Influence of Probiotics-Friendly Pig Production on Meat Quality and Physicochemical Characteristics

In order to evaluate the effects of systemic use of probiotics on meat quality, 80 pigs with an average age of 175±5 d were maintained in conventional probiotic farms equipped with a liquid probiotics application system (40 pigs in each farm). All pigs were Landrace×Yorkshire×Duroc (LYD) three-way crossbred animals and commercially feed-fed according to the regimens of FARMSCO Inc. The diets’ formula and calculated nutrient composition are shown in Table 1. The probiotic farm supplied a large quantity of probiotics to the pigs during all life stages (sows, piglets, growing pigs, and finishing pigs) via drinking and feeding. High quantities of probiotics were prepared by aero-dynamic fermentation with nutrients during 4 d after seeding (3% of the total volume) of Lactobacillus plantarum (2.2×10⁸ CFU/mL) in polystyrene tanks (2 to 3 ton size). The probiotic preparation (2.5×10⁷ CFU/mL) was supplied to pigs by having them drink water directly from the polystyrene tank. The probiotic feed additive was prepared by rotary drum fermentation during 7 d after seeding (2% of the total volume) of the probiotic preparation and mixed with the pig diets (20 kg/ton). The probiotic farm sprayed one to five diluted probiotic preparations inside and outside of the farm facilities instead of chemical disinfectants to prevent the growth of harmful bacteria and pathogens in the immediate environment.

At the end of the experiments, 80 pigs (40 per treatment) were transferred to the slaughterhouse. Feeding was terminated 12 h before slaughter. All pigs were conventionally slaughtered at the average market weight of 115 kg. The individual carcass weight was recorded within 30 min after slaughter and the pig then placed in a chilling room (4°C). The back fat thickness was measured according to the Animal Products Grading Service (2001). After the determination of muscle pH_45min, samples were vacuum-packed at 4°C to subsequently measure meat quality, including muscle pH_24h, color, shear force, WHC, cooking loss, drip loss, and meat composition. The rest of the samples were divided into two parts: one of them was pulverized by liquid nitrogen for nucleotides and free amino-acids and the other one freeze-dried for fatty acids, vitamins, and minerals. The samples of both groups were stored at -70°C until analysis.

Three-color (L*, a* and b*) coordinate measurements per sample were taken at three different locations on the bloomed cut surfaces of the meat sample blocks using a Minolta chromameter (CR-300, Minolta Camera Co., Ltd., Osaka, Japan), with a 1 cm aperture, illuminant D65 and a 2° viewing angle. According to the Commission International de l’Eclairage (CIE) system, color was expressed as CIE L*, a*, and b* (lightness, redness, and yellowness).

We used two different methods to measure WHC: drip loss and cooking loss. Drip loss was assessed by the gravimetric method of Honikel (1998). The samples were trimmed and weighed, then placed in an inflated plastic bag and hung for 48 h at 4°C. After 48 h, the samples were weighed again. Drip loss was calculated as a percentage based on weight before and after hanging. For cooking loss (Honikel, 1998), the samples were weighed and put in a plastic bag, which was placed in an 80°C water bath. When the internal temperature of 75°C was reached, the samples were cooled and weighed again. The difference in weight before and after boiling was expressed as the “percentage cooking loss”.

The Warner-Bratzler shear force (WBSF) was determined by taking meat sample blocks of approximately 300 g. Three representative 1.27 cm diameter cores were removed from each steak parallel to the muscle fiber after cooling. Shear force values were determined with a Warner-Bratzler shear attachment using an Instron Universal Testing Machine (Model 3342; Instron Corporation, USA) with the following operating parameters: load cell, 50 kg, cross-head speed, and 200 mm/min. Each core sample was sheared once across the center of the core perpendicular to the muscle fiber. The shear force value was the mean of the maximum forces required to shear each set of core samples and expressed as kg of force (kgf).

