Influence of sodium reduction and storage temperature on the growth of total microbes and Bacillus cereus in naturally contaminated hamburger patty and loaf bread

Abstract

This study aimed to investigate the influence of sodium reduction and storage temperature on the growth of total microbes and Bacillus cereus in naturally contaminated hamburger patty and loaf bread, respectively. The sodium reduction rate of hamburger patty and loaf bread was 20% and 30%, respectively, and experimental samples were kept at 4 °C, 25 °C, and 40 °C for 60 h. The microbiological analysis included the colony count of total microbes and B. cereus. The water activity (Aw), titratable acidity (TA), and pH were assessed as factors that inhibit microbial growth. In this study, Aw, TA, and pH of all samples were affected by the growth of total microbes and B. cereus during the storage period. Hence, these results suggested that sodium reduction in processed foods should be preferentially applied as a potent inhibition strategy after accurate assessment of inhibitors for different food types.

Introduction

Salt improves the flavor of food products as well as their texture by enhancing the structural consistency (Drüeke, 2016; Kilcast and Angus, 2006). In addition, salt is available at low cost, can inhibit microbial growth, extend the shelf life of food products, and create fermentation conditions for the manufacture of fermented food products (Carbone et al., 2003; He et al., 2012; Kicast and Angus, 2006). On the contrary, excessive intake of sodium has been reported to cause hypertension and various chronic disorders including renal diseases and several cardiovascular diseases, such as heart attack and stroke (O'Donnell et al., 2015). The World Health Organization (WHO), thus, limits the recommended sodium intake at 2000 mg, and advanced countries including the USA, UK, and Japan, have continuously tried to reduce the dietary salt intake (Drüeke, 2016; Gibson et al., 2000; Micklegorough, 2005; Strazzullo et al., 2009). In Korea, the average daily salt intake was estimated to be 4546 mg in 2012, twice the amount recommended by WHO, resulting in country-wide efforts toward the promotion of low salt processed foods and consumer campaigns since March 2012 (MFDS, 2019). However, reducing salt in processed foods increases the risk of food poisoning due to the growth of pathogenic or saprogenic microorganisms (Sofos, 1984; Stanojevic et al., 2009; Wijnker et al., 2006). Moreover, future research should be targeted toward establishing the relevant standards and regulations regarding the production of low salt processed foods. However, to the best of the authors’ knowledge, no studies have performed to confirm the microbial safety of low sodium processed foods at different reduction levels and storage conditions.

Therefore, this present study investigated the influence of sodium reduction by 20–30% and storage temperature at 4 °C, 25 °C, and 40 °C on the growth of total microbes and Bacillus cereus in naturally contaminated processed foods—hamburger patty and loaf bread. For microbiological analysis, two classes of microbes were examined—total microbes, the sanitary-indicative microorganisms, and B. cereus, the food poisoning-causing bacterium widely distributed in nature. For physicochemical analysis, the water activity (Aw), titratable acidity (TA), and pH were also determined.

Materials and methods

Experimental conditions and sample production

The processed foods used in this study were commercially available hamburger patty and loaf bread that were easily found and commonly consumed in everyday life (Collins-Thompson et al., 1984; Collins et al., 1991; Choe et al., 2008; Gracia et al., 2009; O’Donell et al., 2015; MFDS, 2019; Smith et al., 2004). To prepare low salt samples, the salt content in these foods was reduced by 20 and 30%, respectively, compared to that in the original product during the salting stage. Hamburger patties and loaf breads used in the study were ordered and supplied by factories belonging to the Korea Food Industry Association that produce sodium-reduced products. The sodium content of the samples used in this study was based on the sodium reduction policy, which aims to reduce sodium intake by more than 20 percent by 2020 driven by Korea Ministry of Food and Drug Safety, and the unique characteristics of the food were also considered. A total of 240 samples per product were used in the study, and were categorized according to the storage temperature into the control (the original product) and low salt groups. For microbiological analysis, 10 samples from each group were tested. The storage temperature of the samples was set at 4 °C as cold, 25 °C as ambient, and 40 °C as stress condition. Owing to the shelf life, the hamburger patty and loaf bread were assessed for 60 h (2.5 days).

