Meat Science 97 (2014) 174–180

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Antioxidant properties of extracts from Ginkgo biloba leaves in meatballs Joanna Kobus-Cisowska ⁎, Ewa Flaczyk, Magdalena Rudzińska, Dominik Kmiecik Faculty of Food Science and Nutrition, Poznan University of Life Sciences, Wojska Polskiego 31, 60-634 Poznan, Poland

a r t i c l e

i n f o

Article history: Received 7 August 2013 Received in revised form 11 January 2014 Accepted 20 January 2014 Available online 26 January 2014 Keywords: Ginkgo biloba leaf extracts Lipid oxidation Cholesterol oxidation Cholesterol oxidation products Antioxidant Meat

a b s t r a c t The aim was to determine the effect of Ginkgo leaf extracts on the stability of lipids and cholesterol in pork meatballs over 21 days of refrigerated storage. The antioxidants used were characterized by their antioxidant activity towards lipids and cholesterol. Extracts were prepared from green and yellow leaves from Ginkgo biloba L. trees. Water, acetone and ethanol were used as extractants. The extracts showed stabilizing effects on both lipid and cholesterol oxidation processes. The lipid oxidation process of pork meatballs was mostly inhibited by the aqueous and ethanolic extracts of the yellow leaves. Their antioxidant activity was higher than that of BHT. All the extracts had a stabilizing effect on cholesterol and most of them inhibited the formation of oxidized derivatives. The acetone and ethanol extracts of green leaves and the ethanol extract of yellow leaves inhibited the formation of cholesterol oxidation products formation most effectively. © 2014 Published by Elsevier Ltd.

1. Introduction Extracts of Ginkgo biloba L. leaves are interesting because of their multifaceted action. The chemical composition of the anatomical parts of Ginkgo biloba L. was identified by Kawamura, as well as Furkawa. They showed that Gingko leaves are rich in such substances as polyphenols, terpenoids and vitamins (Del Tredici, 1991). These substances belong to a group of natural antioxidants. Therefore, it was decided to investigate this material as a natural antioxidant in stored meat products. A previous study indicated that extracts from yellow and green Ginkgo (Ginkgo biloba) leaves have in vitro model systems with strong antioxidant activity (Kobus et al., 2009, Flaczyk et al., 2009). Positive correlations were found between the flavonol content, chelating activity, reducing power, and DPPH• scavenging activity. In addition, among the analyzed Ginkgo preparations, the yellow ones were richer in flavonol aglycones. Moreover, the extracts with the highest flavonol content demonstrated excellent activity against linoleic oxidation in a βcarotene–linoleate model system. The results from this study suggest that extracts prepared from both yellow and green Ginkgo leaves may constitute a good source of natural antioxidants and could prolong shelf-life of, for example meat. Leaves (green) from Gingko trees are harvested during the growing period. As shown in previous model studies, yellow leaves are also a rich source of antioxidants.

⁎ Corresponding author. Tel.: +48 61 848 73 07; fax: +48 61 848 73 26. E-mail address: [email protected] (J. Kobus-Cisowska). http://dx.doi.org/10.1016/j.meatsci.2014.01.011 0309-1740/© 2014 Published by Elsevier Ltd.

Meat and meat products exhibit varied nutritive qualities because of their different protein and lipid contents, which are dependent on the species, part of the carcass and type of product (Biesalski, 2005). Meat and meat products, due to the presence of fat, are unstable: they deteriorate easily and during storage undergo changes, mainly oxidation. These changes, apart from microbial growth, are the main cause of the limited shelf-life of meat foodstuffs. Moreover, compounds formed as a result of auto-oxidation may have an adverse effect on the quality and flavor of the final product (Ahn, Nam, Du, & Jo, 2001; Ahn & Nam, 2008; Frankel, 2001). At the beginning of the lipid oxidation process, free radicals of lipid hydroperoxides and peroxides are formed (Boselli et al., 2005). Such products often react with each other, forming new compounds, of which the most important are aldehydes, ketones, hydroxy compounds, di- and trihydroxy esters, cyclic compounds and polymers Frankel, 2001; Hur, Park, & Joo, 2007; Kmiecik, Korczak, Rudzinska, GramzaMichałowska, & Hęś, 2009; Kmiecik et al., 2011). Another important component of the meat lipid fraction is cholesterol (3β-cholest-5-en-3-ol), with four conjugated rings, one hydroxyl group and one double bond in the molecule. Oxidation of cholesterol, as with unsaturated fatty acids, occurs in the presence of light and in an environment of singlet oxygen molecules. However, products of this oxidation are scarce (Baggio & Bragagnolo, 2006a,b; Smith, 1996). However, to date no studies have been conducted on the influence of extracts from green and yellow Ginkgo leaves on lipid and cholesterol stability in meat products even though the natural origin of an antioxidant is generally accepted as an important consideration for food use. Therefore, the antioxidant effect of green and yellow Gingko leaf extracts on the oxidation of lipid and cholesterol constituents in a meat model system was investigated.

