African Journal of Microbiology Research Vol. 6(36), pp. 6558-6564, 20 September, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR12.

757 ISSN 1996-0808 2012 Academic Journals

Full Length Research Paper

Survival of microorganisms in high pressure treated minced meat during chilled storage and at pH and temperature mimicking gastrointestinal tract
Sami Bulut
Food Engineering Department, Faculty of Engineering and Architecture, Trakya University, Edirne, Turkey. E-mail: samibulut@trakya.edu.tr. Tel: +90 284 226 1217. Fax: +90 284 226 1225.
Accepted 6 September, 2012

Minced meat samples containing 8.3 10 cfu/g determined by total aerobic count (TAC) were subjected to pressures between 259 to 540 MPa for 3 to 17 min at 6 A day after pressure treatment, no surviva l C. on Plate Count Agar (PCA) was detected in the samples that were treated at and above 300 MPa for 10 min or more after 24 h incubation. However, survivors were detected in the pressure treated samples, when the incubation period was extended to 72 h, indicating repair of injured cells. Compared to control samples, number of surviving microorganisms in pressure treated samples increased significantly after 21 days of storage at 3 thus implicating repair of injured cells was possible at refrigeration C, temperatures. Experiments designed to mimic the temperature and acidity in stomach and lower gastrointestinal tract revealed that microorganisms not inactivated by pressure treatment could not recover or show viability at the pH of the stomach (3.0), but recovery and growth could potentially occur at pH of the lower gastrointestinal tract (6.5). The information could be valuable for the food industry and could imply more stringent controls for increasing the shelf life of foods by high pressure processing (HPP). Key words: High hydrostatic pressure, sublethal injury, chilled storage, gastrointestinal tract, injury recovery, bacterial spores.

INTRODUCTION High hydrostatic pressure (HHP) has proven to be efficient for destruction of vegetative bacteria, viruses, and yeasts, with bacterial spores being more resistant to pressure (Nakayama et al., 1996). HHP can inactivate microorganisms at room temperature or in combination with a low heat treatment while maintaining the main nutritional and organoleptic properties of foods (Knorr, 1993) and extend shelf-life. Research on the effect of high pressure on food was carried out already in the nineteenth century by Hite (1899) who described an increase in shelf-life for products such as milk, fruit and other foods (Rendueles et al., 2011). Intensive research in this field has taken place in the last three decades and as a result, a range of pressure treated food products such as fruit preparations, fruit juices, sauces, rice cakes, sliced cooked meat products, raw squid, guacamole and oysters are now on the market in Japan, USA and Europe. However, due to concerns on safety of utilizing this process for pasteurization or sterilization of food products, its commercialization is rather slow. This is due to the fact that the resistance of microorganisms to HHP is variable depending on many intrinsic and environmental factors. The same microorganism can give different response to HHP in different media. Variation in pressure sensitivity of microorganisms is common for different strains of a microorganism (Patterson and Kilpatrick, 1995). Resistance of microorganisms can be increased when grown or pressure treated in nutritionally rich media containing substances which may provide protection against damage or nutrients essential for repair (Hoover et al., 1989). Higher reductions in the number of Salmonella senftenberg was obtained in phosphate buffer compared to pressure treatment of S. senftenberg in ground chicken (Metrick et al., 1989). The pH of the food or media is another factor influencing the effectiveness of HHP induced inactivation. A reduction in the pH of the media could increase the pressure induced inactivation of