Quantitative culture of total microbes and B. cereus

Microbiological analysis of all the samples was performed according to the microbiological test methods described in Korean Food Standard Code by Korea Ministry of Food and Drug Safety (MFDS, 2019).

Firstly, each food sample was suspended in 225 ml of sterilized phosphorus buffer saline (PBS) (Difco, Sparks, MD, USA) in a sterilized stomacher bag and homogenized for 2 min using a BagMixer stomacher (Interscience, St. Nom, France). Subsequently, 1 ml of the homogenized solution was ten-fold diluted in physiological saline solution 0.85% (sterile) (Sigma, St. Louis, MO, USA), and inoculated onto the Petrifilm™ aerobic count plate (3 M, St. Paul, MN, USA) for quantitative culture of total microbes. After incubated for 48 h at 35 °C, total microbes was enumerated.

And for quantitative culture of B. cereus, 1 ml of each homogenate was serially diluted (tenfold) in phosphate-buffered saline (Sigma), and inoculated onto mannitol–egg yolk–polymyxin B agar (MYP) (Oxoid, Basingstoke, Hampshire, UK) followed by incubation at 30 °C for 24 h. After enumerated suspicious colonies on each plate, five typical colonies were subcultured and confirmed as B. cereus using the VITEK^® 2 GP ID kit (bioMerieux, Marcy l'Etoile, France).

Measurement of Aw, TA and pH

One to five gram of each sample was placed in Aw measuring containers and the Aw meter (AquaLab 4TE, Pullman, WA, USA) was used for the measurement of Aw. For measurement of TA, 10 g sample was added to 100 ml distilled water and homogenized. The homogenized sample was subjected to agitated extraction at 25 °C for 30 min, and the resulting solution was filtered using Whatman™ Grade 4 Qualitative Filter Paper (GE Healthcare, Maidston, UK). The extract was diluted and titrated using 0.1 N NaOH solution (Sigma), and the equation below was used for the calculation.

$$\text{TA\% = 0.0090}\times \text{volume}\text{of}\text{0.1}\text{N}\text{NaOH}\text{used}\times \text{100/weight}\text{of}\text{the}\text{sample.}$$

And for measurement of pH, 10 g sample was diluted ten-fold in distilled water, homogenized, and subjected to agitated extraction at 25 °C for 30 min, followed by filtration using Whatman™ qualitative filter paper: grade 4 circles (GE Healthcare). The pH of the extract was measured at room temperature (25 °C) using a pH meter (Thermo Electron Corp., Milford, MA, USA).

Statistical analysis

All the results were obtained after repeating the procedures three times, and the SPSS Program (ver 18.0, SPSS Inc., Chicago, IL, USA) was used to calculate the mean and standard deviation. Depending on the analytical method, the mean values were compared by one-way ANOVA test (Duncan method). For all the analyses, the significance level was set at p = 0.05.

