PaPer Ital. J. Food Sci., vol. 28 - 2016 139 - Keywords: garlic extracts, color, lipid oxidation, oxidative breakdown products, raw ground beef - EffEcts of garlic Extract on color, lipid oxidation and oxidativE brEakdown products in raw ground bEEf during rEfrigEratEd storagE xinzhuang zhang, Qingxiang MEng, liwEn hE, liping zhao and liping rEn* State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193,China *Corresponding author: Tel. +86 10 62733799, Fax +86 10 62829099, email: renlp@cau.edu.cn AbstrAct the study aims to investigate the effects of garlic extracts on color, lipid oxidation, and oxida- tive breakdown products in raw ground beef during refrigerated storage. the two treatments were: control group (c, with no addition) and experiment group (D, 50 mg garlic extracts added to 100 g beef). Adding garlic extracts significant increased a* value (P A ≤ 0.05), and significant decreased tbArs and PV values (P A ≤ 0.05). the pH and –sH value of D group had a decreasing tendency (P A =0.0522) and an increasing tendency (P A =0.0636) respectively compared to c group. Garlic ex- tracts protected phospholipids, fatty acids and polypeptides from oxidation. the results indicate that garlic extracts have the antioxidant activity, helping maintain the meat color, inhibiting lipid oxidation and protein degradation of raw ground beef during refrigerated storage. mailto:renlp%40cau.edu.cn?subject= 140 Ital. J. Food Sci., vol. 28 - 2016 IntroDuctIon Lipid oxidation is one of the primary mecha- nisms of quality deterioration in meat and meat products. the adverse changes in quality are manifested in flavor, color, texture and nutri- tive value, and the possible production of toxic compounds (cLAuDIA et al., 2014). beef and its products are rich in protein and lipids, which make them most suitable for consumer. Howev- er, beef contains high level of unsaturated fatty acids which are prone to oxidation (tIcHIVAnGA- nA and MorrIssey, 1985). to prevent or reduce lipid oxidation, butylated hydroxytoluene (bHt), butylated hydroxyanisole (bHA), tertiary butyl- hydroquinone (tbHQ), trihydroxybutyrophenone (tHbP), propyl gallate (PG), nordihydroguaiaret- ic acid (nDGA) and ethoxyquin have been ap- plied in meat products (trInDADeA et al. 2014). However, their application had been restricted because of their potential health risks and tox- icity (sun and FukuHArA, 1997). natural anti- oxidants can be used as alternatives to the syn- thetic antioxidants because of their safety and equivalent or greater effect on inhibition of lipid oxidation (AnDrew et al., 2014). Garlic (Allium sativum) has been a favorite additive to enhance the flavor of food as well as herbal medicine for many years in various cultures. It is well known that garlic has anti- microbial, antiprotozoal, antimutagenic, anti- platelet and antihyperlipidemic properties. Gar- lic holds a unique position for therapeutic po- tential due to the antioxidant activity by scav- enging reactive oxygen species (ros), enhanc- ing the cellular antioxidant enzymes, and in- creasing glutathione in the cells. numerous studies have demonstrated that garlic exhibits cardioprotective (rAHMAn and Lowe, 2006), liv- er-protective (wAnG et al., 1998), beneficial ef- fects in diseases such as ischemic-reperfusion arrhythmias and infarction (rIetz et al., 1993), ischemic heart disease (ArorA et al., 1981), hy- pertension (FousHee et al., 1982), hyperlipi- demia (ernst et al., 1985), as well as prevent the processes of cancer (tAnAkA et al., 2006) and aging (LI et al., 2012). A number of researchers reported that garlic and different garlic extracts have antioxidant ac- tivity contributed by organosulfur compounds, flavonoids and phenolic compounds (borek, 2001; otunoLA and AFoLAyAn, 2013). rAHMAn et al. (2012) researched antioxidant properties of raw garlic extract using the 2, 2-diphenyl- 1-picrylhydrazyl (DPPH) scavenging methods. cAo et al. (2013) had studied the