The moisture, protein, fat, and ash content of meat were measured according to the AOAC (1995). Moisture was assessed by the dry-oven method. Protein and fat contents were measured by the Kjeldahl method and the Soxhlet apparatus respectively. Ash content was analyzed using a muffle furnace.

Two different pH meters (pK21 pH meter and pH star probe) were used to measure pH accurately in the slaughter house and laboratory respectively within specific time. The pH values of the 10-rib face longissimus dorsi muscles were measured at 45 min and 24 h post-mortem in triplicates using a portable pH meter (pK21 pH meter, NWK-Binar GmbH, Germany). Afterwards, the longissimus dorsi muscles were excised for further analyses.

The 10-rib face longissimus muscle pH values were assessed at 3 d, 5 d, and 15 d in duplicates using a pH star probe (Horiba 6252-10D, USA).

The TBARS as a lipid oxidation value were measured by a modified method of Buege and Aust (1978). The meat samples were aging 0 d and 20 d respectively at 4°C. Completed aging-period samples were stored at -20°C until analysis. Prior, the samples were thawed at 4°C. A meat sample of 2.5 g was filled into a 50 mL falcon test tube, and homogenized with 7.5 mL deionized distilled water as well as 10 mL of TBA/TCA solution using a Homogenizer (T 25 digital ULTRA-TURRAX^®, Germany) at 12,000 rpm for 20 s. To protect fat oxidation, 25 μL of BHA (butylated hydroxyanisole) was added to each homogenized sample. The homogenate was filled up to 30 mL with distilled water (DW), vortexed, and heated in a 90°C preheated water bath for 15 min to develop color. The heated homogenized samples were cooled in ice-cold water for 15 min and centrifuged at 3,000×g for 15 min at 4°C by using a Hanil Supra 21K Centrifuge Machine (Hanil Science Industrial Co., Ltd., Korea). After centrifugation, the absorbance of the resultant supernatant solution was taken as a duplicate and averaged by using a UV spectrophotometer (Multiskan Go, Thermo Scientific, USA) at 531 nm compared with a blank containing distilled water and a TBA/TCA solution. TBARS were calculated by multiplying the average absorbance value with 5.88 and the results were expressed as mg malondialdehyde (MDA)/kg of meat.

Fatty acids were measured by gas chromatography (GC). Freeze-dried meat samples (0.5 g) were added to 2 mL of boron-trifluoride in methanol and 2 mL of methanol in glass tubes. The tubes were capped with Teflon-lined caps to prevent loss of volume and the samples were placed on a heating block at 80°C. After 10 min, the tubes were vortexed individually every 5 min for 2 h. After 2 h of repeated vortex mixing, the samples were allowed to cool at room temperature and then after adding 3 mL of distilled water and 3 mL of hexane - the tubes were capped again and mixed by vortexing for 15 s. After centrifugation (2,000 rpm, 5 min) to separate the phases, the supernatant was transferred to GC vials for analysis. GC was performed for 1 μL samples on a Shimadzu GC-2014 instrument (Shimadzu Co., USA) using a FAMEWAX column (30 m×0.32 mm i. d., 0.25 μm; column temperature, 250°C) and nitrogen/air as a carrier gas at 53.8 mL/min (split ratio 30:1). The temperature started at 150°C and increased to 250°C with an equilibration time of 3 min.

