because obtained from meat markets in Guadalajara usually shows other types of bacteria were practically absent. Colonies typical of levels of Salmonella contamination close to 100% (8). If raw Salmonella were selected (5-10 per plate) and were screened on pork is handled before preparing salpicon, contamination triple sugar iron and lipine iron agars and confirmed serologically may occur. This type of cross-contamination has been with Salmonella polyvalent antiserum.

^Facultad de Quimica, Universidad Autondma de Queretaro. Selection of raw pork used as a source of contamination Centro Universitario. Apartado postal 494, Queretaro 76010, Twenty-five 1.5-kg samples of raw pork were obtained from Mexico. markets in the city of Guadalajara and 100- to 200-g portions 198 FERNANDEZ-ESCARTIN, SALDANA-LOZANO AND RODRIGUEZ-GARCIA were assayed for Salmonella in order to select the one with the 20 g of salad with 180 ml of 0.1% peptone water. All selected highest concentration of the pathogen. This allowed us to expect dilutions were counted in plate count agar after incubation at 35 °C a good level of Salmonella in the salad. The pork samples were for 48 h. The pH was measured with a Conductronic No. 10 kept under freezing conditions until the selection was made. potentiometer in a 1:5 homogenized sample prepared with distilled Salmonella population was determined by a 3-tube most probable water. number (MPN) procedure (15) using five dilutions. The pork (20 g) was homogenized during 1 min with 180 ml of nutrient broth RESULTS AND DISCUSSION in a Stomacher (Tekman, Model 400). Tubes with preenrichment broth were incubated at 35°C for 24 h and transferred to brilliant Often studies on the fate of pathogenic microorganisms green tetrathionate broth and incubated at 43°C for 24 h. Colonies in foods are done with laboratory-cultured pathogens in were isolated on bismuth sulfite agar. Two typical colonies of oculated into the foods. This approach allows for control of Salmonella were selected from each plate (or one if it was the the number of cells in the inoculum, their distribution in the only one available) screened in triple sugar iron and lipine iron food, and the desired characteristics of the strain(s) used. agars and serotyped. Antisera were obtained from the Instituto Nacional de Diagnostico y Referenda Epidemiologica, Mexico. However, in using this approach, the behavior of the micro organisms does not necessarily reflect what happens under The selected sample contained 9,000 salmonellae per g of pork Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/3/197/1665152/0362-028x-56_3_197.pdf by guest on 24 September 2021 and did not show any abnormal organoleptic feature. natural conditions. Physiology and resistance to different ecologic factors tend to change when the microorganism is Fate of Salmonella during the storage of salpicon transferred from one medium to another. Also, in artificial Beef was cut and cooked according to the procedure de models, the test microorganism is not subjected to the same scribed above. The beef was then contaminated with Salmonella physical and chemical effects as the natural associated by a food handler who had previously handled Salmonella-con- flora. Although using natural contaminants in such studies taminated raw pork. This represents a common food-handling results in less control of the initial number of pathogens in practice in some restaurants and homes in Mexico. Contamination the food, the behavior of the pathogen more closely re was accomplished by cutting in 8-g portions the selected defrosted raw pork (500 g) which contained 9,000 salmonellae per g and sembles what naturally occurs. putting them into a plastic bag. Pork did not show abnormal S. typhymurium grew well in the shredded beef, with organoleptic features. The bag was closed and rubbed by hand for population increments of 1 log10/g at 20°C and near to 4 30 s to homogeneously distribute the Salmonella in the meat. The logl0/g at 30°C after 8 h of storage (Table 1). At zero time raw pork (200 g) was transferred into each of two plastic bags. A the coefficient of variation was 26.4%. Growth of Salmo food handler introduced her previously sanitized hands into one nella in cooked meat is an important contributory factor in bag and handled the meat for 30 s before shredding the cooked salmonellosis outbreaks associated with consumption of beef. The temperature of cooked beef was 40-45 °C at this time. beef (3,19). Hands were then thoroughly washed, disinfected with Iodophor solution (25 ppm), and next rinsed with water. The food handler The raw pork used to contaminate the beef contained handled the raw pork in the second bag using the procedure 9,000 salmonellae per g, including four different serovars. described above. Her hands were rinsed for 1 min in a bag These were Salmonella sandiego, Salmonella worthington, containing 200 ml of sterile nutrient broth. Salmonella in this Salmonella anatum, and Salmonella agona. In addition to suspension were enumerated by the most probable number method. Salmonella sandiego and Salmonella anatum, which were The food handler then prepared the salad by mixing 600 g of detected in both the raw pork and the hands of the food previously contaminated cooked beef, 200 g of minced tomatoes, handler, three other serovars, Salmonella montevideo, 75 g of sliced onions, 250 g of cooked peas, and 250 g of minced Salmonella meleagridis, and Salmonella derby, were avocados. Portions (20-g) of each vegetable ingredient except the isolated from the hands of the food handler. Presumably vinegar were assayed for Salmonella prior to mixing with the contaminated shredded beef. The plastic bags were not sterilized; these serovars also originated from the raw pork. It appears however, the number of microorganisms in 25 representative bags that selecting only two colonies per plate for serotyping was always less than 0.02/100 ml of 0.1% peptone water used to was not enough to provide complete information about all rinse each bag. The salad preparation was divided into approxi the serovars actually present. mately three equal portions, placed in sanitized glass containers, The hands of the food handler preparing the salpicon and covered with cellophane wrap. Commercial vinegar (4.86% contained around 50,000 salmonellae per both hands after acetic acid) was added to the salad at three levels: 0, 2, and 4% handling the raw pork. After handling beef and mixing all vol/wt. The most widely used recipe or salpicon uses around 4% the