This study was conducted to determine the effects of dietary glycine betaine on pork quality and blood characteristics. A total of 80 female pigs (Landrace × Yorkshire × Duroc) were randomly allotted into one of four experimental diet groups. Each group of pigs fed with a commercial diet (control) added with 0.2 g glycine betaine (T1), 0.4 g glycine betaine (T2) and 0.6 g glycine betaine (T3)/kg diet during 40 days. Glycine betaine concentrations in plasma and loin muscle were significantly increased by dietary glycine betaine; however, triglyceride concentration in serum was decreased by dietary glycine betaine. Creatine phosphokinase concentrations in plasma had no significant difference among the dietary groups. Redness (a*) was significantly higher in T2 and T3; however, at the first and seventh days of storage, redness (a*) was not significantly different between dietary groups. Shear force was significantly higher in dietary glycine betaine groups at the first day of storage. Cholesterol content was significantly lower in T2 and T3, whereas T1 was not significantly different compared with the control group. In fatty acid composition, the ratio of saturated fatty acids was increased, whereas unsaturated fatty acids were decreased by dietary glycine betaine.
Glycine betaine is an amino acid (trimethylglycine) present in most organisms, and is an obligatory intermediate in the catabolism of choline. Glycine betaine has been reported to affect some aspects of pork qualities. As a result of this study, dietary glycine betaine should improve meat redness and reduce cholesterol. However, dietary glycine betaine did not influence creatine phosphokinase levels in plasma. These results will be helpful to the pork industry and meat scientists for improving meat qaulity.
Glycine betaine is a naturally occurring product found in many plant and animal species. It is an amino acid (trimethylglycine) present in most organisms, and is an obligatory intermediate in the catabolism of choline (Fernandez-Figares et al. 2002). Glycine betaine is actively accumulated by many mammalian cells under hypertonic conditions, and this process has widespread importance in cell volume regulation with particularly high accumulations in the inner medulla of the mammalian kidney (Lever et al. 2004). Several animal studies suggested that dietary glycine betaine influenced growing performance and carcass quality in pigs. Matthews et al. (2001a,b) reported that the addition of betaine to the diet of finishing pigs may result in improved leanness and carcass quality. Fernandez-Figares et al. (2002) reported that the fat concentration in the carcass was lower in pigs consuming betaine than in controls, and decreased linearly with increasing levels of dietary betaine. Betaine has a decreased backfat thickness (Cardogand et al. 1993) and increased longissimus muscle area in pigs (Smith et al. 1995), whereas Haydon et al. (1995) reported that betaine has increased backfat thickness and decreased longissimus muscle area. However, there is little information on the effects of dietary glycine betaine on blood characteristics, cholesterol, fatty acid and pork quality. Thus, the purpose of this study was to determine the effects of dietary glycine betaine on blood characteristics, cholesterol, fatty acid and pork quality during storage.
MATERIALS AND METHODS
Animal Diet and Experimental Protocol
A total of 80 female pigs (averaging 65 kg in weight; Landrace × Yorkshire × Duroc) were randomly allotted into one of four experimental diet groups. The pigs were allotted to each treatment (four replications of five pigs per replicate) on the basis of weight. Each group of pigs was fed with a commercial diet (control) added with 0.2 g glycine betaine (T1), 0.4 g glycine betaine (T2) and 0.6 g glycine betaine (T3)/kg diet during 40 days. Experimental diet and water were provided on an ad libitum basis throughout the experiment (Table 1). The pigs were slaughtered at approximately 110 kg live weight, and glycine betaine accumulation ratio, blood characteristics, color, shear force, cholesterol and fatty acid composition were measured in loin at 1, 7 and 14 days of cold storage. The pigs were held without food for 12 h before bleeding, and blood samples were taken from the anterior vena cava before slaughter. After collection, the blood samples were placed on ice for 2 h, and then centrifuged for 15 min at 1,500 rpm and 4C. Serum and plasma were collected and frozen (–20C) until subsequent analysis. The 95% glycine betaine was purchased from a commercial biochemical company (CTC Bio, Seoul, Korea).