J. Kobus-Cisowska et al. / Meat Science 97 (2014) 174–180

2. Materials and methods 2.1. Chemicals All solvents and chemicals used were analytical or GC grade. Cholesterol and oxysterol standards, including 5α-cholestan (5α-C), 5cholesten-3β,19-diol (19-OH-C), 5-cholesten-3β,7α-diol (7α-OH-C), 5cholesten-3β,7β-diol (7β-OH-C), 5-cholesten-3β-ol-7-on (7-keto-C), 5α,6α-epoxy-cholestan-3β-ol (α-epoxy-C), 5β,6β-epoxy-cholestan-3βol (β-epoxy-C), cholestan-3β,5α,6β-triol (triol-C), 5-cholesten-3β,25diol (25-OH-C), and 5-cholesten-3β,20-diol (20-OH-C), were purchased either from Sigma-Aldrich (St. Louise, MO, USA) or from Steraloids (New Port, RP, USA). 2.2. Preparation of extracts 2.2.1. Ginkgo leaves Green Ginkgo biloba L. var. Hippocrates leaves were harvested in August (marked as G), and yellow leaves were harvested in October (marked as Y) in 2009. Leaves came from a plantation in Baranowo belonging to the Poznan University of Life Sciences. Leaves, including leaf stalks, were dried at 40 °C until a moisture content of 8% was achieved, and then were ground in a laboratory mill (Retsch, type GM 200, Haan, Germany). The degree of comminution was by use a 0.03–0.8 mm-sized mesh. 2.2.2. Preparation of extracts Twenty grams of ground plant leaves were extracted with 1 L solvent at atmospheric pressure under the following conditions: water at 95 °C during 15 min infusion (GW, YW), acetone–water (3:2 v/v) at 40 °C during 90 min extraction (GA, YA), or ethanol (96%) at 18 °C during 16 h maceration (GE, YE) (Kmiecik, Korczak, Rudzinska, GramzaMichałowska, & Hęś, 2009). The solution was cooled and filtered using a Whatmann 1 filter. All samples were vacuum evaporated and lyophilized. The extracts were stored in a dark, dry and cool place. The bioactive compound composition of the extracts is presented in Table 1. The characterisation of these compounds and the antioxidant activity of the extracts have been analyzed previously (Kobus et al., 2009). 2.3. Meatball preparation and storage The meatballs were prepared as described by Flaczyk, Rudzinska, Wąsowicz, Korczak, and Amarowicz (2006) and Hes, Waszkowiak, and Szymandera-Buszka (2012) with modifications. Samples of raw meat — (pork belly 64.2%, shoulder 35%; all without nitrate and nitrite) were purchased from local stores in Poznan (Poland) and were twice ground in a grinder with mesh sizes of 10 and 5 mm with sodium chloride (0.8%). The meat batter was divided into 8 parts. To each part of the batter the extracts were added. Extracts were added to meat at 500 ppm in relation to the meat batter (previous own studies), while BHT was added at 200 ppm (the maximum limit in meat) (Kobus et al., 2009). A control sample without extract was also prepared. Thus the batters contained 1) GW — water infusion of green Ginkgo leaves, 500 ppm, 2) GA — acetone extract of green Ginkgo leaves, 500 ppm,

3) 4) 5) 6) 7) 8)

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GE — ethanol extract of green Ginkgo leaves, 500 ppm, YW — water infusion of yellow Ginkgo leaves, 500 ppm, YA — acetone extract of yellow Ginkgo leaves, 500 ppm, YE — ethanol extract of yellow Ginkgo leaves, 500 ppm, Control BHT 200 ppm.

The meatballs, approx. 50 g were subjected to thermal processing in a Rational Combi-Dämpfer CCC-6102 convection oven (with hot air circulation, without water vapor, time: 20 min, until 72 °C was recorded inside the meat balls). Samples were then cooled to room temperature and packaged (Multivac, type A 300/16) in vacuum bags (PE/PA, 75 μm thick) and stored for 21 days at 4 ± 1 °C. Analyses were conducted after 1, 7, 14 and 21 days of storage. For each kind of meatball three productions were done and the results averaged. Analysis were performed in three replications. The chemical composition of the meatballs after cooking is presented in Table 2. 2.4. Methods 2.4.1. Lipid extraction The process was with a chloroform:methanol 2:1 v/v mixture. After the separation phase, chloroform was evaporated in a vacuum evaporator and the lipid fraction retained (Flaczyk et al., 2006). The moisture, protein, fat and ash content were determined according to AOAC (1990). 2.4.2. Fatty acid composition The fatty acid composition of the methyl ester derivatives was determined by GC (Wąsowicz, Zawirska-Wojtasiak, & Rudzińska, 2001). Fatty acids were separated using a Hewlett Packard 5890 GC gas chromatograph (Agilent, Wilmington, DE, USA) equipped with Supelcowax10 (Supelco, USA) capillary columns (30 m × 25 mm × 25 μm) and FID. Oven temperature was initially set at 60 °C and increased to 210 °C at 12 °C/min. The injector temperature was set at 240 °C, split ratio at 1:25 and detector temperature at 260 °C. The carrier gas was ultrahigh-purity helium at a flow rate of 1 mL/min. Fatty acids were identified by comparing their retention times with commercially available standards. 2.4.3. Peroxide value (PV) The peroxide value was determined by titration with 0.02 M sodium thiosulfate and was expressed in meq O2/kg (ISO3960, 2005). 2.4.4. Anisidine value (AV) The determination was based on ISO 6885 (2008). The method is based on the reaction of aldehydes present in the sample with a p-anisidine solution in ice-cold acetic acid and measurement of the absorbance of the yellow complex formed at 350 nm (Metertek SP-830, Taiwan). 2.4.5. TBARS determination (TBARS) TBARS was determined according to Pikul, Leszczyński, and Kummerow (1989). Distillates were reacted with thiobarbituric acid (TBA) in boiling water and the absorbance measured at 532 nm

