Pork Loin Treated with High Hydrostatic Pressure as a Food Processing Technology: Subacute Toxicity of the Freeze-Dried Powder and Cytotoxicity of the Methanol Extracts

Abstract

High hydrostatic pressure (HP) treatment is used in food processing owing to its sterilization effect. Meat or meat products are sterilized and become tender by HP processing. Therefore, the variety of HP-processed meat products has increased worldwide. However, little is known about the safety of HP-processed meat products. The aim of this study was to determine the effects of HP processing and HP combined with 0.4 M sodium carbonate treatment (HP-Na) on pork loins and to evaluate the subacute toxicity and cytotoxicity of these processing methods. In an in vivo study, we performed 90- and 180-day feeding tests in mice and did not detect any adverse effects in HP-processed and HP-Na-processed pork loins. In addition, we evaluated the cytotoxicity of HP-processed meats, and did not observe any obvious toxicity associated with pork loin extracts in vitro. These results suggest that HP is not associated with risk factors during processing.

1. Introduction

High hydrostatic pressure (HP) is a food processing technology that uses pressures exceeding 100 MPa. In contrast to other food processing modalities, such as heat, HP has a limited effect on covalent bond and thus preserves the chemical properties of treated targets without degrading flavor, color, and nutritional factors, eg, the vitamin content^1,2,3,4,5). Jam was the first HP-processed food product in the Japanese food market in the 1990s⁶⁾. The primary benefit of HP is its sterilization effect; many studies have reported that HP inhibits the proliferation of toxic microorganisms^7,8,9). For instance, HP can reduce pathogenic microbes, such as Listeria^10,11,12,13), which frequently contaminate meat and meat products. Owing to the additional benefits of HP, such as its effect on texture, the method is used for meat processing worldwide^9,14). However, few scientific studies have evaluated the safety of HP-processed meat products.

Food assessments, including evaluations of food safety, generally follow the guidelines of the Organization for Economic Co-operation and Development (OECD), the World Health Organization (WHO), etc^15,16). These guidelines have shown the animal experiments required for safety assessment using various doses and feeding periods with pathological observations¹⁵⁾.

Consumers are often skeptical of food that is processed by new technologies¹⁷⁾. Bruhn et al. reported that the acceptance of irradiated products is high when consumers are presented with accurate information¹⁸⁾ and that communication plays an important role in the acceptance of a new food-processing technology¹⁷⁾. In the case of HP-processed food products, including meat, awareness regarding this technology among consumers is still low, and these products have been consumed without clear knowledge of their safety¹⁹⁾. The investigation and presentation of scientific information regarding the safety of food products that are processed using a new method is critical.

The aim of this study was to estimate the food safety of HP-processed pork loins based on in vivo and in vitro experiments. We performed 90-day and 180-day feeding tests with mice in line with OECD guidelines for HP-processed pork loins and pork loins processed with HP in combination with sodium bicarbonate treatment (HP-Na processing), respectively. Furthermore, the cytotoxic effects of HP-processed and HP-Na-processed pork loins were evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and a cell cycle analysis with five human cell lines.

2. Materials and Methods

2-1. Chemicals

Solvents were of special grade unless otherwise stated. Methanol (MeOH), dimethyl sulfoxide (DMSO) and sodium carbonate were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Three-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and propidium iodide (PI) were obtained from Dojindo Laboratories (Kumamoto, Japan). Dulbecco’s Modified Eagles Medium (DMEM), Roswell Park Memorial Institute 1640 Medium (RPMI 1640 Medium) and ribonuclease A (RNase) were procured from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Invitrogen (Carlsbad, CA, USA) and penicillin-streptomycin was purchased from Life Technologies SAS (Cergy Pontoise, France).

2-2. Animal Study

2-2-1. Study design

The 90-day and 180-day feeding tests were performed in line with OECD guidelines for Testing Chemicals, No. 408¹⁵⁾.

2-2-2. Animals and housing

Male BALB/c mice of 8-weeks old were obtained from Japan SLC, Inc. (Shizuoka, Japan). The mice were allowed to acclimatize for one week before the initiation of the feeding test. All experiments were approved by the animal experiments committee of Niigata University of Pharmacy and Applied Life Sciences (NUPALS), and tests were performed at the laboratory animal facility of NUPALS in a specific pathogen-free conditioned room with a temperature of 25 ± 3°C and a 12-h light/dark cycle.

