EFFECT OF THERMOPHILIC LACTIC ACID BACTERIA ON THE FATE OF ENTEROBACTER SAKAZAKII DURING PROCESSING AND STORAGE OF PLAIN YOGURT

REYAD R. SHAKER, TAREQ M. OSAILI1 and MUTAMED AYYASH Department of Nutrition and Food Technology Faculty of Agriculture Jordan University of Science and Technology PO Box (3030) Irbid, 22110, Jordan
Accepted for Publication April 30, 2007

ABSTRACT Survival and growth of Enterobacter sakazakii during processing and storage of plain yogurt were investigated. Preheated rehydrated milk was inoculated with a cocktail culture of E. sakazakii (103 cfu/mL of milk) and/or with thermophilic yogurt starter culture of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarius ssp. thermophilus. The inoculated milk was incubated at 40C for 5 h, then the samples were cooled and subsequently stored at 4C for up to 7 days. The results showed that E. sakazakii grew at an early stage of fermentation but declined at the end of the process. There was no signicant difference between the populations of E. sakazakii in the presence or absence of lactic acid bacteria during the rst 4 h of the incubation period but there was signicant difference during the last hour of the incubation period. The populations of E. sakazakii decreased signicantly during cooling and storage of yogurt (pH 4.2-4.7) compared with nonfermented milk samples at 4C. The presence of E. sakazakii did not have a signicant effect on the growth of LAB during fermentation and storage of yogurt. The results obtained from this study indicate that the pH of yogurt and storage temperature were critical to the survival and growth of E. sakazakii in the manufacture of plain yogurt.

PRACTICAL APPLICATIONS Enterobacter sakazakii prevalence in milk products and the production environment has been documented. The results obtained from this study may
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Corresponding author: TEL: +962-02-7201000; FAX: +962-02-7095069; EMAIL: tosaili@ just.edu.jo Journal of Food Safety 28 (2008) 170182. All Rights Reserved. 2008, The Author(s) Journal compilation 2008, Blackwell Publishing

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be of use to dairy producers to manufacture safe products using thermophilic lactic acid bacteria. These bacteria decreased the pH of milk in less than 5 h, resulting in E. sakazakii reduction. pH of yogurt during the fermentation process is a critical control point that should be monitored to produce safe products.

INTRODUCTION Enterobacter sakazakii is a gram-negative motile rod-shaped nonspore forming bacterium belonging to the Enterobacteriaceae family. It is an emerging foodborne pathogen that has been associated with infant meningitis, necrotizing enterocolitis and death among neonates, elderly and immunocompromised adults (Chow et al. 1991; Burwen et al. 1994; Bar-Oz et al. 2001; Van Acker et al. 2001; Iversen and Forsythe 2003; Lehner and Stephan 2004; Forsythe 2005; Kim and Beuchat 2005; Lehner et al. 2005). The International Commission for Microbiological Specication for Foods (ICMSF 2002) has ranked E. sakazakii as a severe hazard for restricted populations, life threatening or substantial chronic sequelae or long duration. E. sakazakii is an ubiquitous organism that has been frequently isolated from different types of food, food manufacturing plants and the environment (Neelam et al. 1987; Cottyn et al. 2001; Gassem 2002; Leclercq et al. 2002; Iversen and Forsythe 2004; Kandhai et al. 2004; Shaker et al. 2006). While E. sakazakii has not been reported to cause a health hazard linked to the consumption of dairy products, the pathogen has been isolated from milk with a long shelf life, powdered milk, cheese products and from the environment in dairy plants (Postupa and Aldova 1984; Muytjens et al. 1988; Skladal et al. 1993; Leclercq et al. 2002; Iversen and Forsythe 2004; Restaino et al. 2006). Therefore, E. sakazakii should be considered as a possible contaminant of milk and the contaminated milk products may represent a potential risk of causing foodborne illnesses in humans. Yogurt is a fermented milk product usually made from cows milk, although milk from other sources, especially powdered milk, can be used for its preparation (Rosenthal et al. 1980; Salji 1991). Yogurt has historically formed a part of the dietary pattern of the Middle Eastern countries. It is a complex ecosystem and a continuous ux in terms of both fermentation conditions and intrinsic factors (Tamime and Robinson 1988). The wide range of biochemical reactions and microbial interactions that take place during milk fermentation presents a concern for keeping the quality of the product high and the safety of the product enhanced through spoilage prevention and inhibition of foodborne pathogens (Presser et al. 1997; Pitt et al. 2000).