Table 1 The bioactive components of Ginkgo leaf extracts. Components [mg/g s.m.]

Green leaves

Yellow leaves

Water infusion

Acetone extract

Ethanol extract

Water infusion

Acetone extract

Ethanol extract

Polyphenols Phenolic acids Flavonols

56.86 ± 2.35 8.77 ± 0.12 0.88 ± 0.02

203.5 ± 4.47 14.16 ± 0.17 1.96 ± 0.03

204.44 ± 0.64 8.17 ± 0.08 0.51 ± 0.02

179.96 ± 3.48 13.80 ± 0.07 3.15 ± 0.05

247.54 ± 1.04 11.45 ± 0.12 4.50 ± 0.03

137.28 ± 1.46 11.12 ± 0.05 1.81 ± 0.03

Results are the mean values of three determinations ± standard deviation.

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J. Kobus-Cisowska et al. / Meat Science 97 (2014) 174–180 Table 2 Chemical composition of pork meat balls after cooking.

2.4.8. Inhibition activity Calculation of the inhibition of lipid oxidation and cholesterol oxidation was performed as follows:

Content

½%  of inhibition ¼ ½100–ðOI extract  100Þ=OI control

Component [%] Moisture Fat Protein Ash

53.54 26.63 19.09 2.65

Fatty acids [%] 14:0 16:0 16:1 18:0 18:1 18:2 18:3

1.47 25.18 3.50 13.91 46.60 8.71 0.61

(Metertek SP-830, Taiwan). The TBARS index was expressed as mg malondialdehyde (MDA)/kg meat. 2.4.6. Cholesterol analysis The content of cholesterol in the meatballs was determined by the procedure of Przygoński, Jeleń, and Wąsowicz (2000). In brief, lipids were extracted using the chloroform/ethanol mixture, then saponified with 1 M KOH in methanol at room temperature and the unsaponifiable fraction was extracted with diethyl ether. The solvent was evaporated under nitrogen and the residue was silylated by BSTFA with 1% TMCS and then analyzed by capillary gas chromatography. A HewlettPackard 6890 gas chromatograph in the split (1:25) mode, with a FID detector and capillary column DB-5 (30 m × 0.25 mm × 0.25 μm, J & W Scientific Inc., Folsom, CA, USA) was used. Analysis parameters were as follows: oven temperature, 290 °C; injector, 310 °C; and the carrier gas was ultra-high-purity helium at 1.6 mL/min. 5α-cholestane was used as an internal standard. 2.4.7. Cholesterol oxidation products (COPs) Cholesterol oxides were determined according to Przygoński et al. (2000). Seven oxysterols (7α-OH-C, 7β-OH-C, 7-keto-C, 25OH-C, α-epoxy-C, β-epoxy-C and triol-C) were identified using the GC/MS instrument. The lipid fraction was extracted using chloroform/methanol with 0.006% BHT. After 1 min of homogenization, the lipid fraction was separated and stored at − 80 °C. A volume of 2 mL of sodium methylate (10%) mixed with methyl-tert-butyl ether (MTBE) (4:6; v/v) was added to the dried sample and left for 1 h. Next, the sample was extracted with chloroform and rinsed with water and a citric acid solution. Then, the chloroform layer was evaporated to dryness under nitrogen. The sample was redissolved in 250 μL of chloroform and transferred onto a SEP-PAK NH2 cartridge (Waters, MA, USA) column, which was conditioned with 10 mL of hexane. Afterwards, the cartridge was washed with 10 mL of hexane, 5 mL hexaneMTBE (5:1; v/v) and 5 mL hexane-MTBE (3:1; v/v). The polar fraction of oxysterols was eluted from the column with 7 mL of acetone. Anhydrous pyridine (100 μL) and BSTFA with 1% TMCS (100 μL) were added to the dried sample and incubated for 4 h at room temperature prior to GC analysis. Cholesterol oxidation products were determined using a HewlettPackard 6890 capillary gas chromatograph. The sample (1 μL) was introduced in splitless mode onto a DB-5 column (30 m × 0.25 mm × 0.25 μm, J & W Scientific Inc., Folsom, CA, USA). The initial oven temperature was set at 110 °C for 1 min, and then increased at 40 °C/min to 270 °C and then at 4 °C/min to 290 °C, which was then maintained for 15 min. The flow rate of the helium carrier gas was 1 mL/min. Compounds were tentatively identified by comparison to retention times of known oxysterol standards. 19-OH-C was used as the internal standard.