Meat samples were made into fine powder with liquid N₂, a mortar and pestle (0.5 g); afterwards, they were mixed with 1 mL of DW and homogenized two times at 30 s intervals. Then, the samples were centrifuged at 13,000 rpm for 10 min at 4°C. The 100 µL supernatants were transferred into a 2 mL tube, mixed with 900 µL formic acid (4% formic acid in 100% ethanol) by vortexing, and incubated at -20°C for 1 h. Then they were again centrifuged at 13,000 rpm for 10 min at 4°C and 100 µL supernatants were mixed with 900 µL formic acid (4% formic acid in 100% ethanol) in a vial. The samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for which 3 µL aliquots were injected on an UPLC system (Waters Xevo TQ-S, Waters Corporation, Milford, MA, USA). The UPLC column was Imtakt Intrada Amino Acid C18 (50×2 mm, 3 µm). The mobile phase buffer A was ACN:100 mM CH₅NO₂=20:80 (v/v) and the buffer B was ACN:THF:25 mM CH₅NO₂:FA=9:75:16:0.3 (v/v/v/v). The flow rate was 0.4 mL/min and established a gradient from 0 to 100% for the B buffer for 17 min. The MS was equipped with an ESI interface operating at an ionization voltage of +3,000 V and a source temperature of 380°C. The capillary voltage, con voltage, and source offset were set at 3 kV, 30 kV, and 30 V respectively. Tandem MS analysis was performed using a multi-reaction-monitoring (MRM) mode. The collision energy from the gas flow of desolvation, cone, and nebulizer were set at 650 L/h, 150 L/h and 7 bar, respectively.

The meat samples were pulverized with liquid N₂, a mortar and pestle (0.3 g), mixed with 5 mL of 0.5 M perchloric acid, kept in ice for 15 min, and then centrifuged at 9,200×g for 5 min at 4°C to extract nucleic acids. The extracted 1 mL nucleic acids were transferred into a 2 mL tube, mixed very carefully with 0.25 mL of 2.1 M potassium hydrogen carbonate (KHCO₃), and kept in ice for 15 min. They were then again centrifuged at 9,200×g for 5 min at 4°C and filtered through a syringe filter (HLB-M, 0.45 µm particle size, 13 mm, Futecs Co., Ltd., Korea). The filtrate was analyzed using high-performance liquid chromatography (HPLC) (Shiseido Nanospace SI-2, Shiseido Co., Ltd. Japan). Regarding the analytical conditions for HPLC, a Imtakt Cadenza CD-C18 reverse phased column (4.6×250 mm, 3 µm, Imtakt Corp., USA) was utilized, with two mobile phases of (1) A, 1,000 mL distilled water+5 mL TBA-OH (tert-butyl ammonium hydroxide) (40%)+1 mL H₃PO₄ (phosphoric acid) and (2) B, 1,000 mL methanol+5 mL TBA-OH (40%)+1 mL H₃PO₄. The mobile phase flow rate was 0.7 mL/min and the injection volume was 5 µL. The column temperature was maintained at 40°C and the detection wave length was 254 nm. The peaks of the individual nucleotides were identified using the retention times for the following standards: hypoxanthine, inosine, inosine-5'-phosphate (IMP), adenosine-5'-phosphate (AMP) (Sigma, USA), and the concentration was calculated using the area for each peak.

Vitamin content was analyzed by LC-MS/MS. A total of 10 mg freeze-dried meat powder was sonicated in 100 μL distilled water and then 900 μL methanol was added, mixed by vortexing, and then again sonicated. After centrifugation, the samples were analyzed using a UPLC system (Waters Xevo TQ-S, Waters Corporation, USA). The UPLC column was a Waters ACQUITY UPLC ^®BEH C18 (2.1×100 mm, 1.7 μm). For the analysis of water-soluble vitamins, buffer A was 0.1% formic acid in water and buffer B 0.1% formic acid in acetonitrile (ACN). For fat-soluble vitamins, buffer A was 0.1% formic acid in water and buffer B 0.1% formic acid in methanol/ACN (40/60, v/v).

Minerals were measured using inductively coupled plasma mass spectrometry (ICP-MS). A 0.05 g sample in 600 μL of 70% nitric acid in a 15 mL conical tube reacted in a fume hood. After reaction for 2 d, sample tubes were incubated at 80°C for 5 h. DW was added to adjust the volume to 10 mL and the sample was diluted with 2% nitric acid from 10¹ to 10⁴ for analysis. Mg, K, Ca, Fe, and Zn were analyzed by ICP-MS (Agilent 7500a, Santa Clara, USA).

