Meat is a staple food, providing proteins of high nutritional value and a high content of essential minerals and B vitamins [1
]. However, meat lacks antioxidants and it is, therefore, susceptible to oxidative changes. Processing, such as grinding, exposes the muscle surface to the air and the lipid membranes to metal oxidation catalysts [2
]. Oxidation processes cause deterioration in the flavor, texture and color of meat, induce the development of toxic compounds and loss of nutrients, and reduce shelf life [3
]. Antioxidants are used to delay, retard, or prevent oxidative reactions in meat products [4
]. The antioxidants added in meat products are mainly synthetic, but due to the current trend to avoid or minimize the use of synthetic food additives, studies to identify novel and natural extracts with potential applications for meat and meat products are needed [5
]. The use of antioxidative plant extracts can be of great benefit also for human health.
Various plant sources have been studied as antioxidants in meat and other products [4
]. However, the information of the potential of Nordic plants such as Nordic berry fruit and leaves, trees, grains, and wild edible plants is scarce. It is known that especially Nordic berry fruit and leaves are excellent sources of phenolic compounds such as phenolic acids, flavonoids, and tannins which can act as both primary and secondary antioxidants [9
]. Coniferous trees are also abundant, but neglected sources of structurally similar polyphenols as in berries. Spruce inner bark contains mainly stilbene glucosides (astringin, isorhapontin and piceid) [12
], while pine heartwood contains mainly stilbene aglycones (pinosylvin and pinosylvin monomethyl ether) [13
Efficient extraction of the antioxidants from their natural sources, along with establishing their in vivo
and in producto
antioxidant activity, has been a great challenge for researchers [9
]. Subcritical water extraction (SWE) is a new, promising extraction method for bioactive compounds. Subcritical water is defined as the water that maintains its liquid state under adequate pressure at temperature between the boiling point 100 °C and critical point 374 °C. Supercritical water has special properties to extract both polar and non-polar analytes. SWE is a green, safe technology which can result in high quality products with lower production cost and higher efficiency [9
The aim of this study was to find new sustainable and effective natural, Nordic antioxidant sources, develop SW extraction methods for the most potential raw materials to extract their antioxidative fractions, and test the effects of the materials and their SW extracts in meat products. To our knowledge, this is the first study to assess the antioxidant effects of SW extracted berry leaves in meat products.
2. Materials and Methods
2.1. Plant Materials
13 different samples were collected during 2015–2016, including blackcurrant (Ribes nigrum), chokeberries (Aronia melanocarpa/mitchurinii), rosehips (Rosa rugosa), blackcurrant juice press cake, the hulls of buckwheat (Fagopyrum escolentum), Scots pine heartwood (Pinus sylvestris), Norway spruce inner bark (Picea abies) and the leaves of sea buckthorn (SBL, Hippophae rhamnoides), lingonberry (Vaccinium vitis-idaea), bilberry (BL, Vaccinium myrtillus), goutweed (Aegopodium podagraria), nettle (Urtica dioica) and dandelion (Taraxacum officinale).
Some wild samples (goutweed and dandelion) were picked in southern Finland. The other samples were donated by various Finnish companies and producers. Nettle leaves, hulls of buckwheat, BL, pine heartwood and spruce inner bark were air-dried, and the other samples were freeze-dried in the laboratory before analyses.
Ethanol (96%) was purchased from Altia (Rajamäki, Finland). Chemicals and reagents used in measuring antioxidant capacity were purchased from Sigma Chemical (Sigma Chemical Co., St. Louis, MO, USA). The chemicals used in the characterization of the spruce inner bark and pine heartwood extracts were pyridine, N,O-Bis(trimethylsilyl)trifluoroacetamide and chlorotrimethylsilane, purchased from Sigma–Aldrich (St. Louis, MO, USA). The standards of phenolic compounds and the chemicals used in the assays were obtained from various manufacturers. Catechin, epicatechin, gallocatechin, epigallocatechin, caffeic acid, chlorogenic acid, ferulic acid, gallic acid, ellagic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, syringic acid, p-coumaric acid, and sinapic acid were obtained from Sigma Chemical. Procyanidin B2 was from Extrasynthese (Lyon, France). Acetonitrile, methanol, concentrated hydrochloric acid (37–38%), and phosphoric acid (85%) were from J. T. Baker (Mallinckrodt Baker Inc., Utrecht, The Netherlands). Cysteamine and formic acid were from Sigma Chemical (Sigma Chemical Co., St. Louis, MO, USA).
2.3. Extraction of Antioxidants with Water and Ethanol-Water at Ambient Temperature
All samples except pine heartwood and spruce inner bark were extracted using water and 50% ethanol (aq) with a solid/liquid ratio 1:10. Extraction mixtures were homogenized with Ultra-Turrax T25 (IKA GmbH, Breisgau, Ger), followed by ultrasound assisted extraction (VWR USC 2100D, VWR International, Helsinki, Fin) for 30 min (45 kHz). Extracts were centrifuged, filtered and stored at −20 °C prior to antioxidant analysis.