2-2-3. Preparation of HP and HP-Na pork-loins

HP-processed and HP-Na-processed pork loin samples were prepared using a previously established method²⁰⁾. Briefly, pork loin samples were shaped into blocks of approximately 1 × 10 × 15 cm after removing the fat. HP-processed and HP-Na-processed pork loins were placed in vacuum-sealed polyethylene bags with deionized water or 0.4 M sodium bicarbonate water, respectively, and the bags were incubated for 40 min at 20°C. Then, the samples were removed from the soaking solution and transferred to the other bags, which were filled with distilled water. Samples were treated for 10 min at 400 MPa at 20°C using a high-pressure food processor (Dr.CHEF; Kobe Steel, Ltd., Kobe, Japan). After pressurization, each packed meat sample was heated for 30 min at 80°C in a standing water bath. After incubation, each sample was removed from the polyethylene bag and freeze-dried for 72 h after cooling. The water content of samples was shown in Table 1. Freeze-dried samples were powdered by ball milling (New Power Mill; Osaka Chemical Co., Ltd. Osaka, Japan) and stored at −20°C until use.

Table 1. Water Content and the Residue of Methanolic Extract

	Water content (%)	Freeze-dried pork sample (g)	80% MeOH extract residue (g)
Pork	70.7	30.0	3.7
HP Pork	70.1	30.0	3.8
HP-Na Pork	74.0	30.0	3.9

Water content of sample was calculated using the following equation:

Water content (%) = (Weight _Raw) − (Weight _Dry) / Weight _Dry × 100.

Pork, unprocessed pork loin; HP Pork, HP-processed pork loin; HP-Na, HP-processed and sodium-treated pork loin (HP-Na processed pork loin)

2-2-4. 90-day and 180-day studies of HP-processed and HP-Na-processed pork loins

After acclimatization, 59 male BALB/c mice were divided into seven groups according to body weight. The HP-processed and HP-Na-processed pork loins were mixed with various amounts of a basic powdered diet (Labo MR Stock; Nosan Co., Kanagawa, Japan) and fed to mice (Table 2). The highest proportion of the dried pork loin samples in diets was set at 5% according to the reported protocols of animal experiments for genetically modified organisms^21,22,23). All the mice were observed for clinical signs, including changes in the fur, pupil size, and unusual breathing patterns twice a day during the experiments. Body weight and dietary consumption were measured once a week and every other day, respectively. The mice were sacrificed, and blood, heart, liver, kidney, spleen, testis, and visceral fat samples were collected at the end of 90 or 180 days. Blood samples were centrifuged at 2,300×g for 15 min at 4°C to collect plasma. We entrusted blood biochemical test to Nagahama Institute of Bio-Science and Technology (Shiga, Japan). Blood biochemical parameters were analyzed by a Hitachi 7180 autoanalyzer (Hitachi, Ltd., Tokyo, Japan) except total bile acid (TBA), which was analyzed by a Hitachi 7170 auto-analyzer (Hitachi Ltd., Tokyo, Japan).

Table 2. Composition of Diets

Group	BALB/c mice (n)		Pork loin treatment	Dietary level (% w/w)
	90 Day	180 Day
Basic diet	12	6	-	0
2% Pork	12	5	Not HP processed	2
5% Pork	12	5	Not HP processed	5
2% HP Pork	6	-	HP processed	2
5% HP Pork	6	-	HP processed	5
2% HP-Na Pork	-	5	HP and 0.4 M Na2CO3 ∙aq	2
5% HP-Na Pork	-	6	HP and 0.4 M Na2CO3 ∙aq	5

Pork, unprocessed pork loin; HP Pork, HP-processed pork loin; HP-Na, HP-processed and sodium-treated pork loin (HP-Na processed pork loin); Na2CO3 aq, aqueous solution of sodium carbonate

2-3.Evaluation of the Cytotoxicity of HP and HP-Na Pork Loin Extracts

2-3-1. Pork loin extract

Thirty grams of freeze-dried HP-processed, HP-Na-processed, and unprocessed pork loins were extracted with 80% methanol (MeOH) for 24 h at 4°C. The extracts were then centrifuged at 13,000g for 15 min to collect supernatants, which were then evaporated (Eyela Rotary Evaporator N-1000;Tokyo Rikakikai, Tokyo, Japan) to dryness under reduced pressure at room temperature. Table 1 shows the amounts of the methanolic extract residues. The residues were dissolved in DMSO and adjusted to 100 mg/mL. The DMSO solutions were filtered through a 0.45-μm pore membrane filter (Merck Millipore Ltd., Carrigtwohill, County Cork, Ireland) and stored at −20°C until use.