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However, yogurt has been linked to a fatal infection caused by a member of the Enterobacteriaceae family, Escherichia coli O157:H7 (Morgan et al. 1993). Survival of pathogenic microorganisms such as E. coli O157:H7, Yersinia enterocolitica and Listeria monocytogenes in yogurt has been reported (Ahmed et al. 1986; Schaack and Marth 1988; Gorden and Small 1993; Aytac and Ozbas 1994; Leyer et al. 1995; Massa et al. 1997; McIngvale et al. 2000; Ogwaro et al. 2002). It is known that survival and growth of foodborne pathogens in fermented foods are inuenced by the ability of microbes to tolerate acidic conditions. E. sakazakii is classied as a moderately acid resistant member of the Enterobacteriaceae; it can withstand exposure to pH 3.5 for >5 h (Edelson-Mammel et al. 2006). Kim and Beuchat (2005) observed that E. sakazakii grew in tomato juice (pH 4.4), watermelon juice (pH 5.1), and cantaloupe juice (pH 6.8), but did not grow in apple juice (pH 4.0) and strawberry juice (pH 3.5) stored at 25C. Because of the possibility that rehydrated milk used for yogurt manufacture could harbor E. sakazakii and the lack of information on the behavior of E. sakazakii in fermented food products such as yogurt, the objective of this study was to investigate the effect of thermophilic lactic acid bacteria (LAB) on the survival and growth of E. sakazakii during plain yogurt processing and storage. MATERIALS AND METHODS Culture Preparation Unless stated otherwise, all growth media and components were obtained from Oxoid (U.K.). Five strains of E. sakazakii one strain American Type Culture Collection (ATCC) 51239, two isolates from dried infant milk, one isolate from dried infant cereal and one isolate from crushed wheat were used in this study. All E. sakazakii isolates were kept individually at -40C in brain heart infusion (BHI) plus 20% of glycerol. To prepare the inocula for the experiments, a loop from each isolate was grown individually in BHI at 37C for 24 h. Three culture transfers were performed to activate each culture. Just before the inoculation of milk, equal volumes of each fresh E. sakazakii culture were combined to form a cocktail culture of E. sakazakii (109 cfu/mL). After that, the cocktail was serially diluted in peptone water (0.1%) (Becton Dickinson, Sparks Glencoe, MD) to decrease the initial level of E. sakazakii to 106 cfu/mL. Yogurt starter culture CH1, direct vat set, containing thermophilic LAB Streptococcus salivarius ssp. thermophilus and Lactobacillus delbrueki ssp.

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bulgaricus was used in this study (CHR Hansen, Inc., Hrsholm, Denmark). The starter came in freeze-dried form and was kept at -18C until use. Yogurt Manufacture Full cream milk powder (Nido, Nestle, Sydney, Australia) used in the study was analyzed and found free of E. sakazakii. Rehydrated milk was prepared by dissolving 130 g of milk powder in 1,000 mL of water using a magnetic stirrer at 37C for 5 min. The milk was batch pasteurized at 90C for 3 min then cooled immediately in a water bath to 40C and tempered at that temperature for 1 h. After that, 1 mL of E. sakazakii cocktail culture and subsequently, 50 mg of freeze-dried yogurt starter culture were added to the milk and stirred for 2 min. Afterward, 30-g samples of the inoculated milk were poured into 100-mL capacity sterile plastic containers and incubated at a temperature of 40C for 5 h (pH approximately 4.6 0.1). The fermented milk (yogurt) was cooled to 4C then stored at the same temperature for 7 days. The milk that was not inoculated with E. sakazakii or LAB was used as controls. The total plate count of the control samples was <10 cfu/mL. Sampling Two samples of the 30 g milk or yogurt in sterile containers were taken randomly every 30 min during incubation, every 1 h during cooling and every 24 h during storage. The samples were used for pH measurements and E. sakazakii and LAB enumerations. Measurement of pH The pH values of milk or yogurt samples were measured by immersing the pH electrode directly in the sample in the sterile container using a pH meter (Cyberscan 500, Eutech instruments, Ayer Rajah Crescent, Singapore). Bacterial Enumeration A 0.1-mL aliquot of milk or yogurt samples and their appropriate serial dilutions (0.1% peptone water) were plated in duplicate on violet red bile salt glucose agar for E. sakazakii enumeration or mixed with de Man, Rogosa & Sharpe (MRS) agar for LAB enumeration. The plates were incubated aerobically at 37C for 24 to 48 h for E. sakazakii or anaerobically (AnaeroGen, Oxoid Ltd., Basingstoke, U.K.) at 37C for 48 to 72 h for LAB. Statistical Analysis All experiments in this study were repeated at least twice and the results are reported as average. Students t-test was used to determine if there is a signicant difference between samples (SAS 8.1, SAS Institute Inc., Cary, NC).