where OI extract = the oxidation index of the sample with extract (PV, AV, TBARS, COPs), and OI control = the oxidation index of the control sample. 2.4.9. Statistical analysis All analyses were conducted on at least three replications. Means and standard deviations were calculated with the use of Microsoft Office Excel 2007 software. Pearson's correlation indexes were calculated with the use of STATISTICA™ PL 7.0 StatSoft. The significance of differences between mean values was determined at p ≤ 0.05 applying the analysis of variance (ANOVA) followed by Tukey's multiple range test. 3. Results Results showing the changes in individual lipid oxidation indexes during storage are presented in Table 3. The peroxide levels in lipids extracted from ground pork meat subjected to heat processing after one day ranged from 0.00 meq O2/kg in samples with an addition of yellow leaf extracts (YW, YA, YE) and BHT to 2.80 meq O2/kg in the control. One week of refrigerated storage had a significant effect on the progress of the oxidation process in all the samples (p ≤ 0.05). Peroxide levels ranged from 1.36 (sample GW) to 3.17 (the control sample), while the anisidine value ranged from 1.68 in sample YA to 5.48 in sample GE. After 2 and 3 weeks of storage, a Table 3 Lipid oxidation indexes in pork meat balls with Ginkgo extracts during 21 days of refrigerated storage. Sample

1 day

7 days

14 days

21 days

Peroxide value [meq O2/kg] GW 1.47b.C ± 0.44 GA 1.31b.A ± 0.10 GE 1.72c.A ± 0.13 YW 0.00a.A ± 0.00 YA 0.00a.A ± 0.00 YE 0.00a.A ± 0.00 Control 2.80d.A ± 0.68 BHT 0.00a.A ± 0.00

1.36a.B ± 0.5 2.45c.B ± 0.51 2.62c.AB ± 0.07 1.66b.C ± 0.8 1.77b.C ± 0.04 1.90b.B ± 0.12 3.17d.B ± 0.2 1.87b.B ± 0.88

1.24ab.A ± 0.24 3.39d.C ± 0.55 3.21d.B ± 0.31 1.03a.B ± 0.08 1.41b.B ± 0.14 2.28c.C ± 0.44 3.59d.BC ± 0.2 2.67c.C ± 0.12

1.96b.D ± 0.03 3.45d.C ± 0.2 3.41d.B ± 0.11 1.23a.BC ± 0.3 1.57ab.BC ± 0.22 2.22c.C ± 0.19 3.76d.C ± 0.16 2.89c.C ± 0.15

Anizidine value GW 6.13dD ± 0.13 GA 3.79abB ± 0.2 GE 6.28dC ± 0.14 YW 5.45cB ± 0.04 YA 4.10bC ± 0.93 YE 3.92bB ± 0.41 Control 6.30dB ± 0.67 BHT 3.50aB ± 0.05

5.01fC ± 0.32 3.16cA ± 0.1 5.48gB ± 0.28 2.98bA ± 0.25 1.68aA ± 0.16 4.00dB ± 0.59 5.09fA ± 0.09 4.45efC ± 1.1

2.16bA ± 0.52 5.64dC ± 0.44 2.12bA ± 0.12 3.13cA ± 0.3 2.06bB ± 0.6 1.24aA ± 0.49 6.12eB ± 0.27 3.06cA ± 0.22

2.81aB ± 0.09 7.90dD ± 0.28 8.52eD ± 0.15 5.41cB ± 0.25 7.09dD ± 0.09 5.32cC ± 0.64 9.60f ± 0.14 4.65bC ± 0.08

TBARS index [mg/kg] GW 1.59eA ± 0.06 GA 1.20cB ± 0.03 GE 1.21cA ± 0.02 YW 0.70aA ± 0.03 YA 0.94bA ± 0.02 YE 0.95bA ± 0.00 Control 1.32dA ± 0.05 BHT 1.86eC ± 0.04

1.72gB ± 0.02 1.12dA ± 0.01 1.36eB ± 0.13 0.82aB ± 0.01 0.94bA ± 0.03 1.08cB ± 0.01 2.29hB ± 0.06 1.55fA ± 0.01

1.71dB ± 0.02 1.93eC ± 0.04 2.28bC ± 0.07 1.61dC ± 0.01 1.34cB ± 0.06 1.10aC ± 0.01 2.41fBC ± 0.06 1.76dB ± 0.03

1.85bC ± 0.01 2.02cC ± 0.08 2.42dD ± 0.01 1.72bC ± 0.01 1.80bC ± 0.01 1.20aD ± 0.01 2.59eC ± 0.02 2.41dD ± 0.06

Abbreviations: GW — water infusion of green Ginkgo leaves, GA — acethone extract of green Ginkgo leaves, GE — ethanol extract of green Ginkgo leaves, YW — water infusion of yellow Ginkgo leaves, YA — acethone extract of yellow Ginkgo leaves, YE — ethanol extract of yellow Ginkgo leaves. Results are the mean values of three determinations ± standard deviation. Index values sharing the same upper case letter in a line are not significantly different (α = 0.05). Values sharing the same lower case letter in a column are not significantly different (α = 0.05).