2.4. Two-Step Extraction of Tree Materials and Determination of Phenolic Compounds
The pine heartwood and spruce inner bark extracts were obtained by two-step extraction using hexane and 95% ethanol (aq) according to the previously optimized protocol [15
]. A stainless steel extraction cell (Dionex Corp., Sunnyvale (CA), USA) was loaded with raw material powder and extracted with n-hexane at 90 °C, and the residue was again extracted with ethanol/H2
O (95:5, v
) at 100 °C using accelerated solvent extraction equipment Dionex ASE-350 (Dionex Corp., Sunnyvale (CA), USA). The extractions were performed as 3 × 5 min static cycles. The ethanolic extracts were further used and concentrated using a rotary evaporator.
The dry solids content of the extracts was determined gravimetrically, and polyphenols were determined by a GC-MS analysis [16
]. Briefly, aliquots of the extracts were evaporated to dryness under an N2
stream and silylated by adding 150 μL of a mixture of pyridine, N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) and trimethylsilyl chloride (TMCS), at a 1:4:1 (v/v/v
) ratio, and the mixture was heated in an oven at 70 °C for 45 min. Betulinol (0.02 mg/mL) and heptadecanoic acid (C17:0, 0.02 mg/mL) served as internal standards. The silylated samples were quantified by GC-MS as described earlier [16
2.5. Subcritical Water Extraction of Berry Leaves and Determination of Phenolic Compounds
SWE was developed for BL and SBL using accelerated solvent extraction equipment Dionex ASE 350 (Dionex Corp., Sunnyvale (CA), USA). SWE conditions, i.e., extraction temperature and static extraction time, were optimized with regard to the antioxidant activity of the extracts using response surface modelling with MODDE (BioPAT®) chemometrics software. The solid/liquid ratio was set at 1:10. The extracts were frozen at −20 °C immediately after extraction and later lyophilized.
Raw materials and the optimized SW extracts were analyzed for the content of major phenolics (i.e., phenolic acids and condensed tannins in BL extract, and ellagitannins and condensed tannins in SBL extract) using previously published high performance liquid chromatographic (HPLC) methods [17
2.6. Antioxidant Activity of Plant Extracts In Vitro
The antioxidant activity of the plant extracts was assessed in aqueous phase as radical scavenging capacity using the ABTS [(2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] decolorization assay [20
] with slight modifications [21
]. The results are expressed as Trolox equivalent antioxidant capacity (TEAC) values, describing the capacity of the samples to scavenge radicals in mg dm/mL in comparison to Trolox.
The susceptibility of the plant extracts to inhibit lipid oxidation was assessed in a lipid phase with a liposome model [22
] with some modifications. Briefly, soybean phosphatidylcholine liposomes were prepared according to Ursini et al. [23
]. Liposomes were stored at 4 °C at least one week prior to the study to increase the lipid hydroperoxide levels. The lipid oxidation reaction was conducted as described earlier [24
]. Briefly, liposomes (100 µL) were mixed with sample, buffer (50 mM K-phosphatebuffer pH 7.4, 100 mM glysine, 450 μM ascorbic acid) and oxidative agent (150 µL of 1 mM ADP in 25 μM FeCl3
) at various sample concentrations. The suspension was allowed to react for 48 h at room temperature in the dark. Consequently, the concentration of the thiobarbituric acid reactive substances (TBARS) formed during the liposome oxidation was determined by a color reaction with thiobarbituric acid (TBA) and butylated hydroxytoluene (BHT). The color reaction was performed by mixing the oxidized liposome suspension with trichloroacetic acid (TCA)/TBA solution (0.375% TBA, 2.25% TCA in 0.25 M HCl) and BHT (2% BHT in Methanol) and consequent incubation in a boiling water bath for 30 min. The solution was cooled to room temperature and centrifuged at 1710× g
for 10 min. Aliquots, 30 µL, of the supernatants, were injected into an Agilent 1100 HPLC-DAD with a SunFire C18 column (4.6 mm × 150 mm, 5 μm particle size, Waters). Samples were eluted with a linear gradient (6–99% in 30 min) of acetonitrile in 0.05% trifluoroacetic acid, and the effluent was monitored at 532 nm. The concentration of malondialdehyde (MDA) was calculated against the MDA-TBA standard curve (12.5–800 μM). Samples were analyzed in triplicates.
Results from the liposome model are presented as an inhibition efficiency ratio (IER) describing the inhibition percentage produced with a sample concentration of 1 µg dm/mL. For the samples with the highest antioxidant potential (spruce inner bark and pine heartwood extracts) and for BL and SBL SW extracts the IC50 values were measured. The IC50 value indicates the concentration of a sample µg dm/mL needed to inhibit 50% of the lipid oxidation in the liposome model. The IC50 value was calculated using a linear regression from a plot inhibition percentage versus sample concentration µg dm/mL.
