Supplementation of thymol powder or green tea extract along with linseed in pig diet increases oxidative stability in blood and pork

Abstract

This trial investigated the influence of dietary extruded linseed combined with plant extract on pig performance, blood lipid profiles, meat quality, oxidative stability and the fatty acid composition of meat and backfat. Four-hundred and eighty crossbred pigs were randomly allocated to four dietary treatments, namely, a control diet (T1), the control diet supplemented with extruded linseed (T2) and identical T2 diet supplemented with thymol powder (T3) or green tea extract (T4). The extruded linseed supplementation decreased blood triglycerides, total cholesterol and LDL cholesterol and improved fatty acid composition of meat and backfat by increasing n-3 PUFA content and decreasing the n-6/n-3 ratio (p < .05). However, it led to decrease the oxidative stability of blood and meat, while supplementation with thymol powder or green tea extract increased the oxidative stability of blood (27.08 and 27.53% in T3 and T4 compared with T2, respectively) and meat (40.15 and 41.21% in T3 and T4 compared with T2, respectively; p < .01). The findings conclude that dietary extruded linseed combined with thymol powder or green tea extract has the potential to produce functional pork.

Highlights

• Feeding pigs an extruded linseed meal supplement reduced blood triglyceride, total cholesterol and LDL cholesterol levels.

• Extruded linseed supplementation increased the n-3 PUFA content of pigs’ meat and backfat.

• Supplementing an enriched n-3 PUFA diet with thymol powder or green tea extract increased the oxidative stability of pigs’ blood and meat.

Keywords:

• Fatty acid composition
• green tea extract
• linseed
• oxidative stability
• thymol powder

Introduction

Pork is the world’s most consumed meat, but conventional pig diets generate pork with a low level of n-3 polyunsaturated fatty acids (PUFA) and a high n-6/n-3 ratio, which exceeds the 4:1 ratio recommended for human nutrition (Dugan et al. 2015). Means of raising the n-3 PUFA content in food have received much attention in recent years because n-3 PUFA have a key role in preventing and reducing the incidence of cardiovascular and inflammatory diseases (Ruxton et al. 2004; Nestel et al. 2015). The n-3 PUFA content of pork, and thus its quality, could be improved by decreasing the n-6/n-3 ratio via dietary enrichment with sources of α-linolenic acid (ALA), such as crushed whole linseed (Okrouhlá et al. 2013) or extruded linseed (Corino et al. 2008). ALA is a precursor of long-chain n-3 PUFA, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Huang et al. 2021). EPA and DHA improve cardiovascular health by altering lipid metabolism, inducing hemodynamic changes, decreasing arrhythmias, modulating platelet function, improving endothelial function and inhibiting inflammatory pathways (Cottin et al. 2011). ALA has cardioprotective effects by lowering lipid and lipoprotein levels and reducing inflammation in the blood vessels (Zhao et al. 2004). Nevertheless, n-3 PUFA are particularly susceptible to oxidisation (Jiang et al. 2017), and the oxidative stability of pork was thus decreased when pigs were fed an n-3 PUFA-enriched diet (Sringarm et al. 2022). Furthermore, a diet high in n-3 PUFA increases oxidative stress (Frankič et al. 2010), which can harm cell constituents including lipids, proteins and deoxyribonucleic acid, and also impairs tissue functions (Su et al. 2018). The oxidative stability of pork deteriorates due to an increase in lipid oxidation, which reduces the shelf life of pork (Rather et al. 2016). For these reasons, there has been much interest in the supplementation of pig diets with antioxidants to prevent lipid oxidation, help to increase pork’s oxidative stability (Rossi et al. 2014; Jiang et al. 2017) and decrease oxidative stress (Frankič et al. 2010; Su et al. 2018). Antioxidants can be synthetic or natural; however, due to the negative consequences of synthetic antioxidants, natural antioxidant demand has grown in the last few years (Shah et al. 2014). Thereby, most recent studies have focussed on identification of natural antioxidants from various plant sources.

Thymol is a phenol discovered in abundance in thyme species, especially in Thymus vulgaris essential oil (Khan et al. 2019), and it was demonstrated to have antioxidant properties (Nagoor Meeran and Stanely Mainzen Prince 2012). Thymol supplementation in broiler chicken diets increased growth performance and chicken meat’s oxidative stability and decreased the level of blood triglycerides and total cholesterol (Zidan et al. 2016). Green tea extract is another natural product shown by in vitro studies to have powerful antioxidative potential (Hu et al. 2009; Norkeaw et al. 2022). The main potent antioxidant components discovered in green tea extract are epicatechin-3-gallate and epigallocatechin-3-gallate (Namal Senanayake 2013). The chicken meat’s oxidative stability was also increased by green tea extract supplementation in broiler chicken diets (Farahat et al. 2016). However, few replace have examined the impact of thymol or green tea extract supplementation in pig diets, and past results addressing the improvement of pork’s oxidative stability have been highly diverse (Augustin et al. 2008; Hossain et al. 2012). Aside from these studies, research on the combination of an n-3 PUFA source with plant extracts in growing-finishing pigs is scarce. We are interested in including extruded linseed as an n-3 PUFA source and thymol powder or green tea extract as natural antioxidants in growing-finishing pig diets to generate functional meat. This trial aims to investigate the influence of dietary extruded linseed combined with thymol powder or green tea extract on pig performance, carcase quality, blood lipid profiles, meat quality, oxidative stability and fatty acid composition of pork.