Table 1. FORMULA OF EXPERIMENT DIET (%, AS-FED BASIS)
Serum triglycerides and plasma creatine phosphokinase (CPK) were determined using enzymatic assay kits (Sigma Diagnostics, St. Louis, MO) as specified by the manufacturer. Glycine betaine measured in plasma by high-performance liquid chromatography (HPLC) (Shimadzu, Tokyo, Japan) based on the method of Lever et al. (1992). Then, 20 µL of the sample was added to 1 mL of extraction solvent (methanol/acetonitrile 1:10 vol/vol) in a 1.5 mL microfuge tube, and mixed thoroughly. Approximately 0.1 g of anhydrous sodium sulfate was added and vortex mixed for 1 h, followed by centrifugation for 5 min at 11,000 rpm. A 250 µL aliquot of supernatant was transferred to a 1.5 mL microfuge tube for derivatization using 2-naphthacyltriflate. First, 50 µL of 2-naphthacyltriflate solution (1 mmol/L in acetonitrile) was added. After 5 min, a 5 µL aliquot of Mg(OH)2 suspension (0.1 g MgO/1 mL H2O) was added, and the sample was mixed for 5 min, followed by centrifugation for 5 min at 11,000 rpm. A 200 µL aliquot of supernatant was transferred into an HPLC vial. Derivatives were separated using an isocratic HPLC system. Separations were made on an Alusphere 250 × 4 mm alumina column (E. Merck, Darmstadt, Germany). The mobile phase consisted of 3.5% vol/vol water in acetonitrile containing triethylammonium succinate buffer (10 mmol/L succinic acid and 3.8 mmol/L triethylamine), and the flow rate was 1 mL/min. Chromatograms were collected and integrated using Delta for Windows version 5 (DataworX Pty Ltd, Brisbane, Australia).
Glycine Betaine Accumulation Ratio
One gram of sample was weighed into a 20 mL test tube, and homogenized with 5 mL of deionized distilled water using the Polytron homogenizer for 5 s at the highest speed. The mixture was vortexed and centrifuged for 5 min at 2,000 rpm. The lower layer was mixed with the same amount of dichloromethane, and then centrifuged for 5 min at 2,000 rpm. Then, 5 µL supernatant solution was mixed with 25 µL acetone and 2-naphthacyl trifluoromethanesulfonate. The mixture was analyzed using an HPLC (Shimadzu). A column was used C18 or Shodex NH2. The mobile phase was acetonitrile/water = 50/50. The flow rate was 1 mL/min, and UV wavelength for detection was 194 nm.
Meat color (CIE a*) was measured by using a Minolta Chroma Meter (Minolta CR 301, Tokyo, Japan). Five random readings were made from the surface of samples.
The samples were cooked in a preheated water bath until the core temperature had reached 80C, and then cooled in running water for 30 min to reach a core temperature below 30C. Shear force was recorded using an Instron (Universal Testing Machine, model 4201, Instron Corp., Canton, MA) until the total break of the cooked sample (1.5 cm in diameter).
Lipids were extracted with chloroform and methanol as described by Folch et al. (1957). Then, 0.1 g of sample was combined with 50 mL of Folch solution (chloroform : methanol, 2:1, vol/vol) and 50 µL of butylhydroxyanisole (50 mL, 10%), and homogenized with a Polytron homogenizer (IKA Labortechnik T25-B, Selangor, Malaysia) for 10 s. The homogenate was filtered with Whatman no. 1 filter paper. The residue and filter paper were blended with 50 mL of the Folch solution, and refiltered. Distilled water (25 mL) was added to the filtered solution and centrifuged at 500 rpm for 10 min. The upper layer (methanol and water layer) was removed using an aspirator, and the bottom layer (chloroform containing lipid extract) was passed through anhydrous sodium sulfate (Na2SO4). The Na2SO4 was rinsed with 30 mL of chloroform. The extracts were concentrated using an evaporator (Zymark TurboVap 500, Hopkinton, MA) at 40C under nitrogen and stored at –40C until required for analysis. Then, 0.1 g of extracted lipid samples was added into 50 mL tube with 10 mL of saponification reagent (30% KOH and ethanol with the ratio of 6:94) and 0.5 mL internal standard (2 mg 5α-cholestane/sample), and then homogenized with a Polytron homogenizer (IKA Labortechnik T25-B) for 10 s, caped and incubated for 1 h at 60C. After cooling the sample, 8 mL of deionized distilled water and 3 mL hexane were added and mixed thoroughly to allow separation. The top layer (hexane layer) was taken out and dried in scintillation vials, and 100 µL of bis-[trimethylsilyl]trifluoroacetamide + 1% trimethylchlorosilane and 200 µL of pyridine were added and mixed, and set overnight and then analyzed by gas chromatography (Shimadzu GC-14A). A ramped oven temperature condition (180C for 2.5 min, increased to 230C at 2.5C/min, then held at 230C for 7.5 min) was used. The temperature of both the inlet and detector was 280C. Helium was the carrier gas at linear flow of 1.1 mL/min. Dector (flame ion detector) air, H2 and makeup gas (He) flows were 350, 35 and 43 mL/min, respectively.
