Evaluation of the Microbiological Status of Raw Beef in Korea: Considering the Suitability of Aerobic Plate Count Guidelines.

This study was conducted to analyze the microbiological contamination status of raw beef distributed in Korea, and evaluate the suitability of current aerobic plate count (APC) guidelines. We analyzed five years (2010-2014) of microbiological monitoring data obtained from the Ministry of Food and Drug Safety and investigated the microbiological status of raw beef collected from meat packing centers and meat shops in the Seoul/Gyeonggi, Gangwon, and Chungcheong regions in August 2015. From 2010-2014, most raw beef (>94%) displayed APC levels of < 1.0 × 106 CFU/g. However, raw beef samples collected from all three regions in August 2015 had comparatively higher APC levels than those reported in previous years. To evaluate the relationship between the APC level and quality, changes in beef loin were evaluated during cold storage for 15 days at 4°C. On day 11, the mean APC level (4.7 × 106 CFU/g) conformed to current guidelines in Korea (1.0 × 107 CFU/g) and the pH value was 5.82. However, the sensory evaluation score for color and overall acceptability was under 3.0, meaning that the beef loin was not acceptable for eating. These results suggest that current APC guideline for raw beef should be lowered to 1.0 × 106 CFU/g to improve both the microbiological safety and palatability of raw beef.

Introduction

Over recent years, meat consumption and production has continually increased. In Korea, annual meat consumption has increased from 36.8 kg/capita in 2009 to 52.3 kg/capita in 2016 (OECD, 2017). Meat is a great source of protein; however, it is easily perishable (Komba et al., 2012). Meat products are readily contaminated by a variety of food hazards, including biological, chemical, physical, and particularly microbial factors (Kim et al., 2016; Ji, 2002). Although the muscles of healthy animals do not contain microorganisms, meat can be contaminated during production and transportation (Ercolini et al., 2006). Contamination easily occurs during processing, via slaughter facility and food handling equipment (belts, grinders, saws, knives, etc.; Eisel et al., 1997). Regulations and guidelines exist to provide safe meat and meat products to consumers. However, despite efforts to serve the safest meat possible to consumers, worries surrounding the contamination of meat with pathogenic microorganisms have increased in recent years (Bae et al., 2011).

As increasing numbers of people adapt a Western-style diet, beef consumption has increased accordingly. As international trade in the meat industry grows, many countries have made efforts to ensure the availability of sanitary and safe meat (Oh and Lee, 2001). Monitoring the microbiological levels of raw meat products is an important facet of sanitary management. The quality and safety of raw beef can be evaluated using indicator microorganisms such as aerobic plate count (APC) and Escherichia coli (Jay, 1992). In the USA, the Food Safety and Inspection Service (FSIS) has conducted the Nationwide Microbiological Baseline study since 1992. Through this, FSIS can monitor the microbial contamination levels (APC, E. coli, Salmonella, etc.) of meat or carcasses, which are regulated by the US Code of Federal Regulations (9 CFR 310.25). The microbiological levels in food are also regulated by law in the European Union (EU; EC No. 2073/2005), and food products that violate regulations must be recalled. In Japan, the Ministry of Health, Labour and Welfare (MHLW) legislated the Food Hygiene Law to control E. coli and pathogen levels in meat products. Many countries control the meat hygiene by regulating or recommending of APC level in meat. Most countries regulate or recommend the APC level in meat at under 5 × 106 CFU/g or cm2 (Table 1). However, the Korean APC guideline of 1.0 × 107 CFU/g is higher than that of other advanced countries (Table 1), and studies have reported that meat of this APC level displayed surface spoilage and off-odor (Gil, 1998; Oh and Lee, 2001; Young et al., 1988). Only the International Commission on Microbiological Specifications for Foods (ICMSF) provided the APC guideline level at 1.0 × 107 CFU/g or cm2 in 1978. Therefore, the APC level recommended by the Ministry of Food and Drug Safety (MFDS) may not meet recent international meat freshness standards. Determination of a practical APC guideline for raw beef is required.