2-3-2. Effect of pork-loin extracts on cell viability

Cell-based assays are often performed to screen cytotoxic activity. The HP-processed and HP-Na-processed 80% MeOH pork loin extracts were evaluated with respect to their cytotoxic activity using an MTT tetrazolium reduction assay.

Five human cell lines were used for the assay. IMR-90 (human fetal lung fibroblast) and PA-1 (human ovarian teratocarcinoma line) were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank, Osaka, Japan), MCF-7 (human breast adenocarcinoma cell line) was obtained from the Health Science Research Resources Bank (HSRRB, Osaka, Japan), and A549 (adenocarcinomic human alveolar basal epithelial cells) and K562 (human leukemic cells) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were seeded at a density of 1.0 × 10⁴ cells/200 µL in 96-well plates (NUNC, Roskilde, Denmark) in DMEM or RPMI medium 1640 containing 10% FBS and 1% penicillin-streptomycin mixture. After the cells were incubated for 24 h at 37°C under a 5% CO₂ atmosphere, the medium was changed to that containing the 80% MeOH extract residues at concentrations of 0, 0.1, 0.25, and 0.5 mg/mL, and the cells were further incubated for 48 h at under the same temperature and atmosphere conditions. At 4 h before the end of the incubation period, 100 µL of 0.05% MTT phosphate-buffered saline (PBS) solution was added to each well. After the cells were solubilized using cell lysis buffer, the MTT formazan product was measured at 595 nm using a microplate reader (iMark™ Microplate Absorbance Reader; Bio-Rad, Laboratories, Hercules, CA, USA).

2-3-3. Effect of pork-loin extracts on the cell cycle

Next, the HP-processed pork loin extracts were assessed for DNA damage and effects on the cell cycle were determined using a flow cytometer. Five human cell lines, IMR90, A549, PA1, MCF7 and K562, were examined. The same cell lines were used to evaluate the effects of the HP- or HP-Na-processed pork loin extracts on cell viability. The cells were cultured in 3.5-cm dishes at a density of 1.0 × 10⁴cells/dish with DMEM or RPMI 1640 containing 10% FBS and 1% penicillin-streptomycin. The HP- or HP-Na-processed pork loin extract residues dissolved in DMSO were added to the cells at a concentration of 0.1, 0.25, or 0.5 mg/mL, and the cells were incubated for 48 h at 37°C under a 5% CO₂ atmosphere. After being incubated, the cells were washed with ice-cold PBS harvested after treatment with 0.05% trypsin-EDTA (Life Technologies, Carlsbad, CA, USA), and fixed with ice-cold 99.5% ethanol. The fixed cells were centrifuged at 2,300×g for 5 min at 4°C and the pellet was resuspended in 0.25% Triton-X on ice after washing twice with ice-cold PBS. After 30 min on ice, the cells were washed twice with 1% bovine serum albumin containing ice-cold PBS, and incubated for 1 h with 100 µL of PI solution (50 µg/mL) containing of 100 µg of RNase. Each sample was diluted with 800 µL of ice-cold PBS and analyzed by flow cytometry (Beckman Coulter, Pasadena, CA, USA).

2-4. Statistics

Data were analyzed by applying unpaired Student’s t-test or Tukey-Kramer test where appropriate. A p-value of <0.05 was considered significant.

3. Results

3-1. Body Weight

All mice survived throughout the feeding period and behavioral abnormalities were not observed. There was no difference in body weight between groups that were fed pork loin samples with or without HP processing (Fig. 1). Additionally, there were no observable clinical signs in any of the mice during the 90-day period. In the 180-day study, there was no significant difference in mouse body weight between groups fed HP-Na-processed and unprocessed pork loin samples, similar to the result of 90-day study.

Fig. 1.

Weight Gain in Mice Fed a Basic Diet, and Unprocessed, HP-processed, and HP-Na-processed Pork Loin During 90- and 180-days of Feeding Study

(A) Effect of feeding with/without HP-processed pork loin on body weight in male BALB/c mice in a 90-day oral toxicity study. Values are means and standard deviations (n = 6).