2.7. Application of Berry Leaves and Their Subcritical Water Extracts in Chicken Marinades and Pork Sausages
The capacities of dried and homogenized BL and SBL and their SW extracts to prevent lipid oxidation in meat products were tested in sausage and marinated chicken leg slices. The concentrations used were selected according to IC50 values and preliminary tests. In the sausage test, the basic sausage mass contained pork meat 75%, water 25%, white pepper 2g/kg mass, salt 16.6 g/kg mass, and diphosphates (E450) 3g/kg mass, and there were eight treatments (Table 1
). Treatment 1 served as a negative control containing only basic mass, treatments 2–7 contained test materials and treatment 8 served as a commercial (positive) control containing NaNO2
and ascorbic acid. Three 400 g sausages were prepared for each treatment by casing in commercial synthetic sausage skin, heat-treated to an inner temperature of 72 °C, cooled in running cold water and stored overnight in a refrigerator below 6 °C. On the following day, the sausages were cut into small cubes (1–1.5 cm3
) and pooled according to the treatments. The amount of 100 g of each treatment were taken for sensory analysis, and the remaining pools were divided into 80 g portions which were stored in plastic bags below 6 °C in a refrigerator until lipid oxidation analysis.
In the marinated chicken leg test, there were also eight treatments (Table 2
). In each treatment, 400 g of chicken leg slices were divided into 70 g portions and mixed in a plastic bag with 30 g of the marinades described in Table 2
. The basic marinade contained rapeseed oil 52%, sucrose 11%, salt 6%, water 21%, and 6% commercial spirit vinegar 10% (aq). Treatment 1 was a negative control containing only the basic marinade, treatments 2–7 contained test materials and in treatment 8 there was no marinade at all. After two hours’ stabilization at 6 °C, 100 g-bags of each treatment were taken for sensory analysis. The remaining samples were stored at 6 °C in a refrigerator until analysis of lipid oxidation.
2.8. Sensory Evaluation
A sensory evaluation of the marinated chicken slices and sausages was conducted readily after preparation by 5 male and 5 female panelists. The samples were labelled with 3-digit random numbers. The sausages were evaluated in two groups of 4 and 5 samples per session. Sausages with 0.2% BL extract were evaluated in both sessions. Marinated chicken slices were fried before sensory evaluation using a Tefal ActiFry low-fat fryer. Sensory evaluation was conducted in two groups of 4 samples per session.
Panelists were given three slices per treatment and asked to evaluate on a scale with fixed extremes from 0 to 5. The evaluated parameters for preference were color (0 = unpleasant, 5 = tempting), flavor and overall acceptability (0 = poor, 5 = excellent). Each point marked was converted to a numerical value as a distance from 0. The most preferred treatments were estimated by ranking the sensory attribute median values. A nonparametric Kruskal-Wallis H test was used to determine if there were statistically significant differences between treatments. The statistical analysis was performed using (IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.).
2.9. Oxidation of Lipids in the Meat Products
The capacity of the BL and SBL and the respective SW extracts to prevent lipid oxidation in the products was assessed by the prevention of TBARS formation during storage. The TBARS in sausage samples were measured after 10 and 20 days of storage. The lipid oxidation status of the marinated and sliced chicken legs was measured after 4 and 8 days of storage. The lipid oxidation statuses as TBARS levels of the sausages and marinated chicken leg slices were measured using a specific HPLC method, described previously in Section 2.4
. Prior to the analysis, the sausage and marinated chicken slice samples were subjected to alkaline hydrolysis to release MDA from meat proteins. First, samples were homogenized using Ultra-Turrax T25 (IKA GmbH, Breisgau, Germany), and four 100 mg subsamples of each homogenized sample were taken for alkaline hydrolysis. The hydrolysis was conducted by mixing the 100 mg subsamples with 200 µL of 1.5 M NaOH and incubating the suspensions in a 60 °C water bath for 30 min. After the hydrolysis, 1 mL of 0.05 M sulfuric acid and 0.5 mL of 20% (w/v
) TCA were added, and the precipitated proteins were separated by centrifugation (3000 rpm, 10 min). The supernatants were then reacted with TBA to form MDA-TBA adducts with pink pigment and analyzed with HPLC, as previously described in Section 2.4
. Chromatographic analyses were performed in duplicate from each of the subsamples (n
≥ 8). Results are expressed as mean ± SD. An independent Student’s t
-test was used to compare the effects of the plant ingredients on the TBARS formation during storage.
The flow diagram of the study is in Figure 1
This study showed that berry leaves, pine heartwood, and spruce inner bark extracts possess superior radical scavenging potential and capacity to inhibit lipid oxidation. SWE emerged as a promising green, chemical-free method for recovering antioxidative compounds from plant materials. BL and SBL, as well as their SW extracts, efficiently prevented lipid oxidation in pork sausage and marinated sliced chicken legs. The results indicate that BL and SBL, and their SW extracts, are potential natural antioxidative agents for preventing lipid oxidation in meat products and therefore provide new possibilities for developing healthier meat products.