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vegetative microorganisms (Koseki and Yamamoto, 2006). Spore populations of Clostridium sporogenes were reduced by 2.5 log when exposed to 404 MPa at 25C, pH 4.0 for 30 min, but the same treatment at pH 7.0 resulted in a less than 0.5 log reduction in spore counts (Stewart et al., 2000). Other studies reported that inactivation of spores of Bacillus subtilis was maximally 80% when the spore suspensions were subjected to pressure treatments at 100 and 600 MPa at 40C over a pH range from 3 to 8 and was not increased at low pH (Wuytack and Michiels, 2001). Cells in exponential growth phase are more sensitive to pressure than those in stationary phase (Saucedo-Reyes et al., 2009). Water activity (aw) of the food is also an important parameter determining the effectiveness of HPP. Goodridge et al. (2006) demonstrated that inactivation of Salmonella enteriditis in almonds increased when almonds were suspended in water and then pressurised. They suggested that the increased aw at the surface of the almond allowed the pressure to impart a more destructive effect on the bacteria. The equipment, pressurization regime and the pressurizing media can also affect the efficiency of microbial destruction during HHP application (Simpson and Gilmour, 1997). HHP could increase the shelf life of foods through inactivating and/or injuring the microorganisms present in food. At standard pressures used in food processing, spores are not inactivated and a pasteurized product is obtained, in which surviving spores have no competitors. In these conditions, the risks posed by pathogenic spores should not be underestimated (Rendueles et al., 2011). After pressure treatments at 100 to 600 MPa, microorganisms could not grow on agar, but still show metabolic activity. This fraction is often described as viable but not culturable cells (Ananta et al., 2004). The presence of spores and non culturable bacteria in food after pressure treatment might be critical in terms of their potential for excreting toxic or food spoiling metabolites. Several studies have reported recovery of HPP treated pathogenic and spoilage bacteria in nutrient broth (Chen and Hoover, 2003; Erkmen and Dogan, 2004), milk (Bozoglu et al., 2004; Bull et al., 2005; De LamoCastellv et al., 2005), phosphate buffer (Koseki and Yamamoto, 2006) and sliced cooked ham (Aymerich et al., 2005; Koseki et al., 2007) by storage between 6 h and 4 weeks at various temperatures. To the best of our knowledge, there is no information on how HPP treated microorganisms, not able to repair themselves and grow at refrigeration temperatures (in a short time), would function in the human gastrointestinal (GI) tract, when ingested with food. An assumption is that, if the microorganisms can endure the low acidity of the stomach without irrecoverable injury or death, they can repair themselves and grow in the nutrient rich environment of the lower GI tract at an optimum temperature (body) and not as low acidity as in the stomach.

In the fast state, intragastric pH in healthy subjects is in the range of 1.3 to 2.5. Eating increases pH to a range of 4.5 to 5.8. About an hour after eating, the pH of the stomach decreases to less than 3.1 (Malagelada et al., 1976; Dressman, 1986; Kong and Singh, 2008). It has been suggested that for bioavailability studies using in vitro gastrointestinal extraction methods, the pH of the stomach should be set in the range of 1 to 4 and the average residence time should be taken as 3 h. Similarly, to simulate small intestine conditions, the samples should be subjected to a pH of 4 to 7.5 for approximately 7 h (Dean and Ma, 2007). The purpose of this study was to investigate the effect of pressure and pressurization time on survival of the natural microbial population in minced meat during 21 days of storage at 3C. Survival and growth of microorganisms from pressure treated samples at conditions of stomach and gastrointestinal tracts acidity and temperature were assessed.
MATERIALS AND METHODS Preparation of minced meat samples for pressure treatment Minced meat obtained from a local market (Kipa, Edirne, Turkey) was mixed by using a food processor (Siemens, Slovenia) in order to homogenize the samples. Using a vacuum packing machine (MV-20, Lipovak, Gebze, Turkey), homogenized samples (10 g) were vacuum packed in sterile plastic films made of Polyamide/Polyethylene (Saran Plastik, Dzce, Turkey) impermeable to air and water. The packed samples were vacuum packed twice to prevent contamination of the pressurization medium in case of rupture during pressurization. Samples were prepared in triplicate for each experimental condition and for untreated control and kept at 3 1 overnight befo re C pressurization. After pressurization, samples were refrigerated at 3 1 until testing on days 1 and 21. C High pressure treatment A 2 L processing unit (Avure Technologies Incorporated, Kent WA, USA) was used to pressurize the samples. Water was used as the pressure medium. Temperature of the pressure transmitting medium inside the pressure chamber was controlled with a cooling jacket surrounding the pressure vessel. Iced water was circulated in the jackets surrounding the pressure chamber to keep the pressure medium (water) in the chamber at about 6 2C. The pressure was increased to the test pressure at a rate of 17 MPa/s and decompression time was less than 5 s. The unit was controlled by a computer program. In order to monitor the temperatures, 2 K-type thermocouple was silver-soldered through the center of the top closure of the pressure chamber and was positioned close to the sample to monitor temperature during pressure treatments.