Table 1.

Comparison of the microbiological limits for carcasses and meat in different countries

Country	Products	Aerobic plate count	E. coli	Institute	Type
Korea	Carcasses (cattle, sheep)	≤1.0 × 105 CFU/cm2	≤102 CFU/cm2	Ministry of Food and Drug Safety 2014-135	Guideline
	Carcasses (pigs)		≤104 CFU/cm2
	Carcasses (chicken, duck)		≤103 CFU/cm2
	Meat (cattle, sheep)	≤1.0 × 107 CFU/g	≤103 CFU/g
	Meat (pigs, chicken, duck)		≤104 CFU/g
USA	Carcasses (steers, heifers, cows, bulls)	-	≤102 CFU/cm2	Food Safety and Inspection Service, Electronic code of federal regulations	Standard
	Carcasses (broiler)	-	≤103 CFU/cm2
	Carcasses (swine)	-	≤104 CFU/cm2
Japan	Meat	≤5.0 × 106 CFU/g	Negative	FCOOP	Self -standard
	Poultry meat	≤5.0 × 106 CFU/g	-
	Raw meat	≤5.0 × 105 CFU/g	Negative	Kinkicoop	Self-standard
	Meat	≤5.0 × 106 CFU/g	Negative
	Meat	≤3.0 × 106 CFU/g	Negative	Kobecoop	Self -standard
	Raw meat	≤3.0 × 106 CFU/g	-	UCOOP	Self- standard
	Meat	≤1.0 × 107 CFU/g	-
Australia	Meat	≤106 CFU/cm2 or g	≤103 CFU/cm2 or g	Meat Standards Committee AS 4696:2007	Guideline
European Union	Carcasses (cattle, sheep, horses)	≤5.0 Log CFU/cm2	≤2.5 Log CFU/cm2	Commission Regulation (EC) No 2073/2005	Standard
	Carcasses (pigs)		≤3.0 Log CFU/cm2
	Meat (MSM)1)	≤5.0 × 106 CFU/g	≤500 CFU/g
ICMSF2)	Meat	≤1.0 × 107 CFU/cm2 or g	-	International Commission on Microbiological Specifications for Foods	Guideline
FAO3)	Meat	≤105 CFU/cm2	≤103 CFU/cm2	United Nations Food and Agriculture Organization	Guideline

1)Mechanically separated meat.

2)International Commission on Microbiological Specifications for Foods.

3)Food and Agriculture Organization of the United Nations.

Therefore, this study was conducted to evaluate the microbiological contamination level of raw beef in Korea by analyzing MFDS surveillance results over five years (2010-2014) and monitoring microbiological contamination levels in raw beef from three regions in Korea. Furthermore, changes in beef quality and microbial levels during cold storage were determined to provide scientific evidence for changes to the APC guideline of raw beef.

Materials and Methods

Analysis of the microbiological contamination of raw beef in Korea from 2010-2014

The APC level of raw beef loin (Longissimus dorsi muscles) is monitored in meat packing centers and meat shops throughout Korea by the MFDS. To analyze the microbiological contamination levels of raw beef in Korea for five years (2010-2014), the APC results for raw beef were obtained from the MFDS. The results are expressed as the distribution of APC (CFU/g).

Analysis of microbiological contamination in raw beef in Korea in August 2015

To monitor the microbiological sanitation status of raw beef throughout Korea, raw beef loin samples (n=21) were collected from meat packing centers (n=3) and meat shops (n=18) in three regions of Korea (the Seoul/Gyeonggi, Gangwon, and Chungcheong regions) in July and August 2015. One meat packing center and six meat shops were randomly selected in each region, and meats on display were purchased. Beef samples were transferred to the laboratory under refrigerated conditions for microbiological analysis.