Materials and methods

Raw materials and plant extracts

Green tea extract was produced by Specialty Natural Product Co., Ltd., Thailand. Thymol powder was obtained from Jiangxi Hairui Natural Plant Co., Ltd, China. The extruded linseed and other raw materials were obtained from Charoen Pokphand Group, Thailand.

Experimental design, animals and diets

In a nine-week trial, 480 crossbred pigs ([Large White × Landrace] × Duroc) with an average beginning body weight of 50.4 ± 5.7 kg were used. The pigs were randomly divided to one of four treatments based on sex and body weight. Each treatment included 6 pens (replicates) of 20 pigs per pen (3 pens of female and 3 pens of male). A two-period feeding program was applied in this experiment (Table 1), including grower (week 0 to 4) and finisher (week 5 to 9) periods. For each period, the pigs were fed four different experimental diets: a control diet (T1, control group), the control diet supplemented with extruded linseed (T2), which is an n-3 PUFA source, the T2 diet supplemented with thymol powder at 500 mg/kg diet (T3) and the T2 diet supplemented with green tea extract at 500 mg/kg diet (T4). Diets T2, T3 and T4 were supplemented with 2.5% extruded linseed in the grower period and 5% extruded linseed in the finisher period. Diet components and nutrient contents are shown in Table 1, and their fatty acid compositions are shown in Table 2. The nutritional standards established by the National Research Council were met or exceeded in all of the experimental diets (NRC 2012). The experiment was conducted in the Feed Research and Innovation Centre of Charoen Pokphand Group, Chonburi, Thailand. The pigs were raised in concrete-floored pens, and each pig was given 1.2 m² of floor area. Each pen had an automatic feeder and nipple drinkers that permitted the pigs to eat and drink as they pleased. Feed consumption and body weight were scaled and recorded at the beginning of the trial, as well as at the end of the fourth and ninth week of the trial to compute the average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR).

Table 1. The ingredient and nutrient composition of experimental diets (as-fed basis).

Item	Grower diets^a (week 0 to 4)				Finisher diets^a (week 5 to 9)
	T1	T2	T3	T4	T1	T2	T3	T4
Ingredients, %
Corn	14.19	10.10	10.05	10.05	25.77	21.71	21.66	21.66
Wheat	15.00	15.00	15.00	15.00	10.00	10.00	10.00	10.00
Barley	5.00	5.00	5.00	5.00	–	–	–	–
Cassava	20.00	20.00	20.00	20.00	20.00	20.00	20.00	20.00
Rice bran fine	15.00	15.00	15.00	15.00	15.00	10.00	10.00	10.00
Rice bran solvent	–	–	–	–	9.00	15.00	15.00	15.00
Soybean hull	–	–	–	–	5.00	5.00	5.00	5.00
Distillers dried grains (DDGS)	8.00	8.00	8.00	8.00	–	–	–	–
Canola meal	6.60	8.00	8.00	8.00	–	–	–	–
Soybean meal	6.30	4.05	4.05	4.05	10.10	7.10	7.10	7.10
Palm kernel meal	5.00	5.00	5.00	5.00	–	–	–	–
Animal protein	2.00	2.00	2.00	2.00	–	–	–	–
Molasses	1.00	1.00	1.00	1.00	2.00	2.00	2.00	2.00
Wheat bran	–	2.28	2.28	2.28	–	–	–	–
Extruded linseed	–	2.50	2.50	2.50	–	5.00	5.00	5.00
Thymol powder	–	–	0.05	–	–	–	0.05	–
Green tea extract powder	–	–	–	0.05	–	–	–	0.05
Limestone	0.70	0.69	0.69	0.69	1.21	1.17	1.17	1.17
Sodium chloride	0.31	0.39	0.39	0.39	0.38	0.45	0.45	0.45
Monocalcium phosphate	0.03	–	–	–	0.54	0.57	0.57	0.57
Vitamin and mineral premix^b	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Nutrient composition
Calculated ME, kcal/kg	3300	3300	3300	3300	3300	3300	3300	3300
Analysed, %
Dry matter	89.05	89.80	89.30	89.70	89.35	89.25	89.65	89.80
Crude protein	16.30	16.50	16.50	16.45	13.05	12.70	12.75	12.75
Crude fat	4.98	5.87	5.77	5.82	4.18	5.31	5.40	5.22
Crude fibre	5.02	5.31	5.47	5.44	5.70	6.25	6.44	6.43
Calcium	0.66	0.67	0.68	0.68	0.70	0.71	0.72	0.72
Total phosphorus	0.67	0.68	0.67	0.67	0.72	0.73	0.75	0.74
Lysine	1.04	1.05	1.03	1.05	0.88	0.89	0.90	0.89
Methionine	0.32	0.33	0.32	0.32	0.27	0.26	0.27	0.27
Methionine + cysteine	0.65	0.67	0.64	0.63	0.53	0.53	0.54	0.53
Threonine	0.66	0.68	0.67	0.67	0.54	0.56	0.57	0.56
Tryptophan	0.19	0.19	0.19	0.19	0.15	0.15	0.15	0.15

Note: ME: metabolisable energy.