(B) Effect of feeding with/without HP-Na-processed pork loin on body weight in male BALB/c mice in a 180-day oral toxicity study. Values are means and standard deviations (n = 5−6).

3-2. Organ Weight

Mice were sacrificed at the end of the 90-day and 180-day feeding studies. Necropsy observations indicated no pathological abnormalities in any of the mice. Individual organ weights and observations indicated that HP did not affect the growth or shape of organs, including the heart, liver, spleen, testes, kidneys, and abdominal fat (Table 3).

Table 3. Absolute Organ Weights of Male BALB/c Mice Fed a Basic Diet, or Unprocessed, High Pressure (HP)-processed, or HP-Na-processed Pork Loin Diet

A
Organ		Basic diet (g)	2% Pork (g)	2% HP Pork (g)	5% Pork (g)	5% HP Pork (g)
Heart		0.17 (±0.01)	0.16 (±0.01)	0.14 (±0.01)	0.15 (±0.01)	0.16 (±0.01)
Liver		1.04 (±0.05)	1.05 (±0.06)	0.09 (±0.09)	1.05 (±0.15)	1.09 (±0.09)
Spleen		0.08 (±0.00)	0.08 (±0.00)	0.09 (±0.01)	0.09 (±0.01)	0.09 (±0.01)
Testis	L	0.09 (±0.01)	0.09 (±0.01)	0.09 (±0.02)	0.09 (±0.00)	0.08 (±0.02)
	R	0.09 (±0.01)	0.09 (±0.01)	0.08 (±0.02)	0.11 (±0.03)	0.90 (±0.02)
Kidney	L	0.23 (±0.03)	0.23 (±0.02)	0.20 (±0.02)	0.22 (±0.02)	0.24 (±0.02)
	R	0.23 (±0.02)	0.22 (±0.02)	0.21 (±0.02)	0.23 (±0.02)	0.24 (±0.02)
Visceral fat		0.49 (±0.16)	0.46 (±0.13)	0.29 (±0.15)	0.43 (±0.10)	0.38 (±0.10)
*: p < 0.05 (Pork vs. HP Pork)
B
Organ		Basic diet (g)	2% Pork (g)	2% HP-Na Pork (g)	5% Pork (g)	5% HP-Na Pork (g)
Heart		0.20 (±0.04)	0.20 (±0.03)	0.21 (±0.04)	0.19 (±0.05)	0.19 (±0.03)
Liver		1.25 (±0.12)	1.16 (±0.06)	1.30 (±0.23)	1.17 (±0.02)	1.23 (±0.12)
Spleen		0.13 (±0.02)	0.10 (±0.01)	0.12 (±0.03)	0.09 (±0.01)	0.09 (±0.02)
Testis	L	0.09 (±0.01)	0.09 (±0.01)	0.09 (±0.00)	0.09 (±0.01)	0.09 (±0.01)
	R	0.10 (±0.01)	0.10 (±0.01)	0.10 (±0.00)	0.09 (±0.02)	0.09 (±0.01)
Kidney	L	0.28 (±0.02)	0.28 (±0.03)	0.28 (±0.01)	0.26 (±0.01)	0.27 (±0.02)
	R	0.29 (±0.02)	0.31 (±0.03)	0.28 (±0.03)	0.27 (±0.02)	0.28 (±0.02)
Visceral fat		0.56 (±0.16)	0.61 (±0.17)	0.52 (±0.17)	0.57 (±0.26)	0.52 (±0.19)
*: p < 0.05 (Pork vs. HP-Na Pork)

(A) Absolute organ weights of male BALB/c mice fed a basic diet, or unprocessed or HP-processed pork loin diet in 90-day feeding test. Data are presented as mean values ± standard deviations (n = 6).

(B) Absolute organ weights for male BALB/c mice fed a basic diet, or unprocessed or HP-processed pork loin diet in 180-day feeding test. Data are presented as mean values ± standard deviation (n = 5−6).

* p < 0.05. Pork, unprocessed pork loin; HP, HP-processed pork loin; HP-Na, HP-Na-processed pork loin

3-3. Biochemical Analysis of Mouse Plasma

Toxic effects of HP-processed pork loins in mice were examined using plasma markers after feeding for 90 and 180 days (Table 4). In the 90-day study, iron, amylase, and triglycerides (TG) tended to be lower in mice fed HP-processed pork loins than those fed unprocessed pork loins, but this difference was significant only between mice fed 2% HP-processed and those fed 2% unprocessed pork loins. Blood urea nitrogen (BUN) was significantly higher in mice fed 2% HP-processed pork loins than that in mice fed 2% unprocessed pork loins, but this was not noted between mice fed 5% HP-processed pork loins and those fed 5% unprocessed pork loins (Table 4A). The plasma albumin/globulin (A/G) ratio and total cholesterol (T-CHO) in mice fed 5% HP-processed pork loins were statistically different from those in mice fed 5% unprocessed pork loins. However, these parameters did not differ significantly from those in the basic diet group (Table 4A).

