Distribution of psychrophilic microorganisms in a beef slaughterhouse in Japan after cleaning.

The purpose of this study was to investigate the abundance and distribution of psychrophilic microorganisms associated with spoilage in beef slaughterhouse environments after cleaning. The processing lines and equipment used in slaughtering and boning were swabbed, and the microbial count was determined using a TSA and MRS medium and Chromocult® Coliform agar incubated at 15ºC and 37ºC, respectively. As a result, the brisket saw (handle side) and trolley hook were the most heavily contaminated with microorganisms, with each having a microbial adhesion rate of 66.7%. The microbial adhesion rates of the apron and milling cutter (edge side) were 50%, respectively, and those of the foot cutter (edge and handle side), splitting saw (edge side), and knife (handle side) were 33.3%, respectively. Next, four colonies were randomly isolated from the petri dish used for the bacterial count measurement to identify the predominant microbial species of the microorganisms attached to each equipment. As a result of Sanger sequencing analysis, yeasts such as Candida zeylanoides and Rhodotorula sp. and bacteria including Pseudomonas sp. and Rhodococcus sp. were identified from the equipment used in the slaughtering line, and it was assumed that these microorganisms were of environmental origin. In contrast, only Pseudomonas sp. and Candida zeylanoides were isolated from the boning line. Despite the use of cleaning operations, this study identified some equipment was contaminated with microorganisms. Since this equipment frequently comes into direct contact with the carcass, it is critical to thoroughly remove the microorganisms through accurate cleaning to prevent the spread of microbial contamination on the carcasses.

Introduction

Beef carcasses become contaminated with microorganisms during the slaughter and subsequent deboning processes. Many pathogenic bacteria are present in cattle skin and intestinal contents, and the contamination spreads by adhering to the carcass surface after skinning [1-3]. Since the slaughterhouse is the furthest upstream from food distribution, strict hygiene management is required to prevent the spread of harmful bacteria downstream. Process control by the Hazard Analysis and Critical Control Point (HACCP) system is one measure to prevent the contamination of carcasses by such harmful bacteria. In Japan, following the EU and the United States, introducing the HACCP system in slaughterhouses became mandatory from June 2021 [4]. In addition to the HACCP system, compliance with Good Manufacturing Practice (GMP) is essential. Careful hygiene management, such as maintenance and inspection of equipment and machinery, is required to produce safe meat.

Bacteria associated with food poisoning, such as enterohemorrhagic Escherichia coli and Salmonella are among the hazardous bacteria subject to critical control point (CCP) management in cattle slaughterhouses [5]. These food-poisoning bacteria are mesophilic facultative anaerobes in the cattle’s intestinal tract. When feces or intestinal contents adhere to the carcass, trimming to completely remove the contaminated section effectively controls pathogenic bacteria. Furthermore, the chilling process after processing the carcass is recognized as CCP to prevent the growth of harmful bacteria by carefully controlling the low temperature [6].

In recent years, the international trading volume of beef has been increasing annually, and beef with a long expiration date has become advantageous in terms of sales strategy. The international beef trade is transported by refrigeration and freezing [7], but transportation under refrigerated conditions is preferable for parts with high unit prices, such as steak meat, because freezing conditions degrade beef quality [8]. However, long transportation periods can allow some low-temperature-growing microorganisms to grow and degrade beef quality [9, 10]. The number of bacteria that attach to the beef at the initial stage is the most important factor in producing beef that can withstand long-term transportation in a refrigerated state. If the number of initial bacteria in beef can be reduced as much as possible, the period until spoilage can be extended.

After skinning, the surface of beef carcasses has a low bacterial count, but microbial contamination accumulates through the equipment, lines, employee gloves, and aprons used in the subsequent process [11, 12]. In slaughterhouses with good hygiene management, the general bacterial count is investigated as part of the hygiene inspection after cleaning the equipment and lines. However, as described above, these are frequently targeted at medium-temperature bacteria that can be pathogenic, and psychrophilic microorganisms that cause spoilage during refrigerated distribution have received little attention. It is critical to prevent psychrophilic microorganisms from adhering to the carcass to produce beef with a long-term shelf life. However, to the best of our knowledge, no studies have been conducted on the distribution of psychrophilic microorganisms in slaughterhouse equipment and lines. The purpose of this study was to understand the actual contamination of psychrophilic microorganisms in the equipment used in the slaughtering line and the boning line.

Materials and methods

Swab sampling

Sampling was carried out at the slaughterhouse of the Federation of Hida Meat Agricultural Cooperative Association. The equipment was tested after the cleaning operation. At this facility, a boning line is attached to the slaughtering line, and a series of tasks are performed from the slaughter line to process the body into carcasses, followed by cutting and packaging at the boning line. Cleaning was carried out after each line was used, and the equipment was wiped the day after cleaning. Between 2019 and 2021, a total of six samplings on the slaughter line (Trial 1–6; 7/31/2019, 8/7/2019, 7/29/2020, 8/5/2020, 1/7/2021, 1/13/2021), and seven samplings on the boning line (Trial 1–7; 8/7/2019, 12/12/2019, 12/18/2019, 7/29/2020, 8/5/2020, 1/7/2021, 1/13/2021) were carried out. The knife, foot cutter, brisket saw, splitting saw, milling cutter, dehider, trolley hook, and aprons used in the slaughtering line were wiped off (S1 Fig). In the boning line, the large division conveyor belt, boning conveyor belt, turntable, meat holder, and electric saw were swabbed (S2 Fig). The saws were sampled twice: once on the edge side and once on the handle side. For the wiping inspection, a commercially available swab (Elmex, Tokyo, Japan) was used, and the entire surface was swabbed for those with complicated shapes, and 100 cm2 was swabbed for equipment with flat surfaces such as aprons, large division conveyor belt, boning conveyor belt, and the turntable.

Measurement of bacterial counts

The number of bacteria in each swab sample was measured on three types of media: trypticase soy agar (TSA; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) for general bacteria, de Man, Rogosa, and Sharpe (MRS) agar (Merck KGaA, Darmstadt, Germany) for lactic acid bacteria, and Chromocult® Coliform Agar (Merck) for coliform bacteria and E. coli. The swab sample was diluted ten-fold with saline solution, and 100 μL of each original solution and diluent were spread onto each agar medium. TSA and MRS media were incubated at 15°C for 96 h, while Chromocult® Coliform Agar was incubated at 37°C for 24 h. After incubation, the number of colonies were counted. The number of bacteria per 1 cm2 was calculated for aprons, the large division conveyor belt, the boning conveyor belt, and the turntable, while the number of bacteria per 1 mL of the swab was calculated for other equipment with complicated shapes. All experiments were duplicated, and those with an average number of colonies of 5 were regarded as the detection limit for reliability (5 CFU / cm2 or 5.0 × 101 CFU/swab).