Each meat sample (10 g) was placed into a sterile Stomacher bag, and sterile water (90 mL) was added to make a 10−1 dilution. The contents were homogenized using a BagMixer 400 Stomacher (Interscience, France) for 2 min, and further diluted using sterile water. Each diluent (1 mL) was plated onto APC or E. coli Petrifilm (3M Microbiology, USA). The films were incubated aerobically at 37°C for 48 h. Blue colonies with bubbles were counted as E. coli.

Evaluating changes in beef loin quality during storage at 4°C

To evaluate changes in beef loin quality during storage, the pH, microbiological, and sensory characteristics were investigated. Hanwoo (grade 1) beef loins (Longissimus dorsi muscles) were purchased from the local meat packing center 24 h postmortem and sliced to 1.5 cm thickness. Each sample was wrapped with low-density polyethylene film and stored at 4 ± 2°C for 15 d. The oxygen transmission rate of low-density polyethylene film was 35,273 cc/m2 /24 h at 1 atmosphere, 0.01 mm of thickness.

The pH was measured using a pH meter (Orion 230A, Thermo Fisher Scientific, Inc., USA) after 10 g of beef sample was homogenized with 90 mL of distilled water for 30 s.

For microbiological analysis, beef loin (10 g) was homogenized with sterile water (90 mL) using a BagMixer 400 Stomacher (Interscience, France) for 2 min, and 1 mL of diluent was plated onto APC Petrifilm (3M Microbiology, USA), which was incubated aerobically at 37°C for 48 h.

A 15-member sensory panel conducted the sensory evaluation of raw beef. The panelists were asked to score the raw beef for color, aroma, drip loss, and overall acceptability. The color, aroma, and overall acceptability were scored using a 9-point scale system from extremely like (9 points) to extremely dislike (1 point). Drip loss was the amount of water extracted from the meat; it was expressed from very high (9 point) to very low (1 point).

Statistical analysis

Analysis of variance was used for statistical analysis using SAS software, version 9.4 (SAS Institute Inc., USA). Duncan’s multiple range test was used to compare means, with a 5% level of significance (p<0.05). All analyses were repeated at least 3 times and are expressed as means ± standard error (SE).

Results and Discussion

The APC and E. coli levels of raw meat are important microorganism indicators, which ensure the safety of meat by detecting microbial growth during transportation, storage, and retail sale (Ahmad et al., 2013). To maintain sanitary conditions, many countries recommend or regulate that the APC level of meat be ≤105-107 CFU/g and the E. coli level be ≤102-104 CFU/g (Table 1). In Korea, the MFDS recommends that the levels for raw beef in meat packing centers and meat shops be ≤1.0 × 107 and 1.0 × 103 CFU/g for APC and E. coli, respectively (Notice 2014-135, MFDS).

To analyze the microbiological contamination level of raw beef in Korea from 2010-2014, APC results were obtained from the MFDS. The distribution of APC in raw beef in meat packing centers and meat shops is shown in Table 2. In meat packing centers, 2,796 raw beef samples were investigated, and the microbial levels of 2,761 (98.75%) samples were ≤1.0 × 106 CFU/g. In meat shops, 4,335 raw beef samples were investigated, and the microbial levels of 4,106 (94.72%) samples were ≤1.0 × 106 CFU/g. Two (0.07%) and 22 (0.51%) samples exceeded the APC guideline level in meat packing centers and meat shops, respectively. This suggests that less than 1% of the beef in both meat packing centers and meat shops was contaminated, indicating successful management of beef contamination between 2010 and 2014. The contamination level in meat shops was higher than in meat packing centers, consistent with a study by Park et al. (2002), which reported that Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus were detected at 2.9, 10.2, and 10.2%, respectively in beef samples from meat shop. L. monocytogenes, and S. aureus were detected at 2.8 and 2.8%, respectively in beef samples from meat packing center. Importantly, 80% of beef served to the consumer passes through a meat shop (Ko et al., 2013). Therefore, the microbial contamination of raw beef in meat shops requires more careful control.