Results and discussion

Hamburger patty

The low salt group of hamburger patty contained samples with the salt content reduced by 20% compared to that in the original product. The sample was stored at 4 °C, 25 °C, and 40 °C for 60 h, during which the counts of total microbes and B. cereus were determined respectively (Tables 1, 2). On day 0, the count of total microbes in the low salt group was 2.91–2.93 log CFU/g, slightly higher than that in the control group, while B. cereus was not detected in both groups. The results according to the storage temperature showed that during 0–36 h of storage at 4 °C, no change in the total microbial count was noted in both groups. However, at 48 h of storage, the counts of total microbes in both groups increased to 3.65 log CFU/g and 2.87 log CFU/g, respectively, with significantly higher counts in the low salt group than in the control group (p < 0.05). At 60 h, close to the end of experiment, both groups exhibited around 3 log CFU/g without significant difference. During 0–48 h storage at 25 °C, the counts from both groups were maintained at a level around 2–3 log CFU/g, which increased to around 5 log CFU/g after 60 h. The count of total microbes in the low salt group stored at 40 °C was maintained at around 3 log CFU/g during 0–12 h, when B. cereus was not detected. However, after 24 h, the count of the total microbes increased to a level above 5 log CFU/g in both groups, and B. cereus began to appear in the low salt group. After 48 h, B. cereus was detected in both groups, with significantly higher count in the low salt group (p < 0.05). And after 60 h, the count of total microbes in the low salt group exceeded 6 log CFU/g. This level of microbial contamination begins to decompose and spoil foods (Nottingham, 1982; Solberg et al., 1990; Stewart, 1987). The level was also significantly higher than that in the control group (p < 0.05). The count of B. cereus was around 3 log CFU/g in both groups. The initial pH of the hamburger patty was reported to be 5.89–5.99 in previous studies (Choe et al., 2008; Garcia et al., 2009). In this study, the initial pH was 5.87 and 5.90, respectively. In the low salt and control groups, consistent with that reported in the previous study (Garcia et al., 2009; Labots, 1981). The pH variation according to the storage temperature showed that when stored at 4 °C, the pHs of both groups were maintained within a stable range of 5.8–6.0 without significant difference between the groups (p > 0.05). The pH of the low salt and control groups that were stored at 25 °C and 40 °C decreased after 24 and 48 h, respectively, without significant difference throughout the experiment. TA of the hamburger patty in both groups was extremely low within the range of 0.08–0.11%. The growth of pathogenic microorganisms is prohibited at a pH lower than 4.0 (Baird-Parker and Freame, 1967; Collins-Thompson et al., 1984; Hamad, 2012). In other words, the pH of hamburger patty is not effective as a factor that inhibits the growth of microorganisms, and a drastic reduction in salt may have a negative effect on microbiological safety of various foods including hamburger patty. This was consistent with report of Taormina (2010). Sodium chloride could inhibit the growth and toxin production by Clostridium botulinum in processed meats and cheeses, and hence, sodium salts was contributed to prevent the spoilage and/or growth of microorganisms in various foods. Therefore, salt reduction should be considered according to the characteristics of each food.

Table 1.

The total microbial count in the hamburger patty stored at 4 °C, 25 °C, and 40 °C

Storage temperature (°C)	Storage period (h)	The total microbial count (log CFU/g)1)
		Control samples	Sodium reduction samples
4	0	2.09 ± 0.12A	2.91 ± 0.08B
	12	2.08 ± 0.15A	2.89 ± 0.27B
	24	2.11 ± 0.23A	3.05 ± 0.38B
	36	2.16 ± 0.49A	3.02 ± 0.36B
	48	2.87 ± 0.40A	3.65 ± 0.31B
	60	3.49 ± 0.37A	3.58 ± 0.54A
25	0	2.16 ± 0.19A	2.92 ± 0.23B
	12	2.20 ± 0.35A	2.95 ± 0.21B
	24	2.17 ± 0.26A	2.97 ± 0.23B
	36	2.97 ± 0.39A	3.15 ± 0.15A
	48	3.73 ± 0.65A	3.33 ± 0.56A
	60	4.97 ± 0.59A	5.32 ± 0.62A
40	0	2.36 ± 0.18A	2.93 ± 0.13B
	12	2.99 ± 0.21A	3.17 ± 0.33A
	24	5.06 ± 0.62A	5.28 ± 0.51A
	36	5.08 ± 0.44A	5.50 ± 0.38B
	48	5.14 ± 0.57A	5.77 ± 0.81B
	60	5.54 ± 0.48A	6.13 ± 0.65B

A total of 360 samples were used in the experiment and 10 samples per group were measured every 12 h. All data are expressed as mean ± standard deviation. The low salt group of hamburger patty contained samples with the salt content reduced by 20% compared to that in the original product

Different letters within a column indicate a significant difference (p < 0.05)

Table 2.

The total Bacillus cereus count in the hamburger patty stored at 4 °C, 25 °C, and 40 °C

Storage temperature (°C)	Storage period (h)	The total Bacillus cereus count (log CFU/g)1)
		Control samples	Sodium reduction samples
4	0	ND	ND
	12	ND	ND
	24	ND	ND
	36	ND	ND
	48	ND	ND
	60	ND	ND
25	0	ND	ND
	12	ND	ND
	24	ND	ND
	36	ND	ND
	48	ND	ND
	60	ND	ND
40	0	ND	ND
	12	ND	ND
	24	ND	0.70 ± 0.14
	36	ND	0.81 ± 0.46
	48	2.22 ± 0.45A	3.10 ± 0.28B
	60	3.00 ± 0.62A	3.22 ± 0.51A