Data were analyzed by using the SAS software (SAS Version 9.3). All data were analyzed using an ANOVA (analysis of variance) procedure to test the statistical significance among treatments. The results are presented as the mean±SE and statistical significance was indicated at p<0.05, based on t-tests.

The physical properties in porcine longissimus dorsi muscles fed with probiotics are shown in Table 2. The color (CIE L* and a*) did not show any significant difference between the groups, but yellowness (CIE b*) was significantly higher in the PRO group. The shear force of the PRO-treated group was significantly lower compared to the control group. Drip loss of the probiotics treatment group increased at 1 d and 3 d and reduced at 15 d compared to the controls. There was no significant difference in backfat thickness, carcass weight, water holding capacity, and cooking loss between the control and probiotics treatment groups. Liu et al. (2013) reported that the supplementation with probiotics (yeasts, lactic acid-producing bacteria, and Bacillus subtilis) reduced the drip loss and cooking loss of pork, but had no effect on pH and shear force. In another experiment, Yang et al. (2010) stated that probiotic C. butyricum with a broiler diet decreased shear force, which indicated improved tenderness. However, experiments reporting the use of L. plantarum as a probiotic on shear force in pigs are limited. Also, Sudikas et al. (2010) published that 0.04% probiotic (Bacillus licheniformis and Bacillus subtilis) supplementation had no influence on the carcass quality but that the carcass weight increased 0.06% compared to controls. Feeding 0.5% probiotics (Lactobacillus CAU6001) improved meat quality (Li and Zhang, 2007). Some previous studies have stated the positive effects of probiotics on pigs meat quality (Alexopoulos et al., 2004; Meng et al., 2010) but differ Quadros et al. (2001). These variable results about the effect of probiotics may be due to several aspects such as bacteria strains, level of supplementation, composition of diet and interaction with other dietary additives (Meng et al., 2010). Furthermore, dietary probiotics significantly improved the pork quality, enhanced WHC, reduced drip loss, and produced a more vivid meat color from finishing pigs (Jiang, 2011; Ma, 2011). Therefore, probiotics influenced meat quality traits like color, shear force, and drip loss.

Compared with the control group (Table 3), dietary supplementation with probiotics significantly reduced ash, salinity, and pH (at 5 and 15 d). Probiotics also increased protein, reduced moisture and fat, but did not reach significance levels (p>0.05). There was no significant effect on TBARS values, while the treatment group’s decreased at 20 d. In a previous study, Sudikas et al. (2010) found that diets with 0.04% and 0.06% probiotic supplementation had no effect on the chemical composition of meat compared to controls. A similar response on pH was observed by Rybarczyk et al. (2016) and they stated that a diet containing bokashi probiotics (Saccharomyces cerevisiae, Lactobacillus casei and Lactobacillus plantarum) decreased at pH₂₄ and pH₄₈, while they increased at pH₁₂₀. On the basis of this research, pH appears to have a great influence on the quality of food: Freshness, water retention, tenderness, color, and texture are greatly influenced by the food’s pH. Commonly, pH is a direct reflection of muscle’s acid content and it affects meat color, shear force, and drip loss (Chen et al., 2009). Bendall and Swatland (1988) reported that the pH of pork was affected by the rate and extent of post-operative action which decreases from the time of bleeding and then reaches the final pH. Li and Chen (2009) showed that probiotics significantly reduced the MDA content of muscles, inhibited muscle lipid peroxidation, reduced the rate of water loss, stabilized color, and improved the meat quality. Due to reducing drip loss, probiotics lowered lipid peroxidation in the muscles by maintaining the cell membranes’ integrity (Liu et al., 2013). As a similar response, Ko et al. (2008) reported that pigs fed diets containing 0.5% green tea probiotic supplementation had lowered meat TBA values compared to finishing pigs fed a 0.5% green tea by-product (p<0.05).