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varied and disproportional increase in the levels of both PV and AV was observed. The lowest peroxide value, both after 14 and 21 days of storage, was recorded in samples with an addition of water extracts from green leaves (1.24 and 1.96 meqO2/kg respectively) and from yellow leaves (1.03 and 1.23 meqO2/kg respectively), while the highest was in the control (3.59 and 3.76 meqO2/kg). Similarly, the anisidine value, after 2 as well as 3 weeks was highest in the control, amounting to 6.12 and 9.60, respectively. It was found that water extract from green leaves inhibited the formation of secondary oxidation products to the highest degree, as manifested by the lowest levels of the anisidine value after both 14 and 21 days of refrigerated storage (2.16 and 2.81 respectively). Statistical analysis of the results and calculation of a correlation between PV and AV showed that only in the control sample, without antioxidants, was there an inversely proportional linear correlation coefficient (r = − 0.69). This meant that the formed peroxides were transformed to secondary oxidation products. In the other samples such a dependence was not observed, which meant that an addition of antioxidants from Ginkgo could have inhibited this transformation process. Another index of secondary products formed by oxidation of unsaturated fatty acids in processed pork meatballs stored for 21 days at 4 °C was assessed as the thiobarbituric acid reactive substance index (TBARS). The result, presented in Table 3, showed the presence of these products in all samples on the first day. Their levels ranged from 0.70 mg/kg in sample YW to 1.86 in the sample with BHT. After one week of storage TBARS increased in all samples, except those with GA and BHT. After 14 days of storage TBARS increased in all samples, but extracts YE and YA were the best antioxidants. In their case, TBARS indexes were 1.10 mg/kg and 1.34 mg/kg and these extracts proved to be better antioxidants than BHT (TBARS = 1.76 mg/kg). Generally, TBARS increased in all samples with the storage. After 21 days of storage TBARS was 1.72 mg/kg in sample YW, 1.20 mg/kg in sample YE, 1.80 mg/kg in sample YA and 1.85 mg/kg in sample GW showing a marked inhibition of the formation of secondary oxidation products in comparison to the control (TBARS = 2.59 mg/kg). Changes in cholesterol levels and the loss of cholesterol in each sample are presented in Fig. 1. Cholesterol levels decreased during storage of the meatballs. Its initial amount ranged from 360.7 mg/100 g to 365.0 mg/100 g of lipid. A decrease in cholesterol levels was observed after 14 and 21 days of storage. Smaller changes were observed in those samples with the addition of Ginkgo ethanol extracts, but the smallest changes were noted in

Fig. 1. The loss of cholesterol content in pork meat balls with Ginkgo extracts during refrigerated storage.