Table 2.

Aerobic plate counts of raw meat from meat packing centers and meat shops across the country over five years (2010-2014)

Place	Distribution of aerobic plate counts (CFU/g)
	≤103	103 - ≤ 104	104 - ≤ 105	105 - ≤ 106	106 - ≤ 107	>107	Total
Meat packing center	1,2221) (43.71)2)	933 (33.37)	492 (17.60)	114 (4.08)	33 (1.18)	2 (0.07)	2,796 (100)
Meat shop	1,279 (29.50)	1,209 (27.89)	988 (22.79)	630 (14.53)	207 (4.78)	22 (0.51)	4,335 (100)

1)Number of samples.

2)Percentage in the total number of samples (%).

APC levels of raw beef from three regions of Korea were monitored in August 2015. The mean APC values of beef in meat packing center from the Seoul/Gyeonggi, Gangwon, and Chungcheong regions were 5.9 × 102, 4.4 × 103, and 1.0 × 102 CFU/g, respectively (Table 3). The mean values of E. coli of beef from meat packing centers in the Gangwon and Chungcheong regions were 6.3 × 101 and 3.0 CFU/g, respectively. No E. coli was detected in beef from meat packing centers in the Seoul/Gyeonggi region.

Table 3.

Aerobic plate count and E. coli content of raw beef loin from meat packing centers in three regions in Korea (CFU/g)

Region	Aerobic plate count			E. coli content
	Mean	Minimum	Maximum	Mean	Minimum	Maximum
Seoul/Gyeonggi	5.9 × 102	5.8 × 102	6.0 × 102	ND1)	ND	ND
Gangwon	4.4 × 103	4.0 × 103	5.2 × 103	6.3 × 101	5.0 × 101	8.0 × 101
Chungcheong	1.0 × 102	5.0 × 101	1.3 × 102	3.0	ND	1.0 × 101

1)ND: Not detected.

APC and E. coli levels in raw beef from six meat shops are presented in Table 4. Microbial contamination can occur during transportation, storage, and treatment at meat shops (Ahmad et al., 2013). The maximum APC value detected in a meat shop was 1.2 × 107 CFU/g, and the minimum was 5.2 × 103 CFU/g. Only meat shop B in the Gangwon region exceeded the APC guideline level. E. coli only detected in three shops, at approximately 1.0 × 101 CFU/g.

Table 4.

Aerobic plate count and E. coli content of raw beef loin from meat shops in three regions in Korea (CFU/g)

Region	Market place	Aerobic plate count			E. coli content
		Mean	Minimum	Maximum	Mean	Minimum	Maximum
Seoul/Gyeonggi	A1)	4.2 × 106	4.0 × 106	4.4 × 106	ND2)	ND	ND
	B	8.3 × 103	7.5 × 103	9.4 × 103	ND	ND	ND
	C	1.8 × 105	8.5 × 104	3.0 × 105	1.3 × 101	ND	3.0 × 101
	D	1.0 × 107	1.0 × 107	1.1 × 107	ND	ND	ND
	E	1.3 × 105	7.6 × 104	2.2 × 105	ND	ND	ND
	F	6.8 × 105	3.1 × 105	9.6 × 105	ND	ND	ND
Gangwon	A	1.1 × 106	5.6 × 105	1.5 × 106	2.7 × 101	2.0 × 101	3.0 × 101
	B	1.2 × 107	7.7 × 106	1.4 × 107	ND	ND	ND
	C	1.1 × 105	6.4 × 104	1.9 × 105	3.0	ND	1.0 × 101
	D	1.6 × 104	1.1 × 104	2.3 × 104	ND	ND	ND
	E	1.3 × 106	1.1 × 106	1.5 × 106	ND	ND	ND
	F	2.8 × 104	1.5 × 104	4.1 × 104	ND	ND	ND
Chungcheong	A	1.1 × 106	5.8 × 105	1.6 × 106	ND	ND	ND
	B	7.5 × 103	5.5 × 103	7.4 × 103	ND	ND	ND
	C	6.1 × 105	5.3 × 105	6.8 × 105	ND	ND	ND
	D	5.2 × 103	3.8 × 103	7.2 × 103	ND	ND	ND
	E	2.2 × 104	1.9 × 104	2.5 × 104	ND	ND	ND
	F	4.5 × 105	4.1 × 105	4.7 × 105	ND	ND	ND