^aT1 = control diet; T2 = control diet supplemented with extruded linseed; T3 = T2 diet supplemented with thymol powder; T4 = T2 diet supplemented with green tea extract.

^bVitamin and mineral premix provided the following per kilogram of diet: 100 mg Fe; 90 mg Cu; 60 mg Mn; 0.35 mg I; 0.25 mg Se; 80 mg Zn; 6000 IU vitamin A; 1400 IU vitamin D₃; 17 IU vitamin E; 2.5 mg vitamin K (menadione); 2 mg vitamin B₁; 8 mg vitamin B₂; 0.05 mg vitamin B₁₂; 0.15 mg biotin; 35 mg niacin; 18 mg pantothenic acid.

Table 2. The fatty acid composition of experimental diets.

Fatty acids (g/100g of total fatty acids)	Grower diets^a(week 0 to 4)				Finisher diets^a(week 5 to 9)
	T1	T2	T3	T4	T1	T2	T3	T4
C12:0	5.14	4.55	4.54	4.58	0.31	0.24	0.23	0.23
C14:0	2.02	1.82	1.81	1.82	0.56	0.47	0.46	0.45
C16:0	20.55	18.20	18.11	18.09	18.59	14.21	14.46	14.38
C16:1	0.85	0.76	0.75	0.75	0.60	0.50	0.51	0.50
C18:0	2.13	2.22	2.22	2.22	1.71	1.74	1.67	1.74
C18:1n-9	28.20	26.22	26.24	26.25	32.98	27.17	27.18	27.16
C18:2n-6	36.66	33.21	33.33	33.15	41.22	33.46	33.28	33.27
C18:3n-3	1.89	10.76	10.77	10.91	1.71	20.27	20.30	20.34
C20:0	0.96	0.86	0.83	0.85	0.87	0.72	0.72	0.73
C20:1n-9	0.76	0.67	0.67	0.68	0.75	0.62	0.63	0.62
C20:2	0.87	0.76	0.75	0.75	0.73	0.61	0.61	0.61
Total saturated fatty acids (SFA)	30.78	27.64	27.50	27.55	22.04	17.39	17.52	17.52
Total monounsaturated fatty acids (MUFA)	29.81	27.65	27.66	27.67	34.32	28.29	28.31	28.27
Total polyunsaturated fatty acids (PUFA)	39.42	44.72	44.84	44.80	43.65	54.33	54.18	54.22
PUFA/SFA	1.28	1.62	1.63	1.63	1.98	3.13	3.09	3.10
C18:2n-6/C18:3n-3	19.43	3.09	3.10	3.04	24.10	1.66	1.64	1.64

^aT1 = control diet; T2 = control diet supplemented with extruded linseed; T3 = T2 diet supplemented with thymol powder; T4 = T2 diet supplemented with green tea extract.

Blood lipid profile

At the beginning, fourth and ninth week of the trial, one pig per pen was randomly selected to collect blood samples from jugular vein. The one pig each pen was randomly and unbiasedly chosen as the representative of their respective pens for blood collection. At the end of the fourth and ninth week of the trial, the same pigs were bled again. The whole blood was centrifuging for 15 min at 1700 g with 4 °C. These sera were kept at −20 °C till an automated clinical chemistry analyser (BX-3010, Sysmex, Singapore) measured the blood lipid profile, which included triglycerides, HDL cholesterol, LDL cholesterol and total cholesterol.

Carcase evaluation and sample collection

The 24 pigs were killed in a commercial slaughterhouse according to standard procedures at the end of trial after fasting for 12 h. Slaughter weight was recorded individually. Hot carcases were weighed after slaughtering, and cold carcases were weighed after keeping for 24 h at 4 °C. The cold carcase weight was used to calculate the dressing yield according to the method of Kowalski et al. (2020). The left sides of the carcases were used for carcase evaluation, which included carcase length, average backfat thickness and loin area. The carcase length was measured from the aitch bone’s anterior tip to the first rib’s anterior edge near the vertebrae (Watkins et al. 1990). The surface area of the longissimus thoracis et lumborum (LTL) muscle at the 10th rib was traced to determine the loin eye area (Matthews et al. 2001), and the area was measured using a measuring tool in the Adobe Acrobat Reader DC program. Backfat thickness measurements at the last rib, first rib and last lumbar vertebrae were averaged to establish the average backfat thickness (Jaturasitha et al. 2002). After keeping the carcases for 24 h at 4 °C, the backfat and LTL muscle samples were stored at −20 °C for determination of meat quality, meat oxidative stability and meat and backfat fatty acid composition.