effects of gar- lic on quality and shelf life of stewed-pork dur- ing refrigerated storage. However, less attention has been assigned to garlic extracts used as an antioxidant in beef, especially in identifying the oxidative breakdown products so far. the objec- tive of the present study was to determine the ef- fect of garlic extracts on pH, color stability, lipid oxidation, and oxidative breakdown products of raw ground beef during refrigerated storage in order to provide a scientific basis for using gar- lic extracts as natural antioxidants to maintain the meat quality, extend shelf-life and prevent economic loss. MAterIALs AnD MetHoDs Materials Garlic extracts (10:1 of garlic: garlic extracts, three percent allicin content) was purchased from yuanshen bio-tech Ltd. (Xian, china). Glutathione were obtained from sigma Aldrich Inc. (st. Louis, Mo, usA). ethanol, chloroform, methanol, ammonium thiocyanate, iron (II) chloride, trichloroacetic acid (tcA), thiobarbi- turic acid (tbA), 5, 5'-dithiobis (2-nitrobenzo- ic acid) (Dtnb), cumene hydroperoxide, tetra- ethoxypropane and other reagent were ‘Anal- ar’ grade from china Medicine (Group) beijing chemical reagent corporation (beijing, china). water for ultra-performance liquid chromatog- raphy quadrupole time of flight (uPLc-QtoF) was purified with a Milli-Q Gradient A10 sys- tem (Millipore, beijing, china). Formic acid and acetonitrile were HPLc-grade (Fisher scientific, new Jersey, usA). three simmental crossed cattle (620±15kg, 18 months) were slaughtered at a local commer- cial abattoir (Jinweifuren co. Ltd., beijing, chi- na). transport, slaughtering, or invasive pro- cedures on live animals involving in this study were handled in strict accordance to the guide- lines approved by the Animal welfare committee of china Agricultural university (Permit num- ber: Dk1008). After ageing at 4°c for 72 h, the longissimus dorsi (LD) were excised from 12th and 13th rib of left half side carcass. the mus- cles were minced twice through a 5 mm plate of meat mincer (model Jys-A800, Joyoung, china) after removing the connective tissue and visible fat. the contents of moisture, protein and fat of the ground beef were 73.14%, 22.26%, and 2.83% respectively. Treatments the meat sample of each cattle was formed into two patties (100 g portions) using a meat former and assigned to the following two treat- ments:: control group (c, 100 g ground beef with no addition) and experiment group (D, 50 mg garlic extracts added to 100 g ground beef). to eliminate the influence of microorganism and en- sure thorough mixed, garlic extracts dissolved in 10 mL of a distilled water and ethanol mixture (1:1, v/v) and then mixed with the muscles of the experiment group. the same volume of dis- tilled water and ethanol mixture (with no add- ed ingredients) was added to the control group. Ital. J. Food Sci., vol. 28 - 2016 141 Meat samples were put into a constant tem- perature incubator (MJX-320, Jiangnan, ning- bo, china) at 4 °c for 13 days. Meat samples (three replicates) were collected in 1.5 mL cen- trifuge tube at storage times of 1, 3, 5, 7, 10, 13 days and were frozen rapidly in liquid nitrogen for subsequent analysis. Analysis of pH and color A pH meter (pH spear, eutech Instruments, usA) was used to measure the pH of the ground beef. the color of the ground beef was deter- mined by portable colorimeter (cr400/410, Mi- nolta, Japan). the specifications of the color- imeter are light source: pulsed xenon lamp; Il- luminant: c, D65; illumination area: Φ8/Φ11; Inter instrument agreement: Δe*ab within 0.6; repeatability: within Δe*ab0.07 standard devi- ation. the color results were calculated based on L*, a*, b* (lightness, redness, and yellowness respectively) in the cIeLAb space. A white plate (cIe L*= 97.83, a*=-0.43, b*=1.98) was used for calibration. Analysis of lipid oxidation Lipid oxidation was evaluated by the perox- ide value (PV) and thiobarbituric acid reactive