1)A: Supermarket, B-F: Butcher’s shops.

2)ND: Not detected.

According to previous studies, raw beef in Seoul, Korea had reported mean APC and E. coli values of approximately 1.0 × 105 and 1.0 × 101 CFU/g (Kim et al., 2005; Ko et al., 2013). According to microbial assessment performed in Korea from February-June 2007, 50% of beef samples showed 104-105 CFU/g of APC (Lee et al., 2007). However, in this study, only 16.6% of samples had APC levels of 104-105 CFU/g, and 66.7% displayed levels ≥1.0 × 105 CFU/g. Our results suggest that beef samples collected in 2015 had higher contamination levels compared to previous microbial assessments performed in Korea. This indicates that enhanced regulations are required to ensure the distribution of hygienic meat to the consumer.

Changes in beef quality and microbial levels were measured during cold storage to evaluate the practical APC level for beef. Table 5 shows the changes in pH, microbiological, and sensory evaluation of beef loin samples during storage for 15 days at 4°C. The pH of fresh meat is usually 5.5-5.8, and increases to 8.0 during meat spoilage (James, 1972; Shin et al., 2006). In Korea, meat is considered spoiled at pH values > 6.2 (Notice 2015-13, MFDS). The initial pH value of the beef loin was 5.63, and it had increased significantly by day 15 (p<0.05), exceeding 6.0 on day 14. This is consistent with previous studies (Kook and Kim, 2005; Shin et al., 2006). Increased pH is correlated with increases in microorganisms, and is accompanied by spoilage (Sutherland et al., 1975). Also it is related to the formation and accumulation of amine and ammonia (Maijala et al., 1993).

Table 5.

Changes in pH, microbiological and sensory quality characteristics of raw beef loin (grade 1) during storage at 4°C

Trait		Storage (days)
		1	3	6	9	10	11	12	13	14	15
pH		5.63 ± 0.015d	5.63 ± 0.013d	5.62 ± 0.006d	5.66 ± 0.009d	5.82 ± 0.006c	5.82 ± 0.010c	5.80 ± 0.012c	5.84 ± 0.032c	6.10 ± 0.027b	6.32 ± 0.009a
Aerobic plate count (Log CFU/g)		3.01 ± 0.188h	3.55 ± 0.054g	3.77 ± 0.019g	5.94 ± 0.017f	6.21 ± 0.117e	6.67 ± 0.100d	6.90 ± 0.022d	7.33 ± 0.019c	7.93 ± 0.075b	8.25 ± 0.025a
Sensory evaluation	Color	7.67 ± 0.289a	7.78 ± 0.147a	6.78 ± 0.222b	5.00 ± 0.167c	3.67 ± 0.289d	2.89 ± 0.309e	2.56 ± 0.294e	2.78 ± 0.324e	2.67 ± 0.289e	1.56 ± 0.294f
	Aroma	8.44 ± 0.176a	7.33 ± 0.333b	6.78 ± 0.278b	5.78 ± 0.324c	4.11 ± 0.389d	3.56 ± 0.377d	2.67 ± 0.167e	2.22 ± 0.401e	2.44 ± 0.294e	1.22 ± 0.147f
	Drip loss	1.67 ± 0.236a	1.78 ± 0.222a	1.56 ± 0.242a	1.78 ± 0.278a	1.67 ± 0.289a	1.44 ± 0.242a	1.56 ± 0.242a	1.78 ± 0.324a	1.78 ± 0.278a	1.67 ± 0.289a
	Overall acceptability	7.89 ± 0.261a	7.67 ± 0.167a	6.78 ± 0.401b	5.56 ± 0.176c	3.67 ± 0.167d	3.00 ± 0.236de	2.78 ± 0.147e	2.33 ± 0.289e	2.44 ± 0.242e	1.44 ± 0.242f