Isolation of microorganisms

Four colonies were selected from TSA medium or MRS medium, and the number of colonies that exceeded the detection limit was confirmed. The bacterial species were identified through sequencing analysis. Since the selected colonies may be bacterial or yeast, morphological observations were made prior to DNA extraction with a optical microscope. DNA extraction was carried out under optimal conditions for yeast and bacteria, as described below. Bacterial isolates from TSA and MRS media were cultured at 15°C for 48 h in trypticase soy broth (TSB) (Becton, Dickinson and Company) and MRS broth (Merck KGaA), respectively. The yeast isolates were cultured at 15°C for 48 h in yeast peptone dextrose (YPD) broth, which was prepared with 20 g/L peptone (Becton, Dickinson and Company), 10 g/L yeast extract (Becton, Dickinson and Company), and 20 g/L glucose (Kokusan Chemical Co., Ltd., Tokyo, Japan). After culturing, 1 mL of the enriched bacterial solution was centrifuged at 15,000 × g for 3 min.

Identification of isolated microorganisms

The supernatant was removed to obtain pellets for DNA extraction. Bacterial DNA extraction was performed using Nucleospin (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. After DNA extraction, the 16S rRNA region of the bacteria and the D1 / D2 large-subunit (LSU rRNA region of yeast were amplified using a GeneAmp 9700 Thermal Cycler) (Life Technologies, Carlsbad, CA, USA). For the 16S rRNA region and D1 / D2 region, universal primers 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′), NL1 (5′-GCA TAT CAA TAA GCG GAG GAA AAG -3′), and NL4 (5′-GGT CCG TGT TTC AAG ACG G-3′) were used, respectively. The polymerase chain reaction (PCR) conditions for bacteria were as follows: initial denaturation at 94°C for 4 min, 30 cycles of amplification (94°C for 30 s, 58°C for 1 min, and 72°C for 1 min), and final extension at 72°C for 4 min. The PCR conditions for yeast were as follows: initial denaturation at 94°C for 5 min, 40 cycles of amplification (94°C for 30 s, 51°C for 1 min, and 72°C for 5 min), and final extension at 72°C for 5 min. The PCR products were purified using AM Pure XP (Beckman Coulter, Brea, CA, USA) and sent to Eurofins Genomics (Eurofins Genomics, Tokyo, Japan) for sequencing with the 27F and NL1 primers. The basic local alignment search tool (BLAST) algorithm was used to compare the derived sequences with the 16S rDNA sequences or 26S rRNA sequences in the DNA Data Bank of Japan database (http://blast.ddbj.nig.ac.jp/blastn, Shizuoka, Japan).

Result and discussion

Distribution of psychrophilic microorganisms in equipment and lines

Slaughtering line

Despite being swabbed after cleaning, all equipment used in the slaughtering line, except for the dehider (edge side), Brisket saw (edge side), and milling cutter (handle side), were found to have microbial adherence in at least one trial (Table 1). Escherichia coli and coliform bacteria, both of which are mesophilic bacteria, were not detected in any of the trials from the equipment used in the slaughter and boning lines. Among the equipment used in the slaughtering line, the brisket saw (handle side) and trolley hook were confirmed to be contaminated with microorganisms in the majority of trials, and the positive rate of microorganisms was 66.7% (4 times was positive among 6 time sampling). The microorganism adhesion rate of milling cutters (edge side) and aprons was 50.0%, while those of the foot cutter (edge and handle side), splitting saw (edge side), and knife (handle side) were 33.3%, and those of dehider (handle side), splitting saw (handle side), and knife (edge side) were 16.7%.

Table 1

The number of psychrophilic microorganisms adhering to equipment used in slaughtering lines.

Zone where equipment is used	Sampling point	The side that was wiped off	Bacterial counts in each medium (log CFU/ml) **												Positive rate (%)
			Trial-1		Trial-2		Trial-3		Trial-4		Trial-5		Trial-6
			2019/7/31		2019/8/7		2020/7/29		2020/8/5		2021/1/7		2021/1/13
			TSA	MRS	TSA	MRS	TSA	MRS	TSA	MRS	TSA	MRS	TSA	MRS
Dirty zone	Foot cutter	Edge side	N.D.	N.D.	2.9	N.D.	N.D.	N.D.	N.D.	N.D.	1.4	1.7	N.D.	N.D.	33.3
		Handle side	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	2.8	2.7	4.4	2.3	N.D.	N.D.	33.3
	Dehider	Edge side	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	0.0
		Handle side	N.D.	N.D.	5.1	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	16.7
Clean zone	Brisket saw	Edge side	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	0.0
		Handle side	6.1	N.D.	5.3	N.D.	7.7	N.D.	N.D.	N.D.	3.1	N.D.	N.D.	N.D.	66.7
	Splitting saw	Edge side	N.D.	2.6	1.8	2.0	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	33.3
		Handle side	N.D.	N.D.	2.6	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	16.7
	Milling cutter	Edge side	N.D.	N.D.	N.D.	2.2	N.D.	N.D.	7.4	N.D.	N.D.	N.D.	6.4	N.D.	50.0
		Handle side	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	0.0
	Knife	Edge side	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	5.6	N.D.	16.7
		Handle side	N.D.	N.D.	N.D.	N.D.	7.4	N.D.	N.D.	N.D.	N.D.	N.D.	6.8	N.D.	33.3
Both zone	Apron*	-	N.D.	N.D.	5.5	3.7	6.3	N.D.	1.4	N.D.	N.D.	N.D.	N.D.	N.D.	50.0
	Trolley hook	-	2.4	N.D.	4.2	N.D.	2.0	1.8	N.D.	N.D.	N.D.	N.D.	2.5	N.D.	66.7

*Bacteria counts were calculated as log CFU/cm2

** E. coli and coliform bacteria were not detected in any of the trials from the equipment used in the slaughtering lines.

The Federation of Hida Meat Agricultural Cooperative Association, investigated in this study, was certified for FSSC22000 (certificate of registration no: JMAQA-FC126) and ISO22000: 2018 (certificate of registration no: JMAQA-F005) in the process of slaughtering and dismantling cattle and cutting carcasses. At this facility, wiping inspections of the processing line and equipment are performed once a week after cleaning as part of hygiene management to evaluate cleaning efficiency. The inspection targets medium-temperature bacteria, which can stimulate growth at 30–37°C. If contamination with these bacteria is confirmed, the cleaning operator is alerted, and thorough cleaning and microbial inspection are carried out until no microbial adhesion is detected. By monitoring the results of the wiping inspection for each cleaning operation, it is possible to confirm whether the hygiene management is adequate. Since the medium-temperature bacteria targeted here include food-poisoning bacteria such as pathogenic E. coli and Salmonella, they are critical for food safety. Coliforms were not detected in any of the sampling trials conducted in this study’s equipment or lines. However, residual microorganisms were found in many samples (Table 1). As a result, it was proposed that hygiene management monitoring of psychrophilic bacteria and medium temperature bacteria can be fully utilized to evaluate the cleaning effect. Furthermore, during long-term refrigeration, psychrophilic bacteria can grow in beef and cause spoilage. If psychrophilic bacteria can be removed entirely from the equipment and lines through daily cleaning, it is considered that the adhesion of microorganisms to the carcass can be reduced, allowing the beef’s expiration date to be extended.