Table 4 showed the results of fatty acid content analysis for porcine fed with probiotics. Palmitic acid (C16:0) and stearic acid (C18:0) were the main triglycerides of saturated fatty acid and they did not show significant differences in the treatments. Oleic acid (C18:l n9) and linoleic acid (C18:2 n6) were the major fatty acids among the unsaturated fatty acids. The content of saturated fatty acid (SFA) and unsaturated fatty acid (USFA) was not significantly different between the treatments. The probiotic group meat showed higher polyunsaturated (PUFA) fatty acid contents, with linoleic acid (C18:2) and concentrations significantly (p<0.05) higher compared to the control group, consistent with Ross et al. (2012). However, monounsaturated (MUFA) acids were significantly lower in the probiotic-treated group. The linoleic acid (C18:2 n-6) and linolenic acid (C18:3 n-3) ratio should be approximately 5:1 to promote health and to minimize cardiovascular disease risks. Although the meat of the probiotic group showed linolenic and linoleic acid significantly (p<0.05) higher compared to the control group, the n-3 and n-6 in the present study are higher than that recommended values for human health. The intensified activity of lactic bacteria could positively influence the ingestion and absorption processes as well as indirectly influence the meat fatty acids profile.

Analyses of free amino acid compositions such as serine, lysine, histidine, and arginine revealed significantly lower levels in the PRO group as compared to the CON group (Table 5). Muscles from the probiotic-treated group had higher levels of glycine, alanine, proline, valine, leucine, and isoleucine, whereas glutamic acid, phenylalanine, tyrosine, and threonine were higher in the control group. The treatment group showed a higher nucleotide compound (hypoxanthine, inosine, GMP, IMP) than the controls (Table 6). Glutamic acid, phenylalanine, tyrosine, AMP, IMP, and GMP contribute to meat flavor perceptions and together comprise the umami taste (Kuchiba-Manabe et al., 1991; Lioe et al., 2005; Wood et al., 2004). In contrast, glycine, alanine, lysine, and proline contribute sweet flavors, and other amino acids produce sour or salty tastes (Zhu and Hu, 1993). Remarkably, IMP indirectly contributes to meat flavor through the breakdown of inosine to form hypoxanthine and with free amino acids such as arginine, phenylalanine, valine, leucine, isoleucine, methionine, and histidine, contributes to a bitter taste (Tikk et al., 2006). Cornet and Bousset (1999) stated that an undesirable flavor could result due to accumulation of some free amino acids. However, free amino acids are of great importance in eating quality due to their specific tastes (Nishimura and Kato, 1988).

The content of vitamins and minerals in pork meat are shown in Table 7. The levels of thiamin (B₁) and ascorbic acid (C) significantly differed in the treatment group (p<0.05), whereas riboflavin (B₂), niacin (B₃), pyridoxine (B₆), retinol (A), α- tocopherol (E), and cholecalciferol (D₃) showed no significant differences between the groups. Some natural antioxidants can provide biological protection from oxidative processes (Su et al., 2007) and are added in the pre- and post-slaughter stages to enhance meat shelf life and quality. Vitamin C has good antioxidant properties and Wheeler et al. (1996) demonstrated that injecting vitamin C into beef resulted in improved color stability and retail display color of the meat. Magnesium (Mg) and potassium (K) were higher in the treatment group but did not reach significance levels, while calcium (Ca), iron (Fe), and zinc (Zn), decreased compared to the control group. Sherman and Metha (2009) showed that the potassium content increased the quality of uncooked meat and poultry products. Zinc is essential for performance-related functions, collagen synthesis, and immune development (Shankar and Prasad, 1998). Different genetic lines of pigs did not show differences in the mineral content of the meat (Tomović et al., 2011). Our current study showed that probiotics increased antioxidant activity without significant changes of mineral contents in LYD meat.