Experiments to mimic the gastrointestinal tracts pH and temperature In the present study, it was assumed that the average pH of stomach was 3.0 and the length of time the meat samples spent in stomach was 3 h. We assumed that the pH in lower GI tract (small and large intestines) was about 6.5. In order to imitate the worst

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Table 1. Experiemntal parameters for HPP and survival of microorganisms in minced meat after 1 and 21 days chilled storage at 3C. Results are from average of 3 counts and the standard deviations (StDev) are derived from these 3 counts for each experiment.

HPP parameters Pressure (MPa) Control 259 300 300 400 400 400 400 400 500 500 541
ND Not detected.

Time (min) 10 10 5 10 10 10 3 17 5 15 10

TAC during chilled storage Day 1 Day 21 Log cfu/g StDev Log cfu/g StDev 6.92 0.11 7.94 0.13 4.62 0.08 8.15 0.08 2.42 0.09 8.07 0.07 3.72 0.12 8.12 0.06 2.12 0.10 6.10 0.03 2.18 0.08 5.90 0.11 1.92 0.07 5.60 0.12 3.62 0.12 6.80 0.13 1.72 0.07 2.00 0.09 2.02 0.03 3.26 0.06 ND 3.45 0.09 ND 2.40 0.15

case scenario, the time meat samples spent in lower GI tract was taken as 10 h instead of 7 h as suggested by Dean and Ma (2004). Based on these assumptions, the experimental conditions for testing the effect of pH and temperature on microorganism in the pressure treated samples were as following:

Experiment designed to imitate the stomach and Lower GI tracts acidity were carried out under aseptic conditions. For each set of experiments, a control sample was tested at the conditions of stomach and lower GI tract. No contamination was found in any of the control samples (minimum detection level 50 cfu/g).

Experimental set up for stomachs pH and temperature Samples of pressure treated meat (ca. 10 g) was opened with a sterile knife and emptied into a sterile stomacher bag. About 80 g of a sterile buffer solution at pH 2.6 prepared by adding 57.8 ml of 0.1 M HCl (Merck, Germany) solution into a 1000 ml of 0.1 M Potassium Hydrogen Phthalate (Merck, Germany) and homogenized by a Seward Stomacher 400 Circulator (UK) at 150 rpm for 2 min. Finally, the homogenized samples were placed into a Sanyo MIR-154 Cooled Incubator (Japan) operating at 37C. During the 3 h of incubation period, the stomacher bags were removed from the incubator and stomached at 150 rpm for 1 min every half an hour and then placed back into the incubator.

Enumeration of microorganisms Control and pressure treated samples (10 g) were mixed with 90 ml of PBS pH 7.0 (Oxoid, Basingstoke, UK) and homogenized in the stomacher at 200 rpm for 1 min. A 1 ml sample of suspension from the stomacher bag was taken into a sterile 1.5 ml Eppendorf tube and serial dilutions 10 were made using PBS (pH 7). Surviving cells were enumerated in triplicate on PCA (Biolab, Izmir, Turkey). The plates were incubated at 37 for 24 to 72 h bef ore counting C the colonies. The averages of three counts from each dilution were used for calculation of surviving cells. Microbial reduction was expressed in terms of logarithmic reduction corresponding to the logarithmic difference between the initial number of microorganism before pressure treatment and the number of microorganisms surviving pressure treatment and storage.

Experimental set up for Lower GI tracts pH and temperature After holding for 3 h at the pH and temperature of the stomach as explained earlier, the stomacher bags were removed from incubator, a 1-ml sample aliquot was taken for microbial analysis. Then, 10 g of Tryptic Soy Broth (Biolab, Izmir, Turkey) was added to stomacher bag to provide extra nutrient in order to make the system more realistic. This is because the lower GI tract could always hold semi digested nutrients which would be available to microorganisms. Finally, the pH of the mix was adjusted to 6.5 by predetermined amount of sterile 2.5 M NaOH solution. After homogenizing the mix at 150 rpm for 2 min, the stomacher bags were placed into the incubator and kept at 37C in s tatic condition for 10 h. The stomacher bags were then taken from incubator and homogenized before taking a 1-ml sample of aliquot for microbial analysis.