The foot cutter and dehider are used in the dirty zone of the slaughtering line. At the beginning of the slaughter process, a foot cutter was used to cut the cattle’s paw, and microbial loads of 1.4 to 2.9 log CFU / mL (Trial-2, 5) on TSA medium and 1.7 log CFU / mL (Trial 5) on MRS medium were confirmed on the edge side (Table 1). Furthermore, a microbial load of 2.8 to 4.4 log CFU / mL (Trial-4, 5) was confirmed in TSA medium and 2.3 to 2.7 log CFU / mL (Trial-4, 5) in MRS medium on the handle side of the foot cutter. After the cattle carcass is suspended, a dehider is used to skin the body. Bacteria were not detected from the edge side of the dehider, and 5.1 log CFU / mL was detected in TSA medium only in Trial 2 from the handle side. The microbial load attached to the hide and paw of cattle is high [3, 13]. Therefore, it is assumed that the number of bacteria adhering to the work equipment in the dirty zone was also high. The dehider has a lower microorganism detection rate than the foot cutter, and it is thought that the dehider has a lower chance of directly touching the hide during the skinning process. In addition, in the facility used in this experiment, the edges of knives and saws were sterilized by soaking in hot water at 83°C after each treatment. Although hot water disinfection is also performed on the foot cutter, it is hypothesized that the dehider has less microbial contamination accumulating during the work. As a result, the microbial contamination may be lower after cleaning. In the first step of the slaughtering line, there are many opportunities to come into contact with highly contaminated cattle parts such as paws and hides, and the number of bacteria after washing was high. This type of equipment requires thorough cleaning to prevent microbial contamination.

Next, we concentrated on the equipment used in the slaughtering line’s clean zone (Brisket saw, splitting saw, milling cutter, and knife). Brisket saws are used to make cuts near the sternum of a cow, and no bacteria were detected from the edge side in any of the trials. On the handle side, high microbial contamination was confirmed in the TSA medium, with the number of bacteria ranging from 3.1 to 7.7 log CFU / mL (Trial-1, 2, 3, 5). The splitting saw is a machine that divides whole beef carcasses in half, and the microbial counts of the edge side are 1.8 log CFU / mL (Trial-2) in TSA medium and 2.0 ~ 2.6 log CFU / mL (Trial-1, 2) in MRS medium. On the handle side, a bacterial count of 2.6 log CFU / mL (Trial-1) was recorded on TSA medium. A milling cutter is a machine used to remove the dura mater of the carcass after it has been split. On the edge side, a bacterial count of 2.2 log CFU / mL (Trial-2) was confirmed in MRS medium, and a high bacterial count of 6.4 to 7.4 log CFU / mL (Trial-4, 6) was detected in TSA medium. On the other hand, the number of bacteria on the handle side was below the detection limit in all trials. Knives are typically used for carcass trimming, with 5.6 log CFU / mL detected (Trial-6) in TSA medium on the edge side and 6.8 ~ 7.4 log CFU / mL (Trial-3, 6) on TSA medium on the handle side. A large number of microorganisms were detected on both sides of the knife.

The parts of the instrument where microbial contamination is likely to remain vary. While brisket saws and knives tend to have contamination on the handle side, splitting saws and milling cutters tend to have contamination on the edge side. The shape of the device may be related to the ease of cleaning. The brisket saw’s cutting edge is relatively simple, whereas the shape of the cutting edge of the splitting saw and milling cutter is complicated, and meat residue is easily caught (S1 Fig). This suggests that it is necessary to select an appropriate cleaning method for its shape when cleaning equipment. In addition, for equipment such as brisket saws and knives that tend to remain contaminated on the handle side, measures such as disinfecting after cleaning on the handle side are required.

Bacterial counts of 1.4 to 6.3 log CFU / cm2 (Trial-2, 3, 4) on TSA medium and 3.7 log CFU / cm2 (Trial 2) on MRS medium were measured from the aprons worn by workers on the slaughter line. In particular, Trial 3 had a high bacterial count. Each time, a swab was taken from the aprons of randomly selected workers, but the detection rate was as high as 50%, and the number of bacteria also large. Aprons can be easily washed in a washing machine compared to other equipment. Therefore, it was determined that the bacterial adhesion to the apron was caused by improper cleaning, and thorough employee guidance was required. The trolley hook is used to hook the stunned cattle’s leg and suspend the carcass, and its bacterial count ranges from 2.0 to 4.2 log CFU / mL (Trial-1, 2, 3, 6) in TSA medium and 1.8 log CFU / mL in MRS medium. The bacterial detection rate was the highest at 66.7%, while the number of bacteria was relatively low.

Boning line

Compared to the slaughtering line, the distribution of microorganisms on the boning line was mainly concentrated on the electric saw (handle side) (Table 2). In Trial 6, only 0.8 log CFU / cm2 were detected in the TSA medium from the turntable. An electric saw is a machine used to cut up carcasses into smaller parts, and no bacteria were detected from the edge side in any of the trials. On the other hand, from the handle side, 2.6 to 5.3 log CFU / mL (Trial-1, 4, 7) was detected in TSA medium, and 1.7 to 5.3 log CFU / mL was detected in MRS medium (Trial-1, 4, 5). The electric saw can be cleaned by removing the blade, but the main body, including the handle, cannot be washed with water (S2 Fig). Therefore, it was difficult to remove the microbial contamination accumulated during the work, and it was estimated that the detection rate of microorganisms was as high as 57.1%.

Table 2

The number of psychrophilic microorganisms adhering to equipment used in boning lines.