Meat quality evaluation

The pH of the LTL muscle between the 13th and 14th ribs was measured 45 min and 24 h after death (Jaturasitha et al. 2002) using a portable pH metre (Model 205, Testo, Lenzkirch, Germany). The collected LTL muscles were cut into 2.5-cm-thick slices from the anterior end for meat colour measurement using a Konica Minolta Chroma Metre (model CR-400, Minolta Camera Co., Ltd., Osaka, Japan), which was conducted 48 h after slaughtering (Chaiwang et al. 2021). The 2.5-cm-thick LTL muscles were allowed to oxygenate for 30 min, and then the colour on the surface area of each muscle was measured at three locations. The meat colour was expressed as lightness (L*), redness (a*) and yellowness (b*). The chemical composition (moisture, crude fat and crude protein) of the LTL muscles was analysed according to proximate analysis methods (AOAC 2006).

The LTL muscles’ water holding capacity (WHC) was evaluated as drip loss, thawing loss and cooking loss according to previous methods (Chaiwang et al. 2021). The drip loss was measured on 2.54-cm-thick slices which were scaled and each sample was wrapped in gauze and sealed in a plastic bag individually. All samples were kept in a refrigerator at 4 °C for 24 h, then the bags were unsealed, and each slice was reweighed without the exudate. The thawing loss was evaluated using 2.54-cm-thick slices, which were scaled and sealed in a plastic bag separately, and all samples were frozen at −20 °C until analysis. After freezing, the bags were kept in a refrigerator for 24 h at 4 °C for thawing. Thereafter, the meat slices were individually reweighed without the exudate. Regarding the cooking loss evaluation, 200 g of each LTL sample was scaled, placed in a heat-resistant plastic bag and then cooked in a water bath (WNE 29, Memmert, Germany) at 80 °C until the sample’s internal temperature reached 72 °C. All samples were allocated to one cooking batch and balanced the place for treatment groups. The cooked samples were subsequently permitted to cool at ambient temperature and reweighed for evaluation of cooking loss. In addition, the cooked samples were cut into five cubes (1 × 1 × 1 cm³) for measurement of shear force (Chaiwang et al. 2021) using a texture analyser (model TA.XT plus, Stable Microsystem, Ltd., London, England) with a Warner–Bratzler shearing instrument equipped with V shape blade. The measurement was performed by shearing the samples in a direction vertical to the long axis of the core, and the shear force was evaluated using the curve’s peak force. A 5 kN load cell calibrated to read over the range of 0–100 N was used, along with a blade speed of 200 mm/min.

Measurement of oxidative stability using the malondialdehyde (MDA) assay

Plasma MDA assay

The samples were centrifuged for 15 min at 1700 g and 4 °C to obtain plasma. The plasma was maintained at −80 °C till MDA measurement. Concisely, 0.2 mL of plasma was mixed with 0.75 mL of 0.44 mol/L phosphoric acid, 0.25 mL of 42 mmol/L aqueous thiobarbituric acid (TBA) and 0.3 mL of distilled water. Thereafter, the mixture was heated in a water bath for 60 min at 90 °C and then cooled on ice. After cooling, 0.5 mL mixture was taken and mixed with 0.5 mL of alkaline methanol (50 mL methanol and 4.5 mL of 1 mol/L NaOH). The mixture was centrifuged for 3 min at 2500 g, and 20 µL of sample was injected into the HPLC system (Khoschsorur et al. 2000). The analytical HPLC was performed on an Agilent 1220 Infinity II liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA) equipped with Ultra Aqueous C18 column (250 × 4.6 mm, 5 µm) (RESTEK, Bellefonte, PA, USA). The mobile phase consisted of 50 mmol/L phosphate buffer (pH 6.8) and methanol (40:60 v/v) at a flow rate of 0.8 mL/min. The fluorescence detector was set at Ex = 515 nm and Em = 543 nm. 1,1,3,3-tetraethoxypropane (TEP) was prepared for MDA standard solution.

Meat MDA assay

The oxidative stability of the LTL muscles was measured after storage in a refrigerator at 4 °C for zero, three and six days. Lipid oxidation was measured using the MDA assay in accordance with the technique of Bergamo et al. (1998); 2 g meat sample was transferred to a centrifuge tube and homogenised in a solution comprising 4.75 mL of distilled water and 0.25 mL of 1000 mg/L ethanolic BHT. Thereafter, 0.5 mL of the homogenate was removed and added to 0.5 mL of ice-cold 10% trichloroacetic acid (TCA). The sample was vigorously mixed for 3 min and centrifuged for 5 min at 10,000 g to remove proteins. A 0.3-mL sample of the supernatant was removed and mixed with 0.7 mL of the TBA solution, which consisted of 0.4% TBA in 2 mol/L acetate buffer at pH 3. The mixture was then degassed and heated in a water bath for 30 min at 90 °C. After heating, the sample was cooled and centrifuged for 5 min at 10,000 g to remove the particulate material. Eventually, 20 µL of sample was injected into the HPLC system. Analytical HPLC was carried out according to Sringarm et al. (2022).