Table 4. Biochemical Analysis of Plasma Markers in Male BALB/c Mice Fed a Basic Diet, or Unprocessed, HP-processed, or HP-Na-processed Pork Loin Diet

A
	Methods		Basic diet	2% Pork	2% HP Pork	5% Pork	5% HP Pork
TP (g/dL)	Biuret		4.80 (±0.22)	4.78 (±0.34)	4.45 (±0.14)	4.72 (±0.43)	4.68 (±0.15)
ALB (g/dL)	BCG		2.78 (±0.13)	2.71 (±0.17)	2.50 (±0.16)	2.58 (±0.27)	2.82 (±012)
A/G	BCG / Biuret		1.40 (±0.14)	1.32 (±0.07)	1.24 (±0.15)	1.22 (±0.13)	1.44 (±0.08)*
BUN (mg/dL)	Enzymatic		26.33 (±3.61)	27.33 (±2.16)	39.55 (±8.67)*	31.42 (±10.59)	33.72 (±8.96)
CRE (mg/dL)	Enzymatic		0.13 (±0.01)	0.13 (±0.02)	0.14 (±0.03)	0.13 (±0.02)	0.15 (±0.02)
Na (mEq/L)	ISE		151.33 (±4.46)	152.33 (±2.05)	155.17 (±2.03)	154.17 (±2.96)	155.67 (±1.97)
K (mEq/L)	ISE		4.98 (±0.93)	4.85 (±0.93)	5.87 (±1.66)	6.22 (±2.38)	5.60 (±1.27)
Cl (mEq/L)	ISE		109.83 (±5.58)	112.33 (±1.24)	112.33 (±8.14)	115.50 (±4.03)	113.00 (±7.79)
Ca (mg/dL)	OCPC		6.47 (±0.20)	6.53 (±0.46)	6.48 (±0.33)	6.68 (±0.58)	6.74 (±0.34)
Fe (μg/dL)	Nitroso-PSAP		129.83 (±10.92)	146.00 (±12.66)	118.67 (±20.20)*	119.17 (±13.12)	125.17 (±21.24)
AST (IU/L)	JSCC		59.67 (±12.63)	67.00 (±12.36)	109.17 (±65.63)	73.67 (±16.28)	98.83 (±41.63)
ALT (IU/L)	JSCC		43.00 (±18.23)	39.50 (±8.40)	50.00 (±13.17)	43.50 (±10.95)	58.67 (±30.96)
ALP (IU/L)	JSCC		219.83 (±14.97)	222.33 (±19.13)	262.33 (±40.27)	219.83 (±55.76)	230.17 (±17.54)
LDH (IU/L)	JSCC		172.83 (±46.39)	202.50 (±25.35)	262.33 (±40.27)	200.33 (±33.14)	424.67 (±322.89)
LAP (IU/L)	GSCC		57.33 (±2.92)	55.67 (±3.90)	55.33 (±3.82)	60.50 (±10.27)	57.50 (±5.44)
AMY (IU/L)	JSCC		2623.00(±371.50)	2689.67 (±254.70)	2179.00 (±439.67)*	2153.33 (±274.17)	2449.33 (±162.57)
ChE (IU/L)	JSCC		21.83 (±1.57)	21.67 (±2.05)	22.00 (±2.83)	26.33 (±7.54)	25.17 (±3.13)
T-CHO (mg/dL)	Enzymatic		91.00 (±7.66)	85.67 (±11.32)	81.33 (±5.91)	107.00 (±8.64)	94.00 (±7.53)*
E/T (%)	Enzymatic		77.50 (±1.38)	76.33 (±1.60)	76.17 (±1.95)	75.67 (±0.47)	75.67 (±0.94)
TG (mg/dL)	Enzymatic		22.17 (±10.67)	28.50 (±7.83)	12.83 (±8.71)*	25.00 (±11.43)	18.00 (±7.26)
LDL-C (mg/dL)	Direct method		1.00 (±0.00)	1.00 (±0.00)	2.60 (±1.50)	1.50 (±0.50)	1.33 (±0.00)
HDL-C (mg/dL)	Direct method		54.00 (±5.16)	49.67 (±5.79)	45.50 (±3.10)	59.17 (±4.52)	53.83 (±4.45)
T-BIL (mg/dL)	Enzymatic		ND	ND	ND	ND	ND
TBA (μmol/L)	Enzyme cycling		3.67 (±1.25)	3.00 (±0.89)	3.33 (±1.34)	4.67 (±4.68)	3.67 (±2.43)
GLU (mg/dL)	Enzymatic		158.17 (±11.43)	159.17 (±11.39)	121.83 (±40.82)	148.50 (±34.39)	166.50 (±17.26)