Sampling point	Bacterial counts in each medium (log CFU/ml) **														Positive rate (%)
	Trial1		Trial2		Trial3		Trial4		Trial5		Trial6		Trial7
	2019/8/7		2019/12/12		2019/12/18		2020/7/29		2020/8/5		2021/1/7		2021/1/13
	TSA	MRS	TSA	MRS	TSA	MRS	TSA	MRS	TSA	MRS	TSA	MRS	TSA	MRS
Large division conveyor belt*	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	0.0
Boning conveyor belt*	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	0.0
Turntable*	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	0.8	N.D.	N.D.	N.D.	14.3
Meat holder	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	0.0
Electric saw-Edge side	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	0.0
Electric saw-handle side	5.3	5.3	N.D.	N.D.	N.D.	N.D.	3.0	2.8	N.D.	1.7	N.D.	N.D.	2.6	N.D.	57.1

* Bacteria counts were calculated as log CFU/cm2

** E. coli and coliform bacteria were not detected in any of the trials from the equipment used in the boning lines.

Identification of psychrophilic microorganisms isolated from equipment and lines

Candida zeylanoides was identified in all isolated colonies on the edge side of the foot cutter used in the dirty zone of the slaughtering line (Table 3). In Trial 5, all isolated colonies on the handle side were identified as C. zeylanoides. Pseudomonas sp., Chryseobacterium sp., Meyerozyma guilliermondii, and Rhodotorula sp., on the other hand, were identified as microorganisms that remained on the handle side of the foot cutter in Trial 4. According to these findings, C. zeylanoides were mainly distributed in the foot cutter regardless of the edge or handle sides. Yeasts such as M. guilliermondii and Rhodotorula sp. were also found. Previous studies have also found yeasts such as C. zeylanoides and Rhodotorula sp. isolated from slaughterhouse lines and equipment [14]. These yeasts are known to be derived from soil and livestock [15]. In addition, all isolated colonies from the dehider used in the cattle skinning process were identified as Rhodococcus sp. is a gram-positive bacterium that is widely distributed on grazing farms and is frequently isolated from the feces of wild animals and livestock, such as pigs, cattle, and horses [16]. Thus, microorganisms derived from cattle and soil are mainly detected in the equipment used in the dirty zone of the slaughtering line. It has been demonstrated that they are not entirely removed by the washing process and remain on the equipment’s surface.

Table 3

Identification results of psychrophilic microorganisms adhering to equipment used in slaughtering line.

Zone where equipment is used	Sampling point	Sampling side	Trial	Isolation medium	Identified genus/ species	The number of isolated colonies
Dirty zone	Foot cutter	Edge side	Trial 2	TSA	Candida zeylanoides	4/4
			Trial 5	TSA	Candida zeylanoides	4/4
				MRS	Candida zeylanoides	4/4
		Handle side	Trial 4	TSA	Pseudomonas sp.	1/4
					Chryseobacterium sp.	1/4
					Meyerozyma guilliermondii	1/4
					Rhodotula sp.	1/4
				MRS	Rhodotula sp.	4/4
			Trial 5	TSA	Candida zeylanoides	4/4
				MRS	Candida zeylanoides	4/4
	Dehider	Handle side	Trial 2	TSA	Rhodococcus sp.	4/4
Clean zone	Brisket saw	Handle side	Trial 1	TSA	Pseudomonas sp.	4/4
			Trial 2	TSA	Pseudomonas sp.	4/4
			Trial 3	TSA	Pseudomonas sp.	4/4
			Trial 5	TSA	Pseudomonas sp.	4/4
	Splitting saw	Edge side	Trial 1	MRS	Rhodotorula sp.	4/4
			Trial 2	TSA	Candida zeylanoides	3/4
					Moraxella osloensis	1/4
				MRS	Candida zeylanoides	4/4
		Handle side	Trial 2	TSA	Candida zeylanoides	3/4
					Rhodococcus sp.	1/4
	Milling cutter	Edge side	Trial 2	MRS	Candida parapsilosis	4/4
			Trial 4	TSA	Pseudomonas sp.	4/4
			Trial 6	TSA	Pseudomonas sp.	4/4
	Knife	Edge side	Trial 6	TSA	Pseudomonas sp.	4/4
		Handle side	Trial 3	TSA	Pseudomonas sp.	4/4
			Trial 6	TSA	Pseudomonas sp.	4/4
Both zone	Apron	-	Trial 2	TSA	Pseudomonas sp.	4/4
				MRS	Yarrowia galli	4/4
			Trial 3	TSA	Pseudomonas sp.	4/4
			Trial 4	TSA	Acinetobacter junii	1/4
					Pseudomonas sp.	3/4
	Trolley hook	-	Trial 1	TSA	Rhodococcus sp.	4/4
			Trial 2	TSA	Rhodococcus sp.	4/4
			Trial 3	TSA	Candida zeylanoides	4/4
				MRS	Sporidiobolus salmomnicolor	2/4
					Candida zeylanoides	2/4
			Trial 6	TSA	Candida zeylanoides	4/4

Isolated colonies on the handle side of the brisket saw used in the clean zone were identified as Pseudomonas sp. in all samples. Yeasts such as C. zeylanoides, Rhodotorula sp., and Candida parapsilosis were frequently found on the edge side of the splitting saw (for splitting carcasses), as well as on the foot cutter. Therefore, it was considered that yeast may have survived on the edge side of the splitting saw after washing. In addition, three of the four colonies isolated on the handle side of the splitting saw were C. zeylanoides, indicating that yeast contamination was distributed on both sides. On the edge side of the milling cutter, C. parapsilosis was predominant in Trial 2, while Pseudomonas sp. was predominant in Trials 4 and 6. All colonies isolated from the edge and handle sides of the knife used for carcass trimming were identified as Pseudomonas sp.

Although this sampling was performed after the washing treatment, yeasts such as Rhodotorula sp. and C. zeylanoides and bacteria including Moraxella osloensis and Pseudomonas sp. were detected from the edge side of the splitting saw, milling cutter, and knife. So far, cleaning plans have focused on gram-negative bacteria that cause food poisoning, but it has been demonstrated that environmental microorganisms, such as yeast, may not be entirely removed by the cleaning process and may remain on the equipment.

Since the edge side of the saw comes into direct contact with the carcass, there is a high possibility that microbial contamination will spread from the carcass. Therefore, it was determined that the cleaning and disinfection method for the edge side needed to be changed to completely remove microorganisms, including yeast. Pseudomonas sp. was found in all samples from the aprons used in the slaughtering process and bacterial groups such as Yarrowia galli and Acinetobacter jejunii. Trolley hooks have been found to contain a wide variety of microorganisms, including C. zeylanoides, Rhodococcus sp., and Sporidiobolus salmonicolor. With the exception of the brisket saw-handle side of Trial-5, all four colonies isolated from each sample were identified as Pseudomonas sp. (Brisket saw-handle side, milling cutter-edge side, knife-both side, apron), and the bacterial count was as high as 5.3 log CFU / mL or more. If Pseudomonas with a high number of bacteria is present on the surface of the equipment, a biofilm may form. Previous research has shown that when Pseudomonas forms a biofilm on the surface of a piece of equipment, it promotes pathogen colonization and activity [17, 18]. Furthermore, other studies have found that Pseudomonas sp. was frequently isolated from the instruments used in cattle and sheep slaughter lines, even after they had been washed [19]. Previously, we conducted a long-term preservation test of beef carcasses slaughtered and dismantled at this slaughterhouse [8]. Under aerobic storage conditions, the bacterial counts were reached at 8–9 log CFU/g, and Pseudomonas spp. predominated the beef microbiota at nine weeks. Numerous studies reported that Pseudomonas spp. is related to meat spoilage under low-temperature conditions [20-23]. Hence, we consider it necessary to remove Pseudomonas spp. from the equipment by washing because of its putrefactive activity.