A total of 360 samples were used in the experiment and 10 samples per group were measured every 12 h. All the data are expressed as mean ± standard deviation. The low salt group of hamburger patty contained samples with the salt content reduced by 20% compared to that in the original product

ND not detected

Different letters within a column indicate a significant difference (p < 0.05)

Loaf bread

The low salt group of loaf bread contained samples with the salt content reduced by 30%. The samples were stored at 4 °C, 25 °C, and 40 °C for 60 h, and the count of total microbes was determined (Table 3). B. cereus was not detected in either of the loaf bread groups at any of the storage temperatures throughout the experiment. According to the storage temperature, the results showed that total microbes were not detected in either of the groups when stored at 4 °C. Whereas at 48 h of storage at 25 °C, the count of total microbes in the low salt group was 1.9 log CFU/g. After 60 h, the counts increased to 3 and 1.62 log CFU/g in the low salt and the control groups. In the samples stored at 40 °C, the count of total microbes in the low salt group after 24 h was 3.7 log CFU/g, which was significantly higher than that in the control group (p < 0.05), and the counts after 48 h increased to a level above 5 log CFU/g in both groups. After 60 h, the count of total microbes in the low salt group exceeded 6 log CFU/g, which means a high level of contamination. The influence of storage temperature on Aw of loaf bread was negligible and Aw of loaf bread tested in this study was 0.97, and Aw in both the low salt and control groups was maintained within a stable range of 0.96–0.97. Low Aw prevents microbial growth and decay, and Aw value that inhibits the growth of bacteria and fungi is below 0.60 and 0.80, respectively (Fontana, 2000; Labots, 1981). The microorganisms related to bread decomposition are B. subtilis, B. licheniformis, and B. cereus (Collins et al., 1991). Among them, B. cereus has been reported to be detected in sandwiches (Kim et al., 2011). Furthermore, the spores of Bacillus strains are able to survive through the process of baking as they are thermostable; their spores can germinate at the temperature of 25–30 °C and above 0.95 Aw (Thompson et al., 1993), emphasizing the importance of contamination control against Bacillus spp. in bread. Therefore, although B. cereus was not detected in this study, the effects of sodium reduction on microbiological safety should be considered because loaf bread has high moisture and Aw that facilitate microbial growth (Smith et al., 2004).

Table 3.

The total microbial count in the loaf bread stored at 4 °C, 25 °C, and 40 °C

Storage temperature (°C)	Storage period (h)	The total microbial count (log CFU/g)1)
		Control samples	Sodium reduction samples
4	0	ND	ND
	12	ND	ND
	24	ND	ND
	36	ND	ND
	48	ND	ND
	60	ND	ND
25	0	ND	ND
	12	ND	ND
	24	ND	ND
	36	ND	ND
	48	ND	1.90 ± 0.21
	60	1.62 ± 0.1A	3.07 ± 0.47B
40	0	ND	ND
	12	ND	ND
	24	2.33 ± 0.54A	3.70 ± 0.64B
	36	4.56 ± 0.26A	4.62 ± 0.82A
	48	5.21 ± 0.49A	5.44 ± 0.48A
	60	5.68 ± 0.70A	6.08 ± 0.42A

A total of 360 samples were used in the experiment and 10 samples per group was measured every 12 h. All data are expressed as mean ± standard deviation. The low salt group of loaf bread contained samples with the salt content reduced by 30%

ND not detected

Different letters within a column indicate a significant difference (p < 0.05)

Conclusively, physicochemical properties such as Aw, TA, and pH could act as the major parameters in microbial growth (Presser et al., 1997; Wareing and Fernandes, 2007). In this study, the lack of an inhibitor that can replace salt and inhibit microbial growth in hamburger patty and loaf bread facilitated microbial growth upon sodium reduction. Thus, sodium reduction should be first applied to processed food products that have a physicochemical factor for inhibiting microbial growth, while research should continue to develop inhibition strategies for the advancement of the food processing industry.

Compliance with ethical standard

Conflict of interest

None of the authors of this study has any financial interest or conflict with industries or parties.