*: p < 0.05 (Pork vs. HP Pork)
B
		Methods	Basic diet	2% Pork	2% HP-Na Pork	5% Pork	5% HP-Na Pork
TP (g/dL)		Biuret	5.05 (±0.17)	5.08 (±0.19)	4.70 (±0.33)	4.98 (±0.41)	4.93 (±0.24)
ALB (g/dL)		BCG	2.65 (±0.22)	2.82 (±0.16)	2.50 (±0.42)	2.80 (±0.19)	2.67 (±0.21)
A/G		BCG / Biuret	1.13 (±0.15)	1.24 (±0.05)	1.10 (±0.23)	1.32 (±0.01)	1.18 (±0.11)
BUN (mg/dL)		Enzymatic	22.90 (±5.30)	18.48 (±1.72)	20.90 (±7.85)*	20.36 (±0.96)	18.60 (±1.87)
CRE (mg/dL)		Enzymatic	0.15 (±0.04)	0.12 (±0.02)	0.10 (±0.00)	0.11 (±0.01)	0.12 (±0.01)
Na (mEq/L)		ISE	154.67 (±2.92)	152.40 (±1.20)	151.40 (±3.61)	153.80 (±1.47)	155.17 (±3.24)
K (mEq/L)		ISE	6.44 (±1.39)	5.84 (±2.13)	5.60 (±1.55)	3.78 (±0.27)	4.54 (±0.33)
Cl (mEq/L)		ISE	109.83 (±7.56)	110.80 (±4.66)	114.00 (±3.58)	115.00 (±2.28)	107.67 (±8.12)
Ca (mg/dL)		OCPC	8.80 (±0.52)	8.90 (±0.54)	8.40 (±0.47)	8.28 (±0.44)	8.78 (±0.46)
Fe (μg/dL)		Nitroso-PSAP	152.00 (±21.30)	160.00 (±19.54)	131.40 (±28.18)	144.60 (±15.70)	144.17 (±11.08)
AST (IU/L)		JSCC	74.50 (±15.88)	84.40 (±35.70)	73.80 (±25.05)	62.80 (±7.41)	66.67 (±9.72)
ALT (IU/L)		JSCC	49.00 (±17.68)	44.67 (±12.47)	96.17 (±113.36)	34.20 (±5.49)	40.50 (±15.13)
ALP (IU/L)		JSCC	192.83 (±52.43)	251.00 (±26.59)	194.20 (±61.46)	235.20 (±15.73)	203.67 (±29.72)
LDH (IU/L)		JSCC	295.83 (±64.43)	353.20 (±224.40)	263.40 (±77.85)	232.00 (±49.13)	327.67 (±187.76)
LAP (IU/L)		GSCC	62.50 (±3.40)	52.67 (±4.42)	53.67 (±4.92)	60.60 (±4.32)	58.67 (±4.46)
AMY (IU/L)		JSCC	2669.50(±209.45)	2571.60 (±102.76)	2538.20 (±373.73)	2929.20 (±404.49)	2508.17 (±227.81)
ChE (IU/L)		JSCC	24.33 (±1.37)	24.60 (±2.24)	22.00 (±3.03)	24.00 (±1.10)	24.17 (±2.34)
T-CHO (mg/dL)		Enzymatic	93.50 (±9.69)	94.20 (±3.37)	86.60 (±5.35)*	92.00 (±5.76)	87.50 (±5.28)
E/T (%)		Enzymatic	76.33 (±0.75)	77.40 (±0.80)	75.60 (±1.62)	77.60 (±1.02)	77.17 (±1.21)
TG (mg/dL)		Enzymatic	59.50 (±25.60)	65.00 (±20.82)	34.40 (±12.09)*	57.40 (±22.46)	41.50 (±11.81)
LDL-C (mg/dL)		Direct method	2.60 (±1.02)	1.20 (±0.40)	2.80 (±2.49)	3.00 (±2.92)	3.33 (±3.20)
HDL-C (mg/dL)		Direct method	53.67 (±3.82)	54.40 (±2.42)	48.20 (±5.60)	54.60 (±3.50)	50.00 (±2.83)
T-BIL (mg/dL)		Enzymatic	ND	ND	ND	ND	ND
TBA (μmol/L)		Enzyme cycling	4.67 (±5.59)	3.60 (±1.74)	5.20 (±5.56)	4.20 (±0.98)	2.80 (±0.75)
GLU (mg/dL)		Enzymatic	118.83 (±38.91)	155.60 (±30.28)	131.20 (±26.83)	124.20 (±13.67)	133.33 (±27.96)
*: p < 0.05 (Pork vs. HP-Na Pork)