Analysis of fatty acid composition

Lipids in the experimental diet as well as LTL muscle and backfat samples were extracted using chloroform-methanol (2:1) in according to Folch et al. (1957). Fatty acids were converted to fatty acid methyl esters (FAME) following (Morisson and Smith 1964). The fatty acid composition was analysed by gas chromatography (GC) according to Arjin et al. (2021). Shimadzu GC-2030 gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a Restek RT-2560 wall-coated fused wax capillary column (0.25 mm × 100 m × 0.25 μm, Restek, Bellefonte, PA, USA) were performed. Helium was used as a carrier gas. The injector temperatures were kept at 250 °C. The oven temperature was programmed to begin rising at a rate of 3 °C/min from 100 °C to 240 °C and then remain at 240 °C for 20 min. The 1 μL samples were injected, and the flame ionisation detector was set to 250 °C. The identity of the samples was compared the retention times of their peaks to those of FAME standard mixtures (Restek, Bellefonte, PA, USA).

Statistical analysis

The linear mixed models (LMM) was used to analyse the variance of all data. Tukey’s post hoc test was used to examine differences among treatments, and a statistical significance was considered at the 0.05 level. Results were reported as means with a standard error of mean (SEM). All analyses included dietary treatment as a fixed effect, with the exception of the meat MDA model, which also included storage time as a fixed effect and its interaction with dietary treatment. Sex was included in all models as a random effect. The growth performance and carcase quality were evaluated in six repetitions, while all chemical analyses were performed with three replicates. Analysis of the data was done using the lme4 package (Bates et al. 2015) in the R statistical program (R Core Team 2020).

Results

Growth performance and carcase quality

Throughout the duration of the experiment, all animals stayed healthy. Table 3 demonstrates the growth performance of pigs fed various diets. The initial weight, final weight, ADFI, ADG and FCR were not significantly different (p > .05) across treatment groups in all feeding periods. Regarding the carcase trait parameters (Table 4), the dietary treatments had no effect on the slaughter weight, hot carcase weight, cold carcase weight, dressing yield, carcase length, loin eye area and average backfat thickness (p > .05).

Table 3. The growth performance of growing-finishing pigs fed different diets.

Item	Dietary treatments^a				SEM	p Value
	T1	T2	T3	T4
Grower (week 0 to 4)
Initial weight, kg	50.34	50.35	50.37	50.34	1.158	1.000
Final weight, kg	77.82	77.15	77.96	77.48	1.478	.997
ADFI, g/d	2258	2196	2177	2223	42.29	.739
ADG, g/d	912	893	915	898	14.94	.872
FCR	2.475	2.461	2.381	2.471	0.019	.092
Finisher (week 5 to 9)
Initial weight, kg	77.82	77.15	77.96	77.48	1.478	.997
Final weight, kg	110.59	109.31	109.84	108.35	1.629	.947
ADFI, g/d	2768	2758	2718	2695	56.38	.475
ADG, g/d	931	919	911	881	14.43	.140
FCR	2.971	2.999	2.979	3.059	0.031	.485
Overall (week 0 to 9)
Initial weight, kg	50.34	50.35	50.37	50.34	1.158	1.000
Final weight, kg	110.59	109.31	109.84	108.35	1.629	.947
ADFI, g/d	2531	2499	2467	2476	47.43	.641
ADG, g/d	922	907	913	888	12.58	.208
FCR	2.741	2.753	2.699	2.782	0.021	.236

Note: SEM: standard error of the mean; ADFI: average daily feed intake; ADG: average daily gain; FCR: feed conversion ratio.

^aT1 = control diet; T2 = control diet supplemented with extruded linseed; T3 = T2 diet supplemented with thymol powder; T4 = T2 diet supplemented with green tea extract.

Table 4. The carcase quality of growing-finishing pigs fed different diets.

Item	Dietary treatments^a				SEM	p Value
	T1	T2	T3	T4
Slaughter weight, kg	112.33	111.83	111.67	111.17	1.224	.987
Hot carcase weight, kg	90.15	89.32	89.30	89.12	1.093	.985
Cold carcase weight, kg	87.62	87.07	87.07	86.52	1.098	.986
Dressing yield, %	77.98	77.83	77.97	77.78	0.281	.992
Carcase length, cm	82.67	82.33	82.33	82.00	0.491	.970
Loin eye area, cm^2	57.82	57.72	57.24	57.00	0.806	.976
Average backfat thickness, cm	2.72	2.71	2.61	2.64	0.065	.882

Note: SEM: standard error of the mean.

^aT1 = control diet; T2 = control diet supplemented with extruded linseed; T3 = T2 diet supplemented thymol powder; T4 = T2 diet supplemented with green tea extract.

Blood lipid profiles

Table 5 displays the blood lipid profiles of pigs fed various diets. At the end of the fourth and ninth week of the trial, pigs fed diets supplemented with extruded linseed alone (T2) or blended with thymol powder (T3) or with green tea extract (T4) had significantly lower total cholesterol and LDL cholesterol levels than those of pigs fed a control diet (p < .05). At the end of the fourth week of the trial, pigs in the T2, T3 and T4 groups had a lower (p < .05) triglyceride level than the control group. Triglyceride level was not significantly different (p > .05) among treatment groups at the end of the ninth week of the trial. However, pigs fed the extruded linseed diets showed a downward trend in the triglyceride level compared with the control group. HDL cholesterol levels, on the other hand, were unaffected by dietary interventions over the study periods. In all experimental periods, supplementation with thymol powder or green tea extract did not impact (p > .05) the blood lipid profile of pigs fed extruded linseed (T2, T3 and T4).