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samples with BHT, where cholesterol levels over the entire storage period decreased by only 2.72%. Among the extracts, GE, GA and YE exhibited the best protection potential towards cholesterol, since its levels after 21 days of storage were 5.2%, 6.5% and 7.4% lower respectively, whereas in the control it was approx. 11% lower. The level of cholesterol oxidized products (COPs) in meatball lipids (Table 4) increased with storage time; however, the addition of antioxidants slowed down the process. In the samples almost all the analyzed COPs were detected after 1 day of storage. After 7 days of storage the total amount of cholesterol oxidation products ranged from 0.88 to 0.98 mg/100 g lipid and accounted for 0.24–0.27% of the initial cholesterol content. Dominant oxysterols were: 7β-OH-C, 7-keto-C and 7αOH-C. After 21 days of storage the dominance hierarchy for oxysterols was: 7β-OH-C, then 7-keto-C, and finally 7α-OH-C. Triol-C was not found in any of the samples, including the control. The level of total COPs increased in all samples, but the smallest amounts were found in GE and GA meatballs (respectively 0.92 and 1.28 mg/100 g lipid) and the largest in the control (3.80 mg/100 g lipid). The level of COPs in the sample with BHT amounted to about 1.97 mg/100 g lipid. The percentage of inhibition of the formation of COPs after 7, 14 and 21 days of storage is shown in Fig. 2. The acetone (GA) and ethanol (GE) extracts of green Ginkgo leaves showed the best inhibition against the formation of cholesterol oxidized products (oxysterols). Pearson's correlation coefficients of cholesterol, lipid oxidation indexes and COPs are presented in Table 5. Changes in cholesterol levels were inversely correlated with such indexes as PV, LA and COPs, but formation of COPs was positively correlated with PV and anisidine values. 4. Discussion The lipids in pork meatballs was assessed by the determination of primary (PV) and secondary products formed by the oxidation of unsaturated fatty acids stored over 21 days at 4 °C. Secondary products were investigated, such as the anisidine value (AV) and the thiobarbituric acid reactive substance index (TBARS). The results showed the highest inhibition towards peroxide values in samples with the addition of 500 ppm YW (inhibition 77%), YA (58%) and GW (48%) extracts. However, in the case of the anisidine value the best extracts were GW (inhibition 70%), YW (44%) and YE (45%). The highest inhibition of another index of secondary oxidation products – TBARS – was in samples YE (54%) and YW (44%). Therefore, the highest inhibition of lipid oxidation was in samples with GW, YW and YE extracts. The worst results were in samples with GE extract. Sebranek, Sewalt, Robbins, and Houser (2005) found that a 0.25% addition of rosemary extracts had a significantly stronger effect on the inhibition of malonic aldehyde formation in pork sausage during refrigerated storage, than 0.02% BHT. Racanicci, Danielsen, and Skibsted (2008) investigated the effect of finely ground leaves of yerba matè and rosemary on the stabilization of lipids in cooked meatballs made from chicken breast meat. It was shown that yerba matè leaves added at 0.05 and 0.1% inhibited, to a higher degree, the formation of primary oxidation products in comparison with identical amounts of comminuted rosemary. There are few data concerning the antioxidant action of Ginkgo extracts on the lipid fraction in foodstuffs, particularly meat products. Kobus-Cisowska, Flaczyk, and Jeszka (2010) and Kobus-Cisowska et al. (2010) showed a positive effect of an ethanol extract of green and yellow Ginkgo leaves in meat dumplings on the stability of lipids during refrigerated and frozen storage. Ginkgo leaf extracts (500 ppm), as well as BHT (200 ppm), prolonged the storage time of dumplings without any deterioration in lipid and sensory attributes. In the present studies the tested extracts inhibited the formation of both primary and secondary oxidation products, to varying degrees. The results are probably influenced by the selective actions of extract components throughout the entire oxidation process. The action of active extract substances in processed meat products varies and their

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Table 4 Changes in cholesterol oxidation products (COP) content in pork meat balls with Ginkgo extracts during refrigerated storage. Days

7α-OH-C

7β-OH-C

α-Epoxy-C

β-Epoxy-C

20-OH-C

Triol-C

25-OH-C

7-Keto-C

Total

0.06a ± 0.01 0.05a ± 0.00 0.04a ± 0.00 0.05b ± 0.00 0.04a ± 0.00 0.07a ± 0.01 0.05a ± 0.00 0.06a ± 0.01 0.07a ± 0.01 0.05a ± 0.00 0.09b ± 0.01 0.02a ± 0.00 0.04a ± 0.00 0.08a ± 0.01 0.05a ± 0.00 0.06a ± 0.00 0.11b ± 0.01 0.05a ± 0.00 0.13c ± 0.01 0.03a ± 0.00 0.07b ± 0.01 0.11b ± 0.01 0.10b ± 0.01 0.07a ± 0.01 0.20c ± 0.02 0.17b ± 0.01 0.05a ± 0.00 0.22c ± 0.02 0.06b ± 0.01 0.11b ± 0.01 0.18c ± 0.02 0.11b ± 0.01

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

0.06a ± 0.01 0.06a ± 0.01 0.06a ± 0.00 0.07a ± 0.01 0.08a ± 0.01 0.05a ± 0.00 0.07a ± 0.01 0.05a ± 0.00 0.07a ± 0.01 0.06a ± 0.00 0.06a ± 0.00 0.09b ± 0.01 0.08a ± 0.01 0.06a ± 0.01 0.08a ± 0.01 0.07a ± 0.01 0.08a ± 0.00 0.06a ± 0.01 0.06a ± 0.01 0.13c ± 0.01 0.12b ± 0.01 0.06a ± 0.01 0.12b ± 0.01 0.12b ± 0.01 0.12b ± 0.01 0.09b ± 0.02 0.06a ± 0.01 0.10b ± 0.01 0.20c ± 0.02 0.17b ± 0.02 0.18c ± 0.03 0.18c ± 0.02

0.21a ± 0.04 0.20a ± 0.03 0.21a ± 0.05 0.19a ± 0.04 0.17a ± 0.01 0.17a ± 0.05 0.20a ± 0.05 0.19a ± 0.01 0.29b ± 0.03 0.20a ± 0.02 0.27b ± 0.01 0.70b ± 0.02 0.17a ± 0.01 0.27b ± 0.02 0.21a ± 0.02 0.36c ± 0.02 0.56c ± 0.03 0.21a ± 0.02 0.54c ± 0.02 0.75c ± 0.07 0.21b ± 0.02 0.46c ± 0.01 0.66b ± 0.02 0.24b ± 0.02 1.04d ± 0.09 0.78b ± 0.03 0.22a ± 0.02 1.01d ± 0.05 0.85c ± 0.07 0.29b ± 0.03 1.22c ± 0.11 0.33c ± 0.03