This study did not investigate whether microorganisms present in the meat are the same as those identified on the equipment and surfaces. To clarify this, strain identification using molecular typing methods, such as RAPD, MLST, and ribotyping, need to be performed [24-29]. Further detailed research should be carried out in the future to determine what kinds of equipment and bacterial species are likely to contaminate the surface of carcasses.

All isolated colonies from the turntable on the boning line were identified as C. zeylanoides (Table 4). In addition, C. zeylanoides were identified on the electric saw’s handle side in all trials except Trial 7. In contrast, all Pseudomonas sp. colonies were identified in Trial 7. Only C. zeylanoides and Pseudomonas sp. were detected in the boning line, whereas numerous yeast species were found in the slaughtering line. It is presumed that the reason for this is that a large number of contaminated microorganisms derived from living organisms such as the hide and feces are introduced into the slaughtering line, and the number and types of bacteria adhering to the equipment are numerous. The boning line, on the other hand, is the process of removing bone and shaping the carcass. It is believed that the majority of the bacteria brought in are derived from bacteria attached to the carcass after slaughter.

Table 4

Identification results of psychrophilic microorganisms adhering to equipment used in the boning line.

Sampling point	Trial	Isolation medium	Identified genus/ species	The number of isolated colonies
Turntable	Trial 6	TSA	Candida zeylanoides	4/4
Electric saw-handle side	Trial 2	TSA	Candida zeylanoides	4/4
		MRS	Candida zeylanoides	4/4
	Trial 4	TSA	Candida zeylanoides	4/4
		MRS	Candida zeylanoides	4/4
	Trial 5	MRS	Candida zeylanoides	4/4
	Trial 7	TSA	Pseudomonas sp.	4/4

In the past, the distribution of spoilage yeasts was investigated with instruments in wineries, bakery industries, breweries, yogurt factories or goat cheese industries [30-34]. The processed meats industry also investigated the distribution of yeast across the instruments used in the industry and identified the surface of facilities, including room equipment and production materials, as the main source of yeast contamination of cured meat [35]. In particular, Candida zeylanoides is frequently isolated from cured meat, and considered predominant in the raw material, fresh meat. In this study, Candida zeylanoides was frequently isolated from the equipment used in the boning line, suggesting that the slaughterhouse, which is the most upstream food distribution center, may be a source of yeast contamination of meat.

Microbial contamination was concentrated on the handle side of the electric saw, where microbes were frequently detected in multiple trials. The concentration of disinfectants, required to remove yeast, was higher than that of food-related bacteria [36]. Salo et al. (2005) demonstrated that alcohol-based disinfectants were most effective in decontaminating yeast isolates [37]. In addition, surfactant-based and peroxide-based disinfectants were effective against floating yeast cells, while biofilm carrier tests reported the effectiveness of chlorine-based foam cleaners. On the other hand, disinfectants containing chlorine and persulfates have failed to kill yeast cells in both suspensions and biofilm formation. As the susceptibility of bacteria and yeast to disinfectants is different, a thorough review of cleaning methods for yeast contaminated equipment is warranted.

Conclusion

The purpose of this study was to determine the distribution of psychrophilic microorganisms that remained on the equipment and lines in the slaughtering and boning lines after cleaning. Many of the equipment used in the slaughtering line have complicated shapes. The microbial contamination varies depending on the site, the attached microorganisms are diverse, and they are environment-derived microorganisms. On the other hand, on the boning line, the equipment with residual microbial contamination and their bacterial species showed the same tendency in multiple trials. It is thought that providing information to the meat industry on equipment and parts where microorganisms are difficult to remove by washing treatment can further improve hygiene management.

Photograph of the equipment used in the slaughtering line.

(TIF)

Click here for additional data file.

Photograph of the equipment used in boning line.

(TIF)

Click here for additional data file.

23 Feb 2022

Please submit your revised manuscript by Apr 07 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Guadalupe Virginia Nevárez-Moorillón, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf.

2. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript “Distribution of psychrophilic microorganisms in the beef slaughterhouse after cleaning” aimed to explore the microbial contamination present in a beef slaughterhouse in Japan. The topic is definitely relevant and warranted, but the manuscript is very poorly written. It needs substantial improvements, both on the scientific language and structure.

The goal of the study cannot be met with this experimental design, and the study also lacks discussion of the results – only 18 references given.

Abstract

L19: give details on the methods used to determine microbial contamination

L22: “Petri dish”… how did you select the colonies?

L24: WGS or other sequencing technology? If you also looked for yeasts, you need to correct “the bacterial species” that you mention before.

L27: leave this conclusion for the end… where do you think the overall high contamination comes from and how did you investigate environmental contamination?

Introduction

L41: what about Japan? What is the status quo? You keep mentioning the international point-of-view, but since your samples were collected in Japan (an in only one facility), you should start from there and then compare with the international practices.

L62: cannot be stopped? Then why do we do it?

L66: remove “to zero”

Materials and Methods

L86: “Materials”

L89: why not before?

L93: what is the wiping inspection?

L87: Please provide a schematic figure with all sampling points.

L108: what is the difference between these media e.g., what are they selective for?

L110: which dilution did you plate?

L136: “)” missing

L120: please split this in two subheadings

Results and Discussion

L164: how did you calculate the adhesion rate?

How do you explain the distribution of Pseudomonas sp.? Additionally, do you mean sp. or spp.?

Reviewer #2: The objective of this study was to investigate the abundance and distribution of psychrophilic microorganisms associated with spoilage in beef slaughterhouse environments after cleaning. The topic is interesting, but there are two relevant aspects that weaken the work. The count of psychrophilic was carried out in different sectors of a slaughterhouse, but this does not imply that they are associated with meat contamination and its subsequent alteration. Since the psychrophilic microorganisms present in the meat were not identified, it was not possible to associate whether the contamination found in the slaughterhouse environment had any relevance to the meat quality. On the other hand, the number of colonies isolated for later identification was low and it is not possible to establish the diversity of microorganisms present or their load on the equipment.

Line 30: It is concluded that, despite the cleaning system used in the slaughterhouse, the concentration of psychrophilic was "very high". However, no parameters are given to support that this load was "very high".

The introduction seems to be very long. Authors should focus on the most important aspects that support their work.

Lines 81-82: "The ultimate goal of this study was to reduce contamination of carcasses by microorganisms from slaughter processing lines and equipment". This objective was not addressed in this work.