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RESULTS Figure 1 shows the changes in E. sakazakii populations and pH during incubation of milk in the absence of starter culture or during lactic fermentation at 40C, cooling and subsequent storage at 4C. E. sakazakii showed a similar growth pattern in the rst 4 h of the 5-hour incubation period regardless of the presence of LAB. During this part of incubation, the populations of E. sakazakii in the presence of LAB were higher, but not signicantly, than those
A
6

* *

Log10 E. sakazakii (cfu/ml)

5 4 3 2 1 0 0 1 2 3 4 5 6 7 8

24

48

72

96

120

144

168

B
7 6.5 6 5.5

* * * * *

pH

5 4.5 4 3.5 3 0 1 2 3 4 5 6 7 8

24

48

72

96

120

144

168

Incubation and cooling

Storage

Time (h)
FIG. 1. CHANGES IN (A) ENTEROBACTER SAKAZAKII POPULATIONS AND (B) PH DURING INCUBATION, COOLING AND STORAGE OF MILK IN THE PRESENCE () OR ABSENCE () OF LAB Point of cooling. * Values are signicantly different (P < 0.05).

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Log10 LAB (cfu/ml)

8 7 6 5 4 3 0 1 2 3 4 5 6 7 8

24

48

72

96

120

144

168

Incubation and cooling

Storage

Time (h)
FIG. 2. CHANGES IN LAB POPULATIONS DURING FERMENTATION PROCESS, COOLING AND STORAGE IN THE PRESENCE () OR ABSENCE () OF ENTEROBACTER SAKAZAKII Point of cooling. * Values are signicantly different (P < 0.05).

in the absence of LAB. The pH of milk containing only E. sakazakii did not change during this period (pH 6.3), but as expected, the pH of milk containing E. sakazakii and LAB decreased gradually and signicantly compared with that of milk with E. sakazakii and reached 5.1. The pH of milk containing E. sakazakii and LAB continued decreasing and reached 4.6 at the end of the incubation period. This decrease was synchronized with a signicant decrease in the populations of E. sakazakii compared with that in samples without LAB. In the last hour of incubation, the populations of E. sakazakii in the fermented milk samples decreased 1.3 log (4.37 to 3.08) and increased 0.5 log (4.88 to 5.40) in nonfermented milk samples. During cooling, the count of E. sakazakii in yogurt samples was reduced 0.9 log (3.1 to 2.2) and the pH of the samples was reduced 0.2 unit (4.6 to 4.4). A slight reduction of 0.1 log in E. sakazakii cells occurred in milk samples without LAB and the pH of the milk was maintained at 6.3. Signicant differences in E. sakazakii populations and pH between milk and yogurt samples were observed. Storage of milk and yogurt samples at 4C over a 7-day period showed a similar degree of E. sakazakii reduction with slight changes in pH. However, during the entire storage period, signicant differences were observed between milk and yogurt samples in terms of E. sakazakii populations and pH. Figure 2 shows the changes in LAB populations during incubation of milk, cooling and subsequent storage in the presence or absence of E. saka-

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zakii. Generally, during incubation, cooling and storage of yogurt, the populations of LAB with or without E. sakazakii showed similar behavior without any signicant differences.