0.98a 0.88a 0.97a 0.88a 0.91a 0.95a 0.98a 0.96a 1.06b 0.89a 1.11b 0.91b 1.13b 1.15b 1.58b 1.21b 1.74c 0.90b 1.79c 1.52c 1.47c 1.21c 2.05c 1.68c 3.23d 1.28c 0.92a 3.27d 2.78d 1.94d 3.80d 1.97d

mg/100 g lipid 1

7

14

21

GW GA GE YW YA YE Control BHT GW GA GE YW YA YE Control BHT GW GA GE YW YA YE Control BHT GW GA GE YW YA YE Control BHT

0.15a ± 0.00 0.16a ± 0.01 0.17a ± 0.01 0.15a ± 0.01 0.17a ± 0.01 0.17a ± 0.01 0.16a ± 0.01 0.17a ± 0.01 0.19b ± 0.02 0.16a ± 0.01 0.16a ± 0.01 0.17a ± 0.01 0.22b ± 0.02 0.18a ± 0.02 0.24b ± 0.02 0.19a ± 0.01 0.28c ± 0.02 0.16a ± 0.01 0.29b ± 0.02 0.29b ± 0.02 0.23b ± 0.01 0.27b ± 0.01 0.31c ± 0.02 0.28b ± 0.01 0.48d ± 0.02 0.41b ± 0.04 0.17a ± 0.01 0.48c ± 0.02 0.49c ± 0.04 0.24b ± 0.02 0.52d ± 0.05 0.24b ± 0.02

0.33a ± 0.01 0.31a ± 0.01 0.35a ± 0.01 0.33a ± 0.01 0.34ba ± 0.01 0.35a ± 0.02 0.38a ± 0.01 0.35a ± 0.01 0.36b ± 0.02 0.32a ± 0.01 0.38a ± 0.03 0.40b ± 0.03 0.40b ± 0.03 0.37a ± 0.02 0.61b ± 0.05 0.40b ± 0.03 0.55c ± 0.02 0.32a ± 0.03 0.57b ± 0.05 0.61c ± 0.03 0.50c ± 0.04 0.50b ± 0.04 0.63b ± 0.05 0.50c ± 0.04 1.07d ± 0.05 0.86b ± 0.03 0.33a ± 0.02 1.09d ± 0.10 1.19d ± 0.10 0.61c ± 0.05 1.22c ± 0.11 0.62d ± 0.05

0.08a ± 0.01 0.07a ± 0.00 0.10b ± 0.01 0.08a ± 0.01 0.08a ± 0.01 0.10a ± 0.01 0.09a ± 0.01 0.09a ± 0.01 0.08a ± 0.01 0.07a ± 0.01 0.10a ± 0.01 0.07a ± 0.01 0.13b ± 0.01 0.10a ± 0.01 0.16b ± 0.01 0.09a ± 0.01 0.09a ± 0.01 0.07a ± 0.01 0.11a ± 0.01 0.12b ± 0.01 0.18c ± 0.02 0.10a ± 0.01 0.16b ± 0.01 0.25b ± 0.02 0.19b ± 0.02 0.17b ± 0.02 0.07a ± 0.01 0.21c ± 0.02 0.25d ± 0.02 0.24b ± 0.02 0.34c ± 0.03 0.24b ± 0.02

0.06a ± 0.00 0.06a ± 0.00 0.07a ± 0.01 0.05a ± 0.00 0.07a ± 0.01 0.07a ± 0.01 0.05a ± 0.00 0.06a ± 0.00 0.06a ± 0.00 0.06a ± 0.01 0.08a ± 0.01 0.09b ± 0.01 0.11b ± 0.01 0.08a ± 0.01 0.11b ± 0.01 0.08a ± 0.01 0.11b ± 0.01 0.06a ± 0.01 0.13b ± 0.01 0.14c ± 0.01 0.17c ± 0.02 0.11b ± 0.01 0.13b ± 0.01 0.18b ± 0.02 0.16c ± 0.01 0.15b ± 0.01 0.06a ± 0.01 0.18d ± 0.02 0.20d ± 0.02 0.27c ± 0.02 0.19c ± 0.02 0.28c ± 0.02

Abbreviations as in Table 3. nd — not detectable. Results are the mean values of three determinations ± standard deviation. Values sharing the same letter in a column are not significantly different (α = 0.05).