a-hMeans ± SE with different letters within same row differ significantly at p<0.05.

The initial APC of beef loin was 3.0 Log CFU/g (1.2 × 103 CFU/g), and it significantly increased through the storage period (p<0.05). Meat starts to decay at an APC of 6-7 Log CFU/g and meat with APC values of 8-9 Log CFU/g are not edible (Shin et al., 2006). In this study, at day 13, the APC of beef loin was 7.3 Log CFU/g (2.1 × 107 CFU/g), exceeding the guideline level (1.0 × 107 CFU/g) and it had started to decay.

The color, aroma, drip loss, and overall acceptability of the beef loin during cold storage were evaluated, and if the color, aroma, and overall acceptability score was below 3, the meat was considered inedible. Consumers choose beef through evaluation of its sensory traits, which are used to determine meat quality as well as healthiness (Verbeke and Viaene, 1999). As the storage period increased, the color, aroma, and overall acceptability score of the beef loin decreased (p<0.05). Drip loss did not show significant differences during storage. At day 11, the color and overall acceptability score were under 3 and aroma was 3.6. This means that the panels recognized the beef loin stored for 11 days was not acceptable for eating. Most international APC guidelines for raw beef are approximately 1.0 × 106 CFU/g (Table 1). Only Korea and the International Commission on Microbiological Specifications for Foods (ICMSF) recommend levels up to 1.0 × 107 CFU/g. This guideline was established in 2007 in Korea, and in 1978 by the ICMSF. Therefore, verification of the Korean APC guideline level for beef is needed. The APC level is highly correlated with the sensory qualities of meat. In this study, the APC level at day 11 was 6.7 Log CFU/g (4.7 × 106 CFU/g). Although this was below the APC guideline for beef in Korea, the sensory panel regarded it as unacceptable for eating. To support our results, Moon et al. (2013) reported that APC (R = −0.902, p<0.01) and sensory quality (R = −0.907, p<0.01) were highly correlated with the freshness of raw beef. Tang et al. (2013) revealed that an APC level of 1.0 × 107 CFU/g corresponded with spoilage and growth of APC on chilled pork, and this correlated closely with sensory changes during storage. Shin et al. (2006) detected meat spoilage when the pH was >6.0 and the APC was 6.8 Log CFU/g, and at that time, the sensory panel regarded the meat as unacceptable. Therefore, an APC value of 1.0 × 107 CFU/g is not adequate to indicate the freshness of raw beef. We suggest that this guideline be lowered to 1.0 × 106 CFU/g.

Conclusions

The prevention of microbiological contamination of beef in Korea was quite successful for the five-year period encompassing 2010-2014. However, we discovered that more recent raw beef for retail sale displayed a higher level of APC contamination, suggesting that this requires stricter control. When quality changes in beef loin was evaluated for 15 days at 4°C, although the APC was less than the accepted level in Korea, sensory panels considered the beef unacceptable at day 11. The present APC guideline for raw beef is higher than in other advanced countries and corresponds to the level at which the sensory characteristics of beef can decrease dramatically. Therefore, we suggest that present APC guideline be lowered to ensure meat safety and palatability. Strengthening the APC guideline similarly to the guidelines of other countries will provide safe beef to consumers in the domestic market and help ensure the safe sale of Korean beef in foreign markets.