Table 5. The blood lipid profile of growing-finishing pigs fed different diets.

Item, mg/dL	Dietary treatments^a				SEM	p Value
	T1	T2	T3	T4
Week 0
Triglyceride	60.17	60.33	58.17	59.50	2.184	.983
Total cholesterol	119.17	122.33	118.33	118.33	2.062	.877
HDL cholesterol	46.92	49.40	47.30	45.72	0.909	.483
LDL cholesterol	52.63	59.03	55.80	55.62	1.343	.336
Week 4
Triglyceride	60.33^b	53.50^c	53.17^c	52.83^c	1.260	.034
Total cholesterol	127.33^b	111.00^c	108.50^c	110.17^c	2.283	<.01
HDL cholesterol	46.75	45.47	43.97	43.45	1.011	.535
LDL cholesterol	58.72^b	47.67^c	46.17^c	46.77^c	1.542	<.01
Week 9
Triglyceride	57.67	50.33	49.17	49.67	2.572	.561
Total cholesterol	128.50^b	109.33^c	108.50^c	109.83^c	2.741	<.01
HDL cholesterol	47.10	45.28	43.77	44.72	1.657	.902
LDL cholesterol	58.73^b	46.73^c	45.63^c	47.18^c	1.713	<.01

Note: SEM: standard error of the mean.

^aT1 = control diet; T2 = control diet supplemented with extruded linseed; T3 = T2 diet supplemented with thymol powder; T4 = T2 diet supplemented with green tea extract.

^b,cMeans with different superscripts in the same row differ significantly (p < .05).

Meat quality

In this study, pH values, meat colour, WHC, chemical composition and shear force were used to evaluate meat quality. As shown in Table 6, none of the meat quality parameters of the LTL muscle were affected by the dietary treatments (p > .05).

Table 6. The meat quality of longissimus thoracis et lumborum muscle of growing-finishing pigs fed different diets.

Item	Dietary treatments^a				SEM	p Value
	T1	T2	T3	T4
pH value
pH, 45 min post-mortem	6.44	6.39	6.54	6.38	0.034	.215
pH, 24 h post-mortem	5.71	5.68	5.73	5.64	0.024	.539
Colour
L* (lightness)	52.97	52.66	52.02	52.04	0.255	.366
a* (redness)	5.35	5.49	5.45	5.73	0.168	.865
b* (yellowness)	4.27	4.56	4.38	4.25	0.146	.856
Chemical composition, %
Moisture	73.30	73.18	73.22	73.09	0.072	.751
Crude protein	22.64	22.56	22.68	22.72	0.104	.955
Crude fat	2.17	2.22	2.15	2.12	0.074	.950
Water holding capacity, %
Drip loss	3.87	3.79	3.94	3.98	0.157	.973
Thawing loss	6.40	6.57	6.79	6.91	0.250	.879
Cooking loss	22.20	22.47	22.38	22.77	0.487	.980
Shear force, N/cm^2	38.29	37.42	37.26	37.46	1.336	.993

Note: SEM: standard error of the mean.

^aT1 = control diet; T2 = control diet supplemented with extruded linseed; T3 = T2 diet supplemented with thymol powder; T4 = T2 diet supplemented with green tea extract.

Blood and meat oxidative stability

At the end of the fourth and ninth week of the trial, the pigs fed T2 diets had the highest (p < .01) plasma MDA levels, in comparison with the other treatment groups, whereas the pigs fed T3 or T4 diets had lower (p < .01) plasma MDA levels than the control group (Figure 1). Influence of dietary treatment and storage time on meat oxidative stability is presented in Figure 2. The meat MDA level significantly increased (p < .01) after three days of storage. A significant interaction between dietary treatment and storage time was found (p < .01). At six days of storage time, the highest MDA level (p < .01) was found in the LTL muscle of pigs fed T2 diets, while the LTL muscle of pigs in the T3 and T4 groups had lower (p < .01) MDA levels than the control group.

Figure 1. The blood oxidative stability of growing-finishing pigs fed different diets. MDA = malondialdehyde. T1 = control diet; T2 = control diet supplemented with extruded linseed; T3 = T2 diet supplemented with thymol powder; T4 = T2 diet supplemented with green tea extract. The data are presented as a mean with standard error of the mean. Bars with various letters within a time class are significant different (p < .01).

Figure 2. Influence of dietary treatment and storage time on the meat oxidative stability. MDA = malondialdehyde. T1 = control diet; T2 = control diet supplemented with extruded linseed; T3 = T2 diet supplemented with thymol powder; T4 = T2 diet supplemented with green tea extract. Interactive effect of dietary treatment (T1, T2, T3 and T4) and storage time (D0, D3 and D6) on the MDA levels measured in LTL muscle over refrigerated storage at 4 °C. The data are presented as a mean with standard error of the mean. Bars with various letters are significant different (p < .01).