Lines 190-192: "If psychrophilic bacteria can be removed entirely from the equipment and lines through daily cleaning, it is considered that the adhesion of microorganisms to the carcass can be reduced, allowing the beef’s expiration date to be extended". It is quite obvious that if microorganisms are completely removed from equipment and surfaces in slaughterhouses, contamination of meat will be greatly reduced. However, the goal of a cleaning system is not sterilization but the reduction to acceptable levels. Therefore, observed microorganisms concentration should be compared with expected concentrations to determine their impact. This is not addressed in this work.

Lines 275-276: It is M&M.

Line 321: It just remains to check if the microorganisms present in the meat are the same as those identified in the equipment and surfaces. So, It seems to be a hypothesis (logical) or a speculation.

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

17 Mar 2022

Response to Reviewers

Thank you for inviting us to submit a revised draft of our manuscript entitled, “Distribution of psychrophilic microorganisms in the beef slaughterhouse after cleaning” to PLOS ONE. We also appreciate the time and effort you and each of the reviewers have dedicated to providing insightful feedback on ways to strengthen our paper. Thus, it is with great pleasure that we resubmit our article for further consideration. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We also hope that our edits and the responses we provide below satisfactorily address all the issues and concerns the reviewers have noted.

Dear Reviewer #1:

�  Abstract

L19: give details on the methods used to determine microbial contamination

(Response)

Thank you for the suggestion.

As recommended, I added detailed information about the microbial investigation.

(Page 2, Line 18–21): The processing lines and equipment used in slaughtering and boning were swabbed, and the microbial count was determined by the culture method using a TSA and MRS medium and Chromocult® Coliform agar incubated at 15ºC and 37ºC, respectively.

L22: “Petri dish”… how did you select the colonies?

(Response)

Thank you for your question. I investigated the predominant microbial species attached to the equipment’s surface. I randomly selected four colonies from one plate. I added detailed information to the original manuscript as follows:

(Page 2, Line 23–25) Next, four colonies were randomly isolated from the one petri dish used for the bacterial count measurement to identify the predominant microbial species of the microorganisms attached to each equipment.

L24: WGS or other sequencing technology? If you also looked for yeasts, you need to correct “the bacterial species” that you mention before.

(Response)

Thank you for your accurate instruction. I used sanger sequencing technology and I added the information to the manuscript (Page 2, Line 26). Also, I changed “the bacterial species” to “the microbial species” (Page 2, Line 25).

L27: leave this conclusion for the end… where do you think the overall high contamination comes from and how did you investigate environmental contamination?

(Response)

Thank you for your question. This study did not investigate environmental contamination. However, the Discussion section cites that these yeasts and bacteria were isolated from soil and livestock (Page 20, Line 286–92).

�  Introduction

L41: what about Japan? What is the status quo? You keep mentioning the international point-of-view-, but since your samples were collected in Japan (an in only one facility), you should start from there and then compare with the international practices.

(Response)

Thank you for your insightful feedback. In fact, in Japan, introducing HACCP became mandatory in slaughterhouses from June 2021. I should have included information about this in the manuscript. Thank you for pointing it out. I modified the document as follows:

(Page 4, Line 42–45) Process control by the Hazard Analysis and Critical Control Point (HACCP) system is one measure to prevent the contamination of carcasses by such harmful bacteria. In Japan, following the EU and the United States, introducing the HACCP system in slaughterhouses became mandatory from June 2021.

L62: cannot be stopped? Then why do we do it?

(Response)

I'm sorry that my previous sentence lacked an explanation. Beef chilled shipping exports can be transported while preserving beef quality compared to frozen conditions. However, low-temperature-growing microorganisms can grow and cause quality degradation in beef. It is important to reduce microorganisms that adhere to beef as much as possible to prevent such a scenario. I corrected the sentence as follows:

(Page 5, Page 63–65) However, long transportation periods can allow some low-temperature-growing microorganisms to grow and degrade beef quality.

L66: remove “to zero”

(Response)

Thank you for your instruction. I remove “to zero”.

�  Materials and Methods

L86: “Materials”

(Response)

I apologize for simple mistake. I corrected it.

L89: why not before?

(Response)

Thank you for your critical question.

This study aimed to investigate the distribution of psychrophilic microorganisms in the equipment used in the slaughtering and boning lines and identify the equipment on which microbial contamination is likely to remain after washing. Therefore, we targeted the equipment after cleaning. By feeding back information about difficult-to-clean equipment to the field workers, they can select an appropriate cleaning method and expect further improvement in hygiene management.

L93: what is the wiping inspection?

(Response)

I apologize. My previous sentence was not appropriate. I changed the sentence as follows:

(Page 7, Line 89-90) Cleaning was carried out after each line was used, and the equipment was wiped the day after cleaning.

L87: Please provide a schematic figure with all sampling points.

(Response)

Thank you for your suggestion. This study aimed to investigate the distribution of psychrophilic microorganisms that adhere to equipment after cleaning, and we do not discuss the transmission of microorganisms in slaughterhouse. Therefore, I decided that the schematic figure should not be included. Instead, we added a photo of the equipment targeted in this study to the supplementary figure. In the future, when conducting research such as strain typing to explore the dynamics of microorganisms in the slaughterhouse, I would like to attach a schematic figure as you suggested. Thank you for your valuable opinion.

L108: what is the difference between these media e.g., what are they selective for?

(Response)

Thank you for your question. I added the information regarding the media as follows:

(Page 8, Line 110–114) The number of bacteria in each swab sample was measured on three types of media: trypticase soy agar (TSA; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) for general bacteria, de Man, Rogosa, and Sharpe (MRS) agar (Merck KGaA, Darmstadt, Germany) for lactic acid bacteria, and Chromocult®︎ Coliform Agar (Merck) for coliform bacteria and E. coli.

L110: which dilution did you plate?

thank you for your question. I added the information as follows:

(Response)

(Page 8, Line 108-109) The swab sample was diluted ten-fold with saline solution, and 100 µL of each original solution and diluent were spread onto each agar medium.

L136: “)” missing

(Response)

Thank you for your polite point. I corrected it.

L120: please split this in two subheadings

(Response)

Thank you for your suggestion. I split this paragraph in two subheadings.

�  Results and Discussion

L164: how did you calculate the adhesion rate?

(Response)

Thank you for your question.

We sampled the slaughtering and boning lines six and seven times, respectively. Adhesion rate (positive rate in Tables 1 and 2) indicates the ratio of the number of times that the number of bacteria above the detection limit was confirmed. Due to the previous sentence confusion, I modified it as follows:

(Page 11, Line 161-164) Among the equipment used in the slaughtering line, the brisket saw (handle side) and trolley hook were contaminated with microorganisms in the majority of trials, and the positive rate of microorganisms was 66.7% (four trials were positive among six samplings).

How do you explain the distribution of Pseudomonas sp.?