Statistical analysis The effect of two main variables; pressure from 200 to 500 MPa, holding time from 5 to 15 min and their interactions on the inactivation of microorganisms were designed and studied by the surface response methodology employing Design-Expert v8 Software (Stat-Ease, Inc. Minneapolis, USA) using central composite design. Assessment of error was derived from three times repetition of the centre point of experimental design, which was set as 400 MPa and 10 min. The parameters of the experimental design are shown in Table 1. In order to understand the effect of pressure, pressurization time and their interactions on inactivation of microorganisms, statistical

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7.0 6.0 5.0 4.0 3.0 2.0

Log Reduction (cfu/g)

15 13 11 9 400 7 5 300 350 450

500

Time (min)

Pressure (MPa)

Figure 1. Reduction in the number of microorganisms in HHP treated minced meat samples after 1 day of storage at 3 as a function of press ure and time. Response surface C constructed based on counts obtained on PCA after 72 h of incubation at 37 C.

analyses was conducted on survival data obtained from samples a day after the pressure treatment and incubated for 72 h at 37 By C. using a linear model, numbers of surviving cells determined by TAC a day after HHP treatment were statistically analyzed by DesignExpert software v8. Level of significance was set for P < 0.05 and the significance of each response variable was assessed by F-test statistical analysis.

RESULTS AND DISCUSSION Microbial load of minced meat samples The average number of microorganisms in untreated 6 control samples was 8.3 10 cfu/g and increased to 8.7 7 10 cfu/g after 21 days of storage at 3C.

Effect of pressure and pressurization time on survival of microorganisms A linear model was fitted to data and analysis of variance (ANOVA) showed that pressure (P = 0.0116) and time (P = 0.0444) were significant parameters. The model Pvalue (0.0166) and R-squared value (0.7448) showed that the model was representing the data well. Response surface model produced the following equation for prediction of log reduction in the pressure treated samples a day after pressure treatment: Log reduction (cfu/g) = 4.63 + 1.03A + 0.64B Where, A is pressure in MPa and B is temperature in C.

Survival of microorganisms was reduced by increased pressure at all pressurization times tested. In general, the positive effect of pressure and pressurization time on inactivation of vegetative cells is well documented in the literature (Kalchayanand et al., 1998; Ritz et al., 2000; Dogan and Erkmen, 2004). Increased pressure resulted in higher reduction in number of survivals a day after pressure treatment. There was no growth on PCA after 24 h of incubation at 37 at pressures above 300 M Pa C regardless of the pressurization time, which corresponds to a minimum of 5.2 log reduction based on a 50 cfu/g minimum detection level. However, extending incubation time of PCAs to 72 h resulted in at least 1 colony count on all the samples, except treatments at 500 MPa for 15 min and 541 MPa for 10 min. Response surface showing log reductions in the samples a day after pressure treatment (Figure 1) was constructed based on the counts obtained on PCA after 72 h of incubation at 37 C, as it was not possible to obtain a response surface by only a few survival data after 24 h of incubation at 37 C. It could be noted from Figure 1 that the increased pressurizing time increases log reduction which is in line with the literature (Chen and Hoover, 2003; Gao and Jiang, 2005; Bover-Cid et al., 2012). Change in total aerobic counts during chilled storage In pressure treated samples, the number of surviving microorganisms significantly increased during the storage at 3 After 21 days at 3C, survival of microorga nism in C. samples that were pressure treated at 300 MPa or less