(A) Biochemical parameters of plasma samples from male BALB/c mice fed a basic diet, or unprocessed or HP-processed pork loin diet in 90-day feeding test. Data are presented as mean values ± standard deviations (n=6).

(B) Plasma biochemical parameters in male BALB/c mice fed a basic diet, or unprocessed or HP-Na-processed pork loin diet in 180-day feeding test. Data are presented as mean values ± standard deviation (n=5-6). * p< 0.05. Pork, unprocessed pork loin; HP, HP-processed pork loin; HP-Na, HP-Na-processed pork loin; BCG, Bromocresol green; ISE, Ion selective electrode; OCPC, Orthocresolphthalein complex; Nitroso-PSAP, 2-Nitroso-5-[N-n-propyl-N-(3-sulfopropyl)amino]phenol; JSCC, Japan society of clinical chemistry; GSCC, German society for clinical chemistry; T P, Total protein; A LB, A lbumin; A/G, A lbumin to globulin ratio; BUN, Blood urea nitrogen; CRE, Creatinine; Na, Sodium; K, Potassium; Cl, Chlorine; Fe, Iron; AST, Aspartate aminotransferase; ALT, Alanine aminotransferase; ALP, Alkaline phosphatase; LDH, Lactase dehydrogenase; LAP, Leucine aminopeptidase; AMY, Amylase; ChE, Chorine esterase; T-CHO, Total cholesterol; E/T, ratio of cholesterol to esters; TG, Triglyceride; LDL-C, Low-density lipoprotein cholesterol; HDL-C, High-density lipoprotein cholesterol; T-BIL, Total bilirubin; TBA, Total bile acid; GLU, Glucose

In the 180-day study, BUN, T-CHO and TG in mice fed 2% HP-Na-processed pork loins were significantly different from those in mice fed 2% unprocessed pork loins but the difference was not noted between mice fed 5% HP-Na-processed pork loins and those fed 5% unprocessed pork loins (Table 4B).

3-4. Cell Viability

To examine the cytotoxicity of HP processing, the extracts of HP processed and HP-Na-processed pork loins were examined for their cytotoxicity toward several cell lines using an in vitro MTT assay. As shown in Fig. 2, there was no difference between the HP-processed and unprocessed pork loin extracts with respect to cell viability at concentrations of up to 0.5 mg/mL for all cancer and normal cell lines examined, except for K562 (Fig. 2). A significant difference in cell viability was found between the HP-processed and unprocessed pork loin extracts at 0.25 mg/mL for K562 cells. However, the differences were not dose-dependent (Fig. 2).

Fig. 2.

The Effect of 80% MeOH Extracts of Unprocessed, HP-processed, and HP-Na-processed Pork Loins on Cell Viability at Various Concentrations

Cell viability was measured by an MTT assay for five different cell lines. Cells were treated with the 80% MeOH extracts for 48 h. Vehicle-treated cells were treated with DMSO. Results shown are means ± standard deviation. *p < 0.05.

Pork, 80% MeOH extract of unprocessed pork loin; HP, 80% MeOH extract of HP-processed pork loin; HP-Na, 80% MeOH extract of HP-Na-processed pork loin

3-5. Cell Cycle Distribution

We further examined the effects of HP-processed and HP-Na-processed pork loin extracts on cytotoxicity in detail based on a cell cycle analysis. Both HP-processed and non-HP-processed meat extracts did not significantly alter the cell cycle distribution (including G1, S, and G2/M) or increase the apoptotic cell population as assessed by sub-G1 phase cells (Fig. 3). These results further confirmed that HP processing does not have cytotoxic effects on meat.