(Response)

Since Pseudomonas was widely distributed in this slaughterhouse, we presumed that the original contamination from the living body was large. In other studies I am conducting, Pseudomonas accounts for more than 80% of the flora on the carcass surface during the slaughter process. Hence, we assumed that Pseudomonas from the carcass contaminates the equipment and remains there after cleaning.

Additionally, do you mean sp. or spp.?

(Response)

"sp." is used as the singular and "spp." is used as the plural. In this manuscript, all are written as "sp.".

Dear Reviewer #2:

The objective of this study was to investigate the abundance and distribution of psychrophilic microorganisms associated with spoilage in beef slaughterhouse environments after cleaning. The topic is interesting, but there are two relevant aspects that weaken the work. The count of psychrophilic was carried out in different sectors of a slaughterhouse, but this does not imply that they are associated with meat contamination and its subsequent alteration. Since the psychrophilic microorganisms present in the meat were not identified, it was not possible to associate whether the contamination found in the slaughterhouse environment had any relevance to the meat quality. On the other hand, the number of colonies isolated for later identification was low and it is not possible to establish the diversity of microorganisms present or their load on the equipment.

(Response)

Thank you for your insightful comments.

In past studies, we conducted a long-term preservation test of beef carcasses slaughtered and dismantled at this slaughterhouse (Dynamics of microbiota in Japanese Black beef stored for a long time under chilled conditions. Food microbiology, 2021, 100: 103849). That study reported that Pseudomonas sp. was the predominant bacterial group in the meat when stored in aerobic packaging. It is well reported that the Pseudomonas genus is a psychrophilic spoilage-causing bacterium. This study revealed that Pseudomonas sp. was widely distributed in the equipment used in this slaughterhouse. From these results, we believe that the psychrophilic microorganisms identified in this study may adhere to the meat and are associated with spoilage. I added the following to the Discussion section:

(Page 25, Line 333–339) Previously, we conducted a long-term preservation test of beef carcasses slaughtered and dismantled at this slaughterhouse (8). Under aerobic storage conditions, the bacterial counts were reached at 8–9 log CFU/g, and Pseudomonas sp. predominated the beef microbiota at nine weeks. Numerous studies reported that Pseudomonas sp. is related to meat spoilage under low-temperature conditions (20–23). Hence, we consider it necessary to remove Pseudomonas sp. from the equipment by washing because of its putrefactive activity.

I selected four colonies for later identification. Certainly, the resolution is low for investigating bacterial diversity, but it is sufficient for investigating the predominant bacterial group. There were many test plots in which this study identified all four strains of the four selected colonies as belonging to the same genus.

Line 30: It is concluded that, despite the cleaning system used in the slaughterhouse, the concentration of psychrophilic was "very high". However, no parameters are given to support that this load was "very high".

(Response)

Thank you for your opinion. As you mentioned, I should not use “highly contaminated”. I modified the sentence as follows:

(Page 2, Line 31–32) Despite the cleaning operations, this study identified some equipment contaminated with microorganisms.

The introduction seems to be very long. Authors should focus on the most important aspects that support their work.

(Response)

Thank you for your valuable opinion. PLOS ONE has no limit on the number of characters in the manuscript, and few people are familiar with the hygiene management of beef slaughterhouses, so I wrote the introduction with this in mind. I believe that all the paragraphs are important in explaining the background of this study. Some sentences were changed with the reader’s understanding in mind (Page 4, Line 42–45; Page 5, Line 63–65; Page 6, Line 80-82). It would be helpful if you could proceed without cutting the content.

Lines 81-82: "The ultimate goal of this study was to reduce contamination of carcasses by microorganisms from slaughter processing lines and equipment". This objective was not addressed in this work.

(Response)

Thank you for your critical opinion. The previous sentence was a leap forward. I modified the sentence as follows:

(Page 6, Line 80-82) The purpose of this study was to understand the actual contamination of low-temperature microorganisms in the equipment used in the slaughtering and boning lines.

Lines 190-192: "If psychrophilic bacteria can be removed entirely from the equipment and lines through daily cleaning, it is considered that the adhesion of microorganisms to the carcass can be reduced, allowing the beef’s expiration date to be extended". It is quite obvious that if microorganisms are completely removed from equipment and surfaces in slaughterhouses, contamination of meat will be greatly reduced. However, the goal of a cleaning system is not sterilization but the reduction to acceptable levels. Therefore, observed microorganisms concentration should be compared with expected concentrations to determine their impact. This is not addressed in this work.

(Response)

The cleaning system needs to reduce the number of bacteria on the equipment’s surface to below the detection limit. In fact, at this facility, they perform a wiping inspection of the equipment after cleaning once a week, targeting mesophilic bacteria (35–37ºC). If the number of bacteria is measured, they sanitize the equipment again. This study investigated psychrophilic bacteria and the number of Escherichia coli and coliform bacteria, which are mesophilic bacteria. We measured the number of psychrophilic bacteria but not mesophilic bacteria. From this result, it was assumed that psychrophilic bacteria are more likely to remain on the instrument’s surface than mesophilic bacteria. However, no instrument had a 100% detection rate of psychrophilic bacteria on the instrument’s surface after cleaning, indicating that the number of psychrophilic bacteria could be reduced to below the detection limit in all equipment. Therefore, this study regarded the value as unacceptable when the number of bacteria in the equipment after cleaning was above the detection limit.

Lines 275-276: It is M&M.

(Response)

Thank you for your instruction. I deleted the sentence.

Line 321: It just remains to check if the microorganisms present in the meat are the same as those identified in the equipment and surfaces. So, It seems to be a hypothesis (logical) or a speculation.

(Response)

Thank you for your opinion. As you instructed, I modified the sentence as follows:

(Page 25, Line 316-319) This study did not investigate whether microorganisms present in the meat are the same as those identified on the equipment and surfaces. However, since the edge side of the saw comes into direct contact with the carcass, there is a high possibility that microbial contamination will spread from the carcass.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

6 Apr 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Guadalupe Virginia Nevárez-Moorillón, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Title: replace “the” by “a” … in Japan after cleaning

Abstract:

L19: remove “by the culture method”

L21: remove “in most trials”, otherwise you need to give before how many trials were conducted

L22: remove “confirmed to be”

L24: remove “one”

L26: Sanger, not sanger

Can you give the % of contamination or the number of samples e.g., (10/24) where you found the microorganisms? Gives an idea of the prevalence of contamination, which is an easy an d interesting info to extract directly from the Abstract

Results and discussion:

This section is very poorly discussed. There is indeed a. lot of detail given, but barely any discussion with the available literature. This is not a report, it is a research article.

L335-339: Pseudomonas should be italicized. This section also reflects my previous comment: it should be spp., as you would expect several species of Pseudomonas to be found, not just one specific species (for those cases, use sp., when you are referring to a specific Pseudomonas).