Fatty acid composition

Dietary treatments resulted in considerable alterations in the fatty acid profile of the LTL muscle and backfat as presented in Figures 3 and 4, respectively. Compared to the control group, the LTL muscle and backfat of pigs in the T2, T3 and T4 groups had a greater (p < .05) content of ALA, C20:3n-3 (ESA), C20:5n-3 (EPA), C22:6n-3 (DHA), n-3 PUFA and PUFA, whereas the LTL muscle and backfat of the control group had a higher (p < .05) content of C16:0 and total saturated fatty acids (SFA) than those of pigs in the T2, T3 and T4 groups. The dietary treatments did not impact total monounsaturated fatty acids (MUFA) content of LTL muscle (p > .05), but the backfat of pigs in the T2, T3 and T4 groups had a lower (p < .05) content of C18:1n-9 and MUFA than that of the control group. Since a result of these alterations in the fatty acid content of the LTL muscle and backfat, the PUFA/SFA ratio was increased (p < .05), whereas the n-6/n-3 ratio was evidently decreased (p < .05) in the LTL muscle and backfat of pigs in the T2, T3 and T4 groups (Figure 5). However, the fatty acid composition of the LTL muscle and backfat was not modified (p > .05) by supplementing thymol powder or green tea extract in the extruded linseed diet.

Figure 3. The fatty acid composition of LTL muscle of growing-finishing pigs fed different diets. FA = fatty acid; SFA = total saturated fatty acids; MUFA = total monounsaturated fatty acids; PUFA = total polyunsaturated fatty acids. T1 = control diet; T2 = control diet supplemented with extruded linseed; T3 = T2 diet supplemented with thymol powder; T4 = T2 diet supplemented with green tea extract. The data is presented as a mean with standard error of the mean. Bars with various letters within a fatty acid type are significant different (p < .05).

Figure 4. The fatty acid composition of backfat of growing-finishing pigs fed different diets. FA = fatty acid; SFA = total saturated fatty acids; MUFA = total monounsaturated fatty acids; PUFA = total polyunsaturated fatty acids. T1 = control diet; T2 = control diet supplemented with extruded linseed; T3 = T2 diet supplemented with thymol powder; T4 = T2 diet supplemented with green tea extract. The data are presented as a mean with standard error of the mean. Bars with various letters within a fatty acid type are significant different (p < .05).

Figure 5. The PUFA/SFA and n-6/n-3 ratios of LTL muscle and backfat of growing-finishing pigs fed different diets. T1 = control diet; T2 = control diet supplemented with extruded linseed; T3 = T2 diet supplemented with thymol powder; T4 = T2 diet supplemented with green tea extract. The data is presented as a mean with standard error of the mean. Bars with various letters within a tissue type are significant different (p < .05).

Discussion

Dietary supplementation with extruded linseed did not influence the growth performance, carcase quality and meat quality parameters of growing-finishing pigs. These results agreed with Corino et al. (2008) and Okrouhlá et al. (2013), who reported that linseed in pig diet did not affect the growth performance, carcase quality and physical pork quality. While thymol powder or green tea extract supplementation was similar to several studies that no effects on the growth performance, carcase quality and meat quality (Augustin et al. 2008).

This research demonstrated that supplementing growing-finishing pig diets with extruded linseed significantly decreased blood total cholesterol, LDL cholesterol and triglyceride levels. Similarly, Liu and Kim (2018) found that linseed oil supplementation significantly decreased blood total cholesterol, LDL cholesterol and triglyceride levels. In terms of the mechanisms, Dannenberger et al. (2014) reported that the expression of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), which are the main enzymes in fatty acid biosynthesis, was significantly decreased in the livers of pigs fed diets supplemented with linseed oil.

The level of plasma MDA is commonly employed as an effective marker to evaluate oxidative stress, which can harm cell constituents such as lipids, proteins and DNA. Excess oxidative radicals cause oxidative stress, but these radicals can be defeated by antioxidants consisting of endogenous enzymes and nonenzymatic components (Su et al. 2018). Dietary supplementation of extruded linseed alone significantly increased the level of plasma MDA. This finding agreed with Frankič et al. (2010), who indicated that MDA level in plasma were dramatically raised in pigs fed a diet supplemented with linseed oil. However, dietary supplementation with extruded linseed combined with thymol powder or green tea extract significantly decreased the level of plasma MDA. We assumed the potent antioxidative potential of thymol powder and green tea extract. The antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase were increased by thymol (Nagoor Meeran et al. 2016). Su et al. (2018) found that supplementing weaned pig diet with an essential oil, which contains 13.5% thymol, significantly decreased the serum MDA level. Moreover, green tea extract has high total phenolic and total flavonoid contents, which are considered as the indicators of antioxidant capacity due to the redox properties of phenolic compounds (Norkeaw et al. 2022). Skrzydlewska et al. (2002) found that green tea extract significantly increased the antioxidant enzyme activities and decreased the amount of lipid peroxidation products in rats. These results indicate that dietary supplementation with thymol or green tea extract increases blood oxidative stability and can be used to inhibit the oxidative stress.