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were not statistically different than untreated control samples (P = 0.502), averaging to 8.04 log unit with a standard deviation of 0.092. Survival of microorganisms detected in the samples that were pressure treated at 300 MPa or less were increased by an average of 4.6 log unit after 21 days of storage at 3 whereas, the n umber C of microorganisms in control samples increased by an average of 1.0 log unit, which suggests that the increase in number of microorganisms in pressure treated minced meat samples were mainly due to the recovery of injured cells during storage at 3C. The extent of recovery was higher at moderate pressures up to 300 MPa whereas, the recovery of microorganisms were significantly delayed at samples treated at higher pressures as seen in Table 1. A day after the pressure treatment, no survival of microorganisms was detected in samples that were treated at 500 MPa for 15 min and 541 MPa for 10 min. However, on day 21, the number of microorganisms detected in the samples that were treated at 500 MPa 15 3 2 min and 541 MPa 10 min, were 2.8 10 and 2.5 10 cfu/g, respectively. Compared to control samples, the significant increase in number of survival (2.4 to 5.6 log unit) in pressure treated samples during 21 days of storage at 3 co uld C be considered as an evidence of injury due to pressure treatment. Also, when the incubation time was increased from 24 to 72 h, the number of colonies counted on PCA plates were increased significantly, which supports the presence of injury in pressure treated samples. Pressure is known to inflict sublethal injury in vegetative cells of bacteria (Alpas et al., 2000; Ritz et al., 2001; Ananta et al., 2004; Yuste et al., 2004; Kilimann et al., 2006; Koseki et al., 2008) and sublethally injured bacteria could recover in nutrient rich medium. Bozoglu et al. (2004) stated that primary and secondary injuries could occur after HHP treatment and injured cells may not form visible colonies on selective or non-selective agar, however colonies could first form on non-selective agar and later on selective agar during prolonged storage. Other studies that support the recovery of sublethally injured cells during storage after a pressure treatment of various types of foods are: minced beef (Carlez et al., 1993), sliced cooked ham (Aymerich et al., 2005), poultry meat (Yuste et al., 1999) and smoked salmon (Erkan et al., 2011). In the literature, there are variations in degree of recovery of microorganisms in pressure treated food products and model food systems during storage however, existence of some degree of recovery depending on the parameters of pressure treatment, type of food and temperature of storage is evident. Our view is that the increase in number of microbial counts during chilled storage is due mainly to the recovery and subsequently growth of the injured cells; however, possibility of germination and growth of bacterial spores should also be noted. It has been reported that refrigeration at 4 was sufficient t o prevent C

germination and outgrowth of Clostridium perfringens spores on vacuum-packaged beef surfaces whereas, storage at 37C for 1 day resulted in spore germina tion, outgrowth, and rapid proliferation to levels exceeding 7 log cfu/g (Novak and Yuan, 2004).

Survival of temperature

microorganisms

at

GI

acidity

and

There was no growth in any of the 19 days old HHP treated samples when they were kept at pH 3.0 0.1 and 37C for 3 h with stomaching every an hour. It co uld be assumed that the pressure treated samples may contain: i) completely inactivated vegetative cells; ii) injured vegetative cells (Alpas et al., 2000; Ritz et al., 2001; Ananta et al., 2004; Bozoglu et al., 2004; Yuste et al., 2004; Kilimann et al., 2006; Koseki et al., 2008); iii) germinated spores (Gould and Sale, 1970; Wuytack et al., 2000; Aouadhi et al., 2012; Vercammen et al., 2012); and iv) spores. Injured vegetative cells and newly germinated spores may be stressed by low pH of TSB (pH 3.0) and therefore may not be able show viability on PCA. On the other hand, aerobic and facultative anaerobic bacterial spores could not germinate and/or grow during the 3 h of exposure to low pH of TSB and subsequently on solid surface of PCA during incubation for 18 to 24 h. Inability of bacterial spores germinating at low pHs could be supported by the results of Ciarciaglini et al. (2000) who studied the effects of preservative treatments on germination of Bacillus subtilis spores in nutrient broth with 10 mM L-alanine and observed that the germination was completely inhibited at pH 4.0 when incubated at 30 for up to 6 h. C Survival of microorganisms were observed in the HHP treated samples when the pH of the mix was brought to 6.5 by addition of NaOH and 10 g TSB and then incubated 10 h at 37 following the exposure to C stomach acidity and temperature for 3 h. Average survival of microorganisms in the samples treated at 300, 400 and 541 MPa were 5.8 (0.48 SD), 5.1 (0.30 SD) and 4.8 (0.43 SD) log units, respectively. Significant number of viable cells observed in the samples from the simulation of lower GI tracts acidity and temperature could be explained by either the recovery of the injured vegetative cells or germination of spores in nutritionally rich (due to added TSB and minced meat itself) liquid media and optimum pH (6.5) and temperature (37C). Wheeldon et al. (2008) reported that the optimum pH range for germination of Clostridium difficile spores was 6.5 to 7.5 and the extent of germination decreased at pH 5.5. Our results suggests that vegetative microorganisms that are not completely inactivated during the HHP treatment may not be able to repair the injuries at the simulated conditions of stomach pH and temperature to be able to show viability on PCA. Similarly, bacterial