ingredients, the salmonellae content of 0 and 2% vin vinegar, although the other two concentrations of vinegar are egar salads was 20/g, whereas the figure obtained with 4% sometimes employed. The salad is served at ambient temperature and is not normally refrigerated before consumption. The time vinegar salad was >l,000/g. It is not easy to obtain a between preparation and serving may be up to 8 h. Leftovers uniform distribution of salmonellae in a heterogeneous usually are refrigerated if facilities exist. The three salad prepa food such as salpicon, a salad prepared with shredded beef rations were held at room temperature (24-26°C), and 20 g was sampled at 0, 4, and 8 h after preparation. They were then placed TABLE 1. Behavior of S. typhimurium in shredded cooked beef in a domestic refrigerator (5-7°C) and sampled after 16 and 40 h held at 20 or 30°C. of refrigerated storage. Time of storage No. Salmonella/g at: Microbiological and pH determinations (h) 20°C 30°C At each sampling time, salad preparations were assayed for Salmonella populations and pH. Salmonella was enumerated and 0 220 40 serotyped as indicated for raw pork. Aerobic plate counts were 4 500 1,400 determined only on salad samples with no vinegar by stomaching 8 2,500 180,000 SALMONELLA IN SALPICON 199 and vegetables. However, we consider that the observed lettuce. Although in Mexico now, the law prohibits the use differences in salmonellae counts at zero time of storage of sewage to irrigate crops, unsanitary practices still persists does not affect our conclusions regarding the behavior and does favor fecal contamination of vegetables. Vegetables tendencies to multiply or survive, as shown in Table 2. contaminated by night soil or raw sewage have been in Salmonella grew in salads made with 0 or 2% vinegar volved as vehicles of Salmonella infections (2). and did not grow but survived at low levels in salad made Up to 14 different serovars of Salmonella were de with 4% vinegar (Table 2). Growth of Salmonella occurred tected in the salads during the study. These included S. in the samples with the low concentrations of vinegar in sandiego, S. worthington, S. montevideo, S. agona, S. spite of the large populations of indigenous bacteria (106 to bredeney, S. poona, S. meleagridis, S. derby, S. panama, S. 108 CFU/g) present in the salads. Growth of Salmonella anatum, S. reading, S. give, S. schwarsengrund, and S. most likely occurred while the salads were cooling, i.e., infantis. It is likely that all of them originally came from before they reached final refrigeration temperature (5-7°C). the raw pork, since we did not find Salmonella in any of the The importance of rapid cooling of perishable foods has other ingredients added to the salads and the experimental been established (4,11,17). work at all times was made under aseptic conditions. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/3/197/1665152/0362-028x-56_3_197.pdf by guest on 24 September 2021 The pH 5.3 of the salad containing 4% vinegar was Although the hands of the food handler were not verified inhibitory and perhaps bactericidal to Salmonella. In for absence of salmonellae before handling raw pork, they products like mayonnaise and vinaigrettes, acetic acid in were washed and disinfected in a manner that would re hibits or even kills pathogenic bacteria (10,13,21). These move coliforms from artificially contaminated hands. effects depend on the concentration of the acid and pH of Cooked foods exposed to Salmonella contamination by the food (12). Mayonnaise pH (3.8-4.0) generally is low materials such as raw pork become especially hazardous enough to make it safe. However, if pH is higher, patho because a wide variety of serovars may be present; certain gens such as Salmonella may survive and salmonellosis serovars are more virulent than others (20). outbreaks might occur (16). Unlike mayonnaise, salpicon Raw pork was used to contaminate the beef utilized in pH is higher (5.34) and is consumed within a few hours this study. However, beef available at home may arrive after preparation; the low rate of bacterial inactivation that highly contaminated from the butcher shop. Pork and beef occurs in salpicon does not permit a confident control of are subjected to cross-contamination in local butcher shops the risk associated. Further, the risk of Salmonella multi as suggested by a recent survey in which 65 and 87% of plication in salpicon should be considered, at least in beef and pork samples, respectively (8), were contami specific locations of beef where acid penetration might be nated. Generally, beef is shredded after cooking as soon as slow and even partially neutralized by the buffer activity of its temperature (approximately 50°C) permits; however, the proteins. Another potential source of Salmonella contami shredded beef may be held for several hours at room nation to salpicon would be those vegetables used in its temperature before it is used for preparing salad. If a large preparation. In fact, some salpicon recipes even use minced amount of salad is to be prepared, beef should be cooked the previous day, then rapidly cooled, and stored overnight TABLE 2. Effect of vinegar on behavior of Salmonella in salpicon under refrigeration. It can be shredded the following day to during storage at room temperature followed by refrigeration. minimize the risk of cross-contamination. This study has revealed that salmonellae can multiply Time of Determination % vinegar in salpicon in shredded beef or salpicon, when less than 4 ml of sampling 0 2 4 vinegar is added per 100 g of salad. To prepare and serve (h) salpicon in a safe way, it is necessary to prevent the Storage at room temperature (24-26°C) contamination of cooked meat from raw meat (especially at the time of shredding), to add a minimum concentration of Salmonella/% 20 20 >1,100 vinegar of 4%, mixing the vinegar uniformly into the salad, APC 6.28 rapidly cool the salad once prepared, and maintain the salad pH 5.73 5.55 5.34 at <6°C until served. As other perishable meals (11,17), the Salmonella 50 500 23 salad should not be held unrefrigerated for more than 3 h. APC 8.26 Hygienically shredding the beef, the concentration of vin pH 6.01 5.62 5.39 egar added, and proper refrigeration, all of which can be Salmonella 200 40 23 APC 7.66 monitored, are critical control points for salpicon. pH 6.00 5.55 5.30 ACKNOWLEDGMENT Storage under refrigeration (5-7°C) We greatly appreciate the suggestions made by Dr. Michael P. 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