Reviewer #2: The authors have adequately addressed my previous comments. Although I have a disagreement with the interpretation of some points, it is not a sufficient reason not to accept the modifications made. I only have one comment left that I consider to be a weakness of the study but to be resolved in future works.

I understand and agree with the authors that Pseudomonas are the most representative populations of psychrophilic microorganisms in meat. However, from this study, you cannot assume that the microorganisms present in the equipment are the same as those on the carcasses. For this, it should resort to molecular studies.

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

28 Apr 2022

Response to Reviewers

Thank you for inviting us to submit a revised draft of our manuscript entitled, “Distribution of psychrophilic microorganisms in a beef slaughterhouse in Japan after cleaning” to PLOS ONE. We also appreciate the time and effort you and each of the reviewers have dedicated to providing insightful feedback on ways to strengthen our paper. Thus, it is with great pleasure that we resubmit our article for further consideration. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We also hope that our edits and the responses we provide below satisfactorily address all the issues and concerns the reviewers have noted.

Dear Reviewer #1:

Thank you for your detailed suggestions regarding the Abstract. Thanks to these, the text has been vastly improved. With regard to the Discussion section, because there are currently no published reports of studies similar to the one we have carried out, it was difficult to compare and discuss our results with other findings side by side. However, we have deepened the discussion as much as possible. Thank you for pointing this out.

Title: replace “the” by “a” … in Japan after cleaning

(Response)

Thank you for the suggestion. As recommended, I modified the title.

Abstract:

L19: remove “by the culture method”

(Response)

Thank you for your instruction. I removed “by the culture method”

L21: remove “in most trials”, otherwise you need to give before how many trials were conducted

(Response)

Thank you for your instruction. I removed “in most trials”

L22: remove “confirmed to be”

(Response)

Thank you for your instruction. I removed “confirmed to be”

L24: remove “one”

(Response)

Thank you for your instruction. I removed “one”

L26: Sanger, not sanger

(Response)

Thank you for your correction. I corrected it.

Can you give the % of contamination or the number of samples e.g., (10/24) where you found the microorganisms? Gives an idea of the prevalence of contamination, which is an easy and interesting info to extract directly from the Abstract

(Response)

Thank you for your suggestion. I have added the information about the percentages of contamination, as indicated below. Thanks to you, the Abstract text is now better.

Page 2, Lines 21–25: As a result, the brisket saw (handle side) and trolley hook were the most heavily contaminated with microorganisms, with each having a microbial adhesion rate of 66.7%. The microbial adhesion rates of the apron and milling cutter (edge side) were 50%, respectively, and those of the foot cutter (edge and handle side), splitting saw (edge side), and knife (handle side) were 33.3%, respectively.

Results and discussion:

This section is very poorly discussed. There is indeed a lot of detail given, but barely any discussion with the available literature. This is not a report, it is a research article.

(Response)

Thank you for your helpful suggestions. To the best of our knowledge, there are no published papers on studies that have investigated the distribution of psychrophilic microorganisms on different slaughterhouse equipment after cleaning. Therefore, a discussion based on available literature findings was difficult. However, we have deepened our discussion on the following aspects: why contamination was high in the specific equipment (Lines: 206–218, 238–246, 268-271), the type of bacteria attached (Lines: 286–295, 353–361), and which of those bacteria are involved with putrefactive risks (Lines: 330-342, 361-364). Additionally, in response to your suggestions, we have added following discussion section regarding yeast contamination of food industry and yeast susceptibility against disinfectant. Thank you for your valuable opinion.

Page 27, Line 364-373: In the past, the distribution of spoilage yeasts was investigated with instruments in wineries, bakery industries, breweries, yogurt factories or goat cheese industries. The processed meats industry also investigated the distribution of yeast across the instruments used in the industry and identified the surface of facilities, including room equipment and production materials, as the main source of yeast contamination of cured meat [35]. In particular, Candida zeylanoides is frequently isolated from cured meat, and considered predominant in the raw material, fresh meat. In this study, Candida zeylanoides was frequently isolated from the equipment used in the boning line, suggesting that the slaughterhouse, which is the most upstream food distribution center, may be a source of yeast contamination of meat.

Page 28, Line 375-385: Microbial contamination was concentrated on the handle side of the electric saw, where microbes were frequently detected in multiple trials. The concentration of disinfectants, required to remove yeast, was higher than that of food-related bacteria [36]. Salo et al. (2005) demonstrated that alcohol-based disinfectants were most effective in decontaminating yeast isolates [37]. In addition, surfactant-based and peroxide-based disinfectants were effective against floating yeast cells, while biofilm carrier tests reported the effectiveness of chlorine-based foam cleaners. On the other hand, disinfectants containing chlorine and persulfates have failed to kill yeast cells in both suspensions and biofilm formation. As the susceptibility of bacteria and yeast to disinfectants is different, a thorough review of cleaning methods for yeast contaminated equipment is warranted.

L335-339: Pseudomonas should be italicized. This section also reflects my previous comment: it should be spp., as you would expect several species of Pseudomonas to be found, not just one specific species (for those cases, use sp., when you are referring to a specific Pseudomonas).

(Response)

Thank you for pointing out these errors. I apologize for overlooking the italicization of bacterial names and this has since been corrected. Also, as you pointed out, when multiple Pseudomonas species are being described, they are represented as “spp.”

Dear Reviewer #2:

I understand and agree with the authors that Pseudomonas are the most representative populations of psychrophilic microorganisms in meat. However, from this study, you cannot assume that the microorganisms present in the equipment are the same as those on the carcasses. For this, it should resort to molecular studies.

(Response)

We agree totally with what you say. It is necessary to use molecular typing methods (MLST, RAPD, ribotyping, etc.) to determine whether the bacteria attached to the equipment actually propagate to the carcass surface. With regard to this point, the following has been added in the relevant part as the limitation of this research. Thank you for your valuable feedback. We would like to incorporate your opinions into our Future work.

Page 26, Lines 344-349: This study did not investigate whether microorganisms present in the meat are the same as those identified on the equipment and surfaces. To clarify this, strain identification using molecular typing methods, such as RAPD, MLST, and ribotyping, need to be performed [24-29]. Further detailed research should be carried out in the future to determine what kinds of instruments and bacterial species are likely to contaminate the surface of carcasses.

Submitted filename: renamed_3070f.docx

Click here for additional data file.

29 Apr 2022

Distribution of psychrophilic microorganisms in a beef slaughterhouse in Japan after cleaning

PONE-D-22-01666R2

Dear Dr. Takahashi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Guadalupe Virginia Nevárez-Moorillón, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

8 Jul 2022

PONE-D-22-01666R2

Distribution of psychrophilic microorganisms in a beef slaughterhouse in Japan after cleaning

Dear Dr. Takahashi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Guadalupe Virginia Nevárez-Moorillón

Academic Editor

PLOS ONE