Fatty Acid Analysis
Lipids were extracted with chloroform and methanol as described by Folch et al. (1957). For lipid hydrolysis, an aliquot of lipid extract (30 mg) and 3 mL of 4% H2SO4 in methanol were combined in a screw-capped test tube. The test tube was placed in boiling water (100C) for 20 min, and subsequently cooled at room temperature. The resulting free fatty acids were methylated with 1 mL of 14% boron trifluoride in methanol at room temperature for 30 min. Water (1 mL) and hexane (5 mL) were added. The samples were vortexed and centrifuged at 500 × g for 10 min. The upper organic solvent layer was used to determine fatty acid composition. Fatty acid methyl esters were analyzed on a gas chromatograph (Shimadzu GC-14A) equipped with an on-column injector port and flame ionization detector. A Silar capillary column (30 m × 0.32 mm × 0.25 µm; Shimadzu) was used for the separation of the fatty acid methyl esters. The gas chromatography oven temperature was 140C, and increased at a rate of 2C/min to a final temperature of 230C. The temperatures of injector port and detector temperatures were set at 240 and 250C, respectively. Fatty acid methyl ester (1 µL) was injected onto the split injection port (100:1 split ratio). The flow rate for He carrier gas was 50 mL/min. Each fatty acid was detected by the standards' retention time (Table 2).
Table 2. EFFECT OF DIETARY GLYCINE BETAINE ON FATTY ACID COMPOSITION IN LOIN
The effects of dietary glycine betaine on pork quality and blood characteristics were analyzed using SAS software (SAS, 1996, SAS Institute Inc., Cary, NC) by the generalized linear model procedure; the Student–Newman–Keuls's multiple range test was used to compare the differences among means. Significance was defined at P < 0.05.
RESULTS AND DISCUSSION
As shown in Fig. 1, glycine betaine concentrations in plasma were significantly increased by dietary glycine betaine. Kettunen et al. (2001) also reported that the dietary betaine supplementation increased the plasma betaine concentration approximately threefold. The serum triglyceride content was significantly lower in dietary glycine betaine groups compared with control; however, no significant difference was found between T2 and T3 (Fig. 2). The major involvement of betaine in lipid metabolism is in its lipotropic activity. Barak et al. (1994) reported that the dietary betaine has been shown to stimulate liver lipid mobilization and alter the blood lipoprotein profile. The natural lipids are mainly composed of triglycerides, which form the principal part of depot lipids in meat systems (Fernandez et al. 1998). Thus, dietary glycine betaine decreases fat deposition by reduction of serum triglyceride in animal bodies. The involvement of betaine in lipid metabolism offer an interesting challenge in meat-producing animals because of the current interest in producing lean meat (Fernandez et al. 2000). In general, CPK concentrations in pig can be barometer of stress tolerance because CPK concentrations can be reduced by stress before slaughtering. Warriss et al. (1990) also reported that plasma CPK level was associated with stress-sensitive and meat quality meat such as ultimate pH and pale soft exudative (PSE) meat. In this study, however, CPK concentration in plasma was not significantly different by dietary glycine betaine (Fig. 3). Therefore, dietary glycine betaine should not influence the stress tolerance or PSE of pigs.
Glycine Betaine Concentration
Glycine betaine concentrations of loins were significantly increased with dietary glycine betaine; however, glycine betaine concentration of control group has been shown to trace (Fig. 4). This result was in agreement with Kettunen et al. (2001) who reported that muscle betaine concentration was lower when dietary betaine level was low in chicks, but when the dietary betaine concentration increased, betaine accumulated in the liver and intestinal tissues where it may have a role in osmoregulation. As a result of this study, we assumed that not only plasma glycine betaine level, but also muscle glycine betaine level may be increased by dietary glycine betaine in pigs; therefore, it may influence the pork quality.