DISCUSSION The behavior of E. sakazakii in fermented food products has not been studied. Yet, growth of other members of the Enterobacteriaceae in these products has been investigated extensively (Alm 1983; Ahmed et al. 1986; Nunez et al. 1986; Farrag 1992; Massa et al. 1997; Chang et al. 2000; Govaris et al. 2002; Ogwaro et al. 2002; Gulmez and Guven 2003). Ogwaro et al. (2002) studied the survival of E. coli O157:H7 in traditional African yogurt and reported that the populations increased 34 log during fermentation of milk at temperatures of 25, 30 and 37C for 24 h and decreased at 43C. The growth and survival pattern of E. sakazakii in this study was similar to that observed by Chang et al. (2000) for E. coli O157:H7 during fermentation of skim milk. They found that the organism grew rapidly during fermentation and reached the stationary phase after 1224 h of cultivation at 37C (pH 5.65.8) and declined after 36 h (pH < 5.0). The decline of a viable population of E. coli O157:H7 after 36 h of fermentation was justied by the low pH effect or by production of inhibitory substances by LAB. Also, the results of the present study are, in general, in agreement with the data of Massa et al. (1997), who studied the survival of E. coli O157:H7 during fermentation of yogurt with S. thermophilus and L. bulgaricus. They found that the populations of the pathogen decreased from 3.5 to 3.0 log at the end of the fermentation process of milk (pH 5.1). Like the present ndings, Gulmez and Guven (2003) found that the populations of Yersinia enterocolitica increased during the rst 3 h of the fermentation of yogurt at 43C but decreased at the end of the 5-h process (pH 4.5 1). However, it is difcult to compare the results of the present study and those mentioned earlier because of differences in fermentation temperature and type of fermentative and pathogenic microorganisms. E. sakazakii in this study grew in an ever increasing acidic environment, and it is known that the exposure of microorganisms to moderate acidic environment enhances their tolerance to acid (Schaack and Marth 1988; Gahan et al. 1996); however, Kim and Beuchat 2005 observed that E. sakazakii grew in lower pH values than those reported in this study. They reported that E. sakazakii grew in watermelon juice (pH 5.1) and tomato juice (pH 4.4). Osaili et al. (unpublished work) found that E. sakazakii cells increased 0.1 log but decreased about 0.15 and 0.2 log in BHI broth adjusted to pH 5.5, 5 and 4.5, respectively, with lactic acid during incubation at 37C for 4 h. The differences in the results of the present study and those reported in previous studies

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indicated that factors other than organic acids and pH may affect the growth of E. sakazakii in yogurt. The presence of LAB in yogurt may interfere with E. sakazakii. This interference, which includes the competition of LAB with E. sakazakii for nutrients, might contribute to the decline in the populations of E. sakazakii (Jay et al. 2005). Thus, it may be hypothesized that E. sakazakii used in this study has grown easily in milk during the early stages of fermentation when organic acids and LAB levels are low. When E. sakazakii was grown as monoculture in milk, the growth during incubation was substantial and populations increased 2.4 log. The substantial increase of cells is expected since E. sakazakii was incubated at its optimum temperature (40C). Iversen et al. (2004) reported the optimum growth temperature of E. sakazakii in infant milk formula was between 37 and 43C. During cooling and storage at 4C, the temperature of the samples was shifted away from the optimum growth temperature of E. sakazakii. E. sakazakii growth stopped during cooling and the populations gradually decreased during storage of milk or yogurt at 4C. It has been reported that E. sakazakii did not grow but either survived or died during storage at 4C. Kim and Beuchat (2005) observed that E. sakazakii populations decreased during storage for 7 days at 4C in cantaloupe, watermelon, carrot, cucumber, lettuce, and tomato, apple, strawberry and cabbage juices. Osaili et al. (2008) found that E. sakazakii did not grow in infant milk formula during storage at 4C for up to 8 h. Nazarowec-White and Farber (1997) reported that the minimum growth temperature of E. sakazakii in infant milk formula was 5.5C and the microbe either survived or died at a refrigeration temperature of 4C. Iversen et al. (2004) reported that 6C was the minimum growth temperature of E. sakazakii. The behavior of other members of Enterobacteriaceae during storage of yogurt at 4C has been reported. It has been stated that E. coli O157:H7 recovered in yogurt after storage for 717 days at 4C (Hudson et al. 1997; Massa et al. 1997; Dineen et al. 1998; Govaris et al. 2002). Nunez et al. (1986) stated that Enterobacter cloacae in Burgos cheese manufactured with 1% starter lactic culture decreased during storage at 4, 7, 10, 15 and 20C for 5 and 7 days (pH 4.44.7). Ogwaro et al. (2002) reported that E. coli O157:H7 populations in traditional African yogurt declined 12 log over 4 days of storage at 4C. LAB during fermentation of milk decreased slightly with the presence of E. sakazakii compared with LAB populations in the absence of E. sakazakii. This nding could be explained by the microbial interference phenomenon. McNaught and Mace (2001) and Jay et al. (2005) explained this phenomenon as the inhibition of one microbe by another through competition for nutrients. In conclusion, this study has shown that E. sakazakii can grow at early stages of fermentation during yogurt manufacture but die at later stages of

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fermentation. pH of yogurt during fermentation process is a critical control point that should be monitored to produce a safe product. Storage of yogurt at low temperatures is necessary to inhibit the growth of E. sakazakii. The survivors of E. sakazakii from fermentation steadily decreased during storage of yogurt at 4C.

ACKNOWLEDGMENTS This work was supported by the Deanship of Scientic Research at Jordan University of Science and Technology. The authors thank Dr. Elizabeth Martin at the University of Arkansas, U.S.A. for reviewing the manuscript.

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