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spores that are not inactivated by pressure treatment could not germinate at the conditions mimicking the stomach pH and temperature and show viability on PCA. However, vegetative microorganism could recover from their injuries and bacterial spores could germinate at the conditions of lower GI. In addition, the growth of the recovered microorganisms and germinated spores could also be possible. To the best of our knowledge, there is no study on injury recovery of vegetative cells in GI tract, however there are studies on fate of bacterial spores in GI tract that are supporting our view on possible germination and/or outgrowth of bacterial spores at the conditions mimicking the pH and temperature of lower GI tract. In characterizing spore probiotics, Hoa et al. (2001) studied the persistence and dissemination of B. subtilis spores given orally to mice. Their results showed that the number of spores excreted in the feces of mice was, in some experiments, larger than the original inoculum. This was explained by germination of a proportion of the spore inoculum in the intestinal tract, followed by limited rounds of cell growth and then sporulation again. Using a murine model, Casula and Cutting (2002) showed that spores of Bacillus species can germinate in significant numbers in the jejunum and ileum and may colonize the small intestine. Cartman et al. (2008) showed that orally administered spores of B. subtilis germinated in GI tracts of chicks and 20 h after spores were administered, vegetative cells outnumbered spores throughout the GI tract. It should be noted that our conclusions are not taking into account the complicated processes and interactions, such as effect of digestive enzymes, competitive microflora of the guts, interactions of nutrients, etc. into account. However, our results point out a possible risk of recovery and growth of injured pathogenic microorganisms and germination and/or growth of bacterial spores in GI tract, if the pathogens and spores are not completely inactivated during the HHP treatment. Conclusions Our results showed that despite the absence of growth immediately after a pressure treatment in a nutrient rich environment such as, minced meat, a substantial recovery could be possible during storage. The undetected microbial growth immediately after HHP treatment could be due to injury rather than death of the microorganisms. It is assumed that the injured microorganisms can recover in the GI tract and grow/multiply. Our results suggest that the microorganisms could not recover from injury or grow at stomach acidity and temperature. However, recovery and growth could potentially occur in lower GI tract where the acidity is low. This could have important implications in terms of the safety of HHP treated foods especially when they are consumed by immunologically impaired. It

should be noted that our findings are based on a simplified approach which does not take the complicated processes and interactions, such as effect of digestion enzymes, bile and pancreatic juices, competitive microflora of the guts, interactions of nutrients, etc. into account. Therefore, the degree of recovery and/or growth of microorganisms could be different in vivo. However, the possibility of the risk for recovery of pathogenic microorganisms in GI tract could not be ignored, if the pathogens are not killed but injured by HHP treatment. Further in vitro and in vivo studies are required to better understand the possibility of the recovery of pressure injured microorganisms and differentiate the extent of recovery and growth of injured cells from the extent of germination and growth of bacterial spores in GI tract. ACKNOWLEDGEMENTS This work was supported by Republic of Turkeys Small and Medium Size Enterprises Development Organization (Grant no: 02229). The author thanks Dr. G. A. Evrendilek and A. Yaman for letting them use HHP equipment and for their assistance. Special thanks to N. Oner who assisted with the experimental work throughout the study.
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