Fig. 3.

The Effect of 80% MeOH Extracts of HP-processed and HP-Na-processed Pork Loins on the Cell Cycle Distribution for Five Cell Lines

Five human cell lines were treated with 80% MeOH extracts of the processed pork loins and the cell cycles were measured by flow cytometry after staining with propidium iodide (PI).

HP, 80% MeOH extract of HP-processed pork loin; HP-Na, 80% MeOH extract of HP-Na-processed pork loin

4. Discussion

In general, heat (high temperature or pasteurization) and chemicals are frequently used for sterilization to avoid the proliferation of pathogenic bacteria, and these methods are regarded to guarantee the safety of food products during storage. HP processing is an alternative technology that can be used to kill pathogenic bacteria^{4,8,10,11,12,13)} and is thus useful for food preservation^5,6,7). However, several food-borne pathogenic microorganisms, eg, Clostridium botulinum strains, exhibit some degree of resistance to HP processing. To inactivate these pathogens, the combination of HP with heat shows positive effects^24,25,26). Our HP-processed and HP-Na-processed pork loin samples were treated at 400 MPa at 20°C, and thus, these treatments may not have any influences on C. botulinum viability. Further experiments are required to investigate the safety of pork that is treated at 500–600 MPa and 70–80°C to inactivate these microbes. A unique property of HP processing is that it has a limited effect on covalent bonds; thus, the chemical properties of food ingredients hardly change during HP processing⁴⁾. In the case of meat processing, antibiotic and growth promoter residues area considerable issue for many consumers²⁷⁾. The effective surveillance of meat samples, including analyses of residues and traceability schemes, is necessary. Furthermore, HP not only avoids the loss of nutritional components, but also minimally affects the original color and flavor of food^2,3,4,5). These properties are quite different from those of other methods using heat or chemical treatment. Therefore, HP is currently applied in the production of many foods, including meat. However, few studies have examined the safety of HP-processed meat.

In the present study, we showed that the HP-processed pork loins have no cytotoxicity, based on both in vivo and in vitro experiments. First, we administered HP-processed and HP-Na-processed pork loins to BALB/c mice for 90 days or 180 days, and did not observe any pathological differences between the groups (Fig. 1, Tables 3 and 4). Some significant differences between groups fed the processed pork loins and those fed unprocessed pork loins were observed in blood biochemical parameters, such as BUN, amylase, A/Gand T-CHO (Table 4), but these differences were not dose-dependent (Table 4), suggesting that these biochemical parameters were within normal range. Moreover, we investigated whether 80% MeOH meat extracts of HP-processed and HP-Na-processed pork loins have cytotoxic effects on human-derived cell lines. MTT assay used for it is a good indicator of cell viability, and, therefore, often used to investigate whether the sample has cytotoxic effects²⁸⁾. In this study, the HP-processed and HP-Na-processed pork loin extracts had no effect on cell viability and the cell cycle distribution (Figs. 2 and 3). These in vivo and in vitro results suggest that HP is not associated with risk factors during processing.

Faustman and Cassens reported that food color is an important criterion for consumers²⁹⁾. HP processing inhibits the deterioration of meat or meat products caused by bacteria and/or oxidation. Therefore, the colors, flavor, and texture of food are adequately preserved^4,9). Our research group has recently observed that HP-Na-processed pork loins exhibit improved texture, ie, tenderness, with production of more bioactive peptides and proteins compared to simple HP-processed pork loins²⁰⁾. Several studies have shown that HP tenderizes the meat^30,31); this is an important research area of research for commercial purposes^14,30). The results of the present study showed that HP-processed and HP-Na-processed pork loin samples did not have any effect on animal health, and, therefore, this study provides important evidence to support the expansion of HP processing in the meat product market. Additionally, Hajos et al. have suggested that HP processing reduces the allergenic risk of meat components³²⁾. These studies, including our study, indicate that HP treatment is not only a safe food processing technology, but also a technology that improves food functions. HP treatment would be a beneficial and reliable technology for food processing in meat and meat products.

Acknowledgements

This work was the collaboration with Niigata Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence (CREATE) of the Japan Science and Technology Agency (JST).

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