At the first and seventh days of storage, redness (a*) was significantly higher in T2 and T3; however, at the 14 days of storage, redness (a*) was not significantly different in all dietary groups (Fig. 5). Øverland et al. (1999) reported that subjective color was paler in pigs fed with betaine. However, Matthews et al. (2001a) did not observe any effects on the color of pigs fed with betaine in the loin muscle or biceps femoris muscle. Usually, lipid oxidation may initiate the oxidation of myoglobin to metmyoglobin, and, thus, change meat color from red to unattractive brown. The rate of meat discoloration is closely related to the rate of myoglobin oxidation induced by lipid oxidation (Yin and Faustman 1993). Our previous study showed that lipid oxidation was decreased by dietary glycine betaine (data not shown). Thus, dietary glycine betaine can improve the color stability of loin. It may be because the dietary glycine betaine increases saturated fatty acids (SFAs) and decreases unsaturated fatty acid (USFA), because SFA is more effective in stability of oxidation than USFA.
Shear force was slightly decreased during storage period in all dietary groups (Fig. 6). Higher shear force values were observed in dietary glycine betaine groups. Especially, T2 was the highest shear force value compared with other dietary groups. Fernandez-Figares et al. (2002) reported that the rate of protein deposition in the carcass tended to be linearly related to the dietary betaine content, and lean gain efficiency was also numerically improved by dietary betaine. Saunderson and Mackinley (1990) reported that betaine is effective in reducing body fat and in increasing the protein content of chick breasts at 21 days of age. Lawrence et al. (1995) and Cardogand et al. (1993) also reported that dietary betaine was associated with decreased backfat thickness, although growth performance and carcass traits were not affected. In this study, shear force value may be influenced by dietary glycine betaine because it increased protein contents and decreased fat contents (data not shown).
The cholesterol concentration in loin was significantly influenced by dietary glycine betaine (Fig. 7). Lower concentration contents were observed in T2 and T3, whereas T1 was not significantly different compared with the control group. Betaine is effective in reducing body fat and increasing the protein content of chick breasts at 21 days of age (Saunderson and Mackinley, 1990). Betaine has been suggested for swine for the reduction of carcass backfat and for increasing lean mass, as well as improving feed efficiency (Cromwell et al. 1999). Lawrence et al. (2002) also reported that adipose tissue deposition in gilts may be less readily altered by dietary betaine supplementation because of their lower potential for fat deposition. The mechanism for the reduction of cholesterol contents in muscle has not been understood yet. It may be because the reduction of cholesterol content in loin by dietary glycine betaine might be associated with decreasing of fat deposition and cholesterol reduction, although we tested cholesterol by the same amount of extracted fat.
Dietary glycine betaine increased the ratio of SFAs and decreased USFAs in loins. The proportion of myristic acid and linoleic acid was decreased by dietary glycine betaine, whereas those of stearic acid were increased by dietary glycine betaine. These changes are the main reasons for increased SFA and decreased USFA. This result agrees with Fernandez et al. (1998) who reported that dietary glycine betaine significantly reduced USFA and increased SFA. There are indications that betaine may play a role in fatty acid metabolism. As a result of lipid oxidation, complex mixtures of aldehydes, ketones, hydrocarbons, esters, lactones and alchohols can be produced, and an oxidative off-odor can be generated. The accelerated lipid oxidation was mainly because of increased lipid oxidation associated with USFA. Thus, increased SFA and decreased USFA by dietary glycine betaine may improve the oxidative stability or redness of loin.
Betaine has been shown to affect some aspects of pork quality and blood characteristics, but other reports have shown that betaine has no effect on pork quality. Although betaine may affect growth performance and carcass characteristics of pigs, the effects are variable. It is not clear how betaine would affect blood characteristics and pork quality attributes. As a result of this study, dietary glycine betaine should improve the meat redness and reduced cholesterol. Dietary glycine betaine increased SFA and increased shear force. However, dietary glycine betaine did not influence CPK levels in plasma. Until now, the mechanism for the changes of meat quality and blood characteristics by dietary glycine betaine has not been understood yet. Thus, more research will be needed to determine the effects of dietary glycine betaine on meat quality and blood characteristics.
The authors acknowledge a graduate fellowship provided by the Ministry of Education and Human Resources Development, Korea. Young-Hwa Hwang was supported by a scholarship from the BK21 Program.