rate and direction depend on the technological parameters of the production process, the composition of the fatty acids and the degree of preliminary lipid oxidation. Thus, in relation to the problem of lipid fatty acid oxidation, it can be stated that addition of Ginkgo extracts, to varying degrees, increased the stability of lipids during storage of pork meatballs subjected to heat processing. The application of the ethanol and acetone extracts of yellow leaves and water extract of green Ginkgo

leaves had the greatest effect on the inhibition of oxidative changes in meatballs lipids. Different results were obtained regarding Ginkgo extract activity against cholesterol. Among the extracts, GE inhibited, to the highest degree, the formation of 7α-OH-C, α-epoxy-OH-C and 7-keto-C, as well as 20-OH-C and 25-OH-C. A slightly lower inhibitory effect, in relation to these oxysterols, was found in samples with BHT. Over 14 and 21 days of storage, it was also observed that in meatballs with YA and YE extracts the formation of toxic 25-OH-C occurred more rapidly. Similar pro-oxidant properties in relation to 25-OH-C were observed in samples with BHT, which also promoted the formation of the β-epoxy-C epimer. Possibly, the lower content of the 7β-OH-C derivative in samples with BHT resulted from its rapid transition into the β-epoxy-C form. Moreover, GA extracts also effectively inhibited the process of cholesterol oxidation, particularly in terms of the formation of 20-OH-C and 25-OH-C. Thus, the tested extracts exhibited, to varying degrees, some protective action towards cholesterol and the formation of COPs. To date, there has been no clarification of what changes occur in cholesterol during

Table 5 Pearson's correlation coefficients⁎ of cholesterol, COPs and lipid oxidation indexes in pork meat balls during refrigerated storage.

Fig. 2. The inhibition of the formation of cholesterol oxidation products (COPs) by Ginkgo extracts in pork meat balls during refrigerated storage.

Cholesterol COPs ⁎ p ≤ 0.05.

LOO

LA

TBARS

COPs

−0.43 0.60⁎

−0.72⁎ 0.84⁎

−0.62⁎ 0.44

−0.82⁎ –

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storage in chilled and frozen meat products. A given proportion is oxidized to different derivatives of COPs. There are no data on the use of Ginkgo extracts as potential natural antioxidants in relation to cholesterol. However, work has been published on the effect of other antioxidants on the degradation of cholesterol and formation of COPs in processed meat products. Baggio and Bragagnolo (2006a,b) investigated the level of cholesterol in salami type sausages (stored at room temperature with no access to light) and bologna (stored refrigerated), which contained a commercial mixture of antioxidants (BHT and natural antioxidants in the form of spices: pepper, garlic, rosemary, etc.). No statistically significant differences were found in the content of cholesterol during 3-month storage of the products. Such a strong protective effect was probably caused by the synergistic effect of the added antioxidants. In relation to cholesterol, in heat processed pork meatballs addition of ethanol and acetone extracts of Ginkgo delayed the formation of undesirable COPs. It was also shown that the extracts from green leaves slowed these changes to a greater degree than yellow leaf extracts. This primarily involved such COPs as α-epoxy-C, β-epoxy-C and 25OH-C. Inhibition cholesterol oxidation products by the extracts could be explained by competition between lipid radicals and extract polyphenols for the position with a free electron in the cholesterol molecule. Such a competition could occur between hydroxyl groups of the above mentioned COPs and functional groups of the reactive polyphenols in the extracts. Oxidative changes in lipid and cholesterol occur more rapidly in products exposed to heat than in raw products. This might explain changes in cholesterol levels and the presence of most oxysterols in the first measurement period. However, 3 weeks of refrigerated storage in vacuum packaging did not result in the formation of the most cyto- and angiotoxic product of cholesterol oxidation, i.e. triol-C. Probably storage of samples under refrigeration in vacuum bags prevented the occurrence of such advanced changes. In the first stage of the lipid oxidation reaction, hydroperoxides are formed. They are unstable compounds and easily undergo further multi-directional changes, leading to the formation of secondary oxidation products resulting in an increase in LA and TBARS values. On the other hand, lipid hydroperoxides could undergo decomposition to free radicals in the presence of transition metal ions, such as Cu+ 1 and Fe+2. Traces of these metals form alcoxyl and hydroxyl radicals which probably additionally contributed to cholesterol oxidation and formation of selected COPs. Correlation analysis between oxidation stability indexes of lipids confirmed the above hypotheses. Negative correlations between the level of cholesterol and all analyzed lipid indexes were − 0.43 on the LOO index, − 0.72 on the LA index and − 0.62 on the TBARS index. Thus, the higher the value of the analyzed index was, the less unchanged cholesterol it contained. Analogously the positive correlations between the level of oxysterols and the lipid oxidation indexes were: 0.60 on the LOO index, 0.84 on the LA index and 0.65 on the TBARS index. Thus, each of the lipid oxidation indexes could be a marker of cholesterol oxidation, leading to the formation of COPs. Flaczyk et al. (2006) and Rudzińska, Flaczyk, Amarowicz, Wąsowicz, and Korczak (2007) also found correlations between lipid oxidation indexes and the formation of COPs (Flaczyk et al., 2006; Rudzińska et al., 2007). They tested meatballs with a 2% addition of acid and enzymatic hydrolysates of pork crackling, during refrigerated and frozen storage. They observed a positive correlation between COP content and LN, LA and TBARS values. Also, Zaborowska et al. (2005) found a high correlation between total COPs, and LN and LA values in “bologna” and “beer” type sausages. It can be concluded that the added extracts showed inhibitory action against the formation of most COPs in the lipids of pork meatballs subjected to heat treatment and refrigerated storage. All the

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