After six days of storage, dietary supplementation with extruded linseed alone significantly increased the level of MDA in the LTL muscle. In previous studies, a significant impact of dietary treatments on lipid oxidation in pork was found after three days of refrigerated storage (Rossi et al. 2014). Generally, lipid oxidation in meat during storage increases when the content of PUFA is increased by dietary manipulation (Cheng et al. 2017), because PUFA are especially susceptible to oxidation and could affect meat quality and stability (Jiang et al. 2017). However, dietary supplementation of extruded linseed mixed with thymol powder or green tea extract significantly decreased the LTL muscle MDA level compared to that of pigs fed the control diets. Meat MDA levels in this study were correlated with plasma MDA levels, which can be attributed by the powerful antioxidant properties of thymol powder and green tea extract.

Supplementation of pig diets with extruded linseed evidently increased ALA content and decreased the n-6/n-3 ratio in the LTL muscle and backfat, which was related to the content of ALA in the extruded linseed diets. Dietary extruded linseed also increased the content of ESA, EPA and DHA in the LTL muscle and backfat. ALA is a precursor of long-chain n-3 PUFA, such as EPA and DHA (Huang et al. 2021); therefore, supplementation with linseed allowed ALA to compete more efficiently with linoleic acid (C18:2n-6) for the routes that produce their long-chain fatty acid derivatives (Riley et al. 2000). Similarly, Corino et al. (2008) found that dietary linseed significantly increased the level of ALA, EPA, DHA and DPA (C22:5n-3) and reduced the n-6/n-3 ratio in the muscle and backfat of pigs. The increase in n-3 PUFA content in the LTL muscle and backfat of pigs fed extruded linseed diets resulted in a higher PUFA/SFA ratio and a lower in the n-6/n-3 ratio. According to Riley et al. (2000) and Okrouhlá et al. (2013), linseed supplementation in pig diets significantly increased the level of n-3 PUFA, PUFA/SFA ratio and decreased the n-6/n-3 ratio in the muscle and adipose tissue. In this study, the PUFA/SFA ratio in the LTL muscle and backfat of pigs fed diets supplemented with extruded linseed was more than the UK Department of Health’s minimal recommendation of 0.4 (Dugan et al. 2015). Furthermore, feeding pigs with extruded linseed diets resulted in a significant reduction in the n-6/n-3 ratio in the LTL muscle and the backfat, approaching the recommended ratio of 4:1 (Dugan et al. 2015). A significant reduction in the n-6/n-3 ratio of the muscle and backfat to be under the ratio of 4:1 was found by feeding pigs with a diet containing 10% linseed for 60 days prior to slaughter (Huang et al. 2008). This study revealed that the C18:1n-9 level in the backfat of pigs fed extruded linseed diets decreased, but this reduction was not observed in the LTL muscle. This is because stearoyl-CoA-desaturase, which is an enzyme that catalyses the synthesis of monounsaturated fatty acids from saturated fatty acids. Kouba et al. (2003) indicated that the decrease in C18:1n-9 content in adipose tissue of pigs given a linseed diet was due to a reduction in stearoyl-CoA-desaturase activity, whereas no influence on stearoyl-CoA-desaturase activity in muscle. Regarding the effect of extruded linseed combined with thymol powder or green tea extract on fatty acid composition, neither plant extract affected the fatty acid composition of the LTL muscle and backfat compared to dietary extruded linseed alone. In addition, we discovered that the application of green tea and thymol extract has the potential to increase the oxidative stability of pigs fed a high PUFA diet. Our research indicated that green tea is more practical than thymol due to its lower cost. The only difference between green tea and thymol is their cost; both ingredients are equally beneficial when added to pig feed to promote oxidative stability.

Conclusions

Dietary supplementation with extruded linseed combined with thymol or green tea extract can be used to produce functional pork that is high in n-3 fatty acids and low in n-6/n-3 ratio, which is good for human health in preventing and reducing the incidence of cardiovascular and inflammatory diseases, without affecting growth performance, carcase traits or meat quality parameters. Due to the utilisation of only one plant extract inclusion level, the optimal dietary inclusion level of these two potential plant extracts must be further optimised to reduce meat production costs. Future studies are also needed to evaluate the potential impact on sensory characteristics, which are essential when developing any type of modified product. Extruded linseed mixed with thymol or green tea extract can be added to the diets of commercial pigs to improve the quality of the pork and raise the price of the product.

Ethical approval

The experiment was approved by the Animal Care and Use Committee Chiang Mai University (2564/AG-0001).

Author contributions

RN conceptualisation, methodology, data curation, formal analysis, investigation, writing-original draft, writing-review and editing. CA: conceptualisation, methodology, formal analysis, investigation, writing-original draft, writing-review and editing. AS: methodology, investigation. PH: investigation. NC: validation. SM: writing-review and editing. TY: formal analysis. KS: conceptualisation, project administration, writing-review and editing.

Acknowledgements

The authors would like to acknowledge Charoen Pokphand Group for providing the animals and feed.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Additional information

Funding

This research is supported by a grant from Post-Doctoral Fellowship, Chiang Mai University and National Research Council of Thailand [grant number GSCMU(NRCT)/09/2564].