substances (tbArs) according to the method of rIcHArDs et al. (2003) (rIcHArDs and Dett - MAnn, 2003) and free thiol groups (-sH) was analyzed following the method proposed by eGeLAnDsDAL et al. (2011) with minor modi- fications. Determination of PV: Approximately 0.3 g of sample was homogenized for 30 s with 5 mL of cold chloroform/methanol (1:1) in a 50 mL glass tube using a refiner (FJ-200, Jintan, chi- na). subsequently, the glass tube was rinsed for 30 s again with 5 mL of cold chloroform/ methanol (1:1). the homogenate and wash so- lution were then combined in a 25 mL glass test tube. Adding 3.08 mL of 0.5% nacl then mixed for 10 s with a vortex (G560e, scientif- ic Industries Inc., new york, usA). next, the mixture was centrifuged at 1821g (DL-6000b, shanghai Anting, china) for 6 min at 4°c. two milliliters of the lower chloroform layer was re- moved and transferred to a tube by a glass sy- ringe before 1.3 mL chloroform/methanol (1:1) was added to the 2 mL sample. then 25 μL of 3.94 mol/L ammonium thiocyanate and 25 μL of 18 mmol/L iron (II) chloride were added to the tube, mixing thoroughly after each addition. Finally, the sample was incubated at room tem- perature for 20 min and the absorbance at 500 nm was measured using a spectrophotometer (uV1102, shanghai tianmei, china). A stand- ard curve was constructed using cumene hy- droperoxide and the PV in the sample was ex- pressed as µmol/kg. Determination of tbArs: mixing 50% trichlo- roacetic acid (tcA) with 1.3 % thiobarbituric acid (tbA) on the day of use and then heating to 65°c. Approximately 0.12 g of the muscle sample was added to 1.2 mL the tcA - tbA mixture and mixed via a vortex (G560e, scientific Industries Inc., new york, usA) and then heated at 45°c for 60 min using a vapor-bathing constant tem- perature vibrator (HQ45, chinese Academy of sciences, wuhan, china) before the sample was centrifuged at 12,000 g for 5 min (Mikro 200r, Hettich, Germany). the absorbance of the super- natants at 532 nm was determined. A standard curve was prepared using tetraethoxypropane, and the concentration of tbArs in the samples was expressed as µmol/kg. Determination of -sH: approximately 0.1 g of the muscle sample was weighed into a 10 mL centrifuge tube then 1700 μL phosphate-buff- ered saline (pH=8.2) and 100 μL Dtnb were added. the mixture was heated at 45 °c away from light for 60 min using a vapor-bathing constant temperature vibrator (MJX-320, Jin- gbo Jiangnan, china). eight milliliters of meth- anol was added to the tube which was vor - texed for 30 s before centrifuging at 1821g for 20 min at 4°c. subsequently, 1 mL supernate was transferred to a new centrifuge tube, dilut- ed with 3 mL methanol and then the absorb- ance was measured at 412 nm. Glutathione was used for generating a standard curve and concentration of -sH in samples was expressed as mmol/kg. Analysis of oxidative breakdown products sample preparation: 50 mg muscle sample from each treatment at 13 days was weighed into a 2 mL eppendorf tube. the first step: 1.5 mL cold water/methanol (1:1) and 0.5 g 1 mm zirmil ceramic beads were added to the tube and then mixed thoroughly using a tissue ho- mogenizer (Precellys 24, bertin, France) for two cycles at 6500 Hz, 40 s for each cycle. the mix- ture was centrifuged at 12,000 g (Mikro 200r) for 10 min at 4°c. supernate (400 μL) was col- lected in a new eppendorf tube and stored at 4°c for further use. the second step: the residual sediment from step one was extracted by 1.5 mL cold chloroform/methanol (3:1) again with the same procedure of homogenization and centrif- ugation and the same volume of supernate col- lected in the eppendorf tube. the mixed sam- ples from two steps, were then concentrated in a centrifugal concentration meter (zLs-1, Her- exi, Hunan, china). water/methanol (9:1; 120 μL) was used to dissolve the samples with vor- tex oscillation for 40 s. supernate (100 μL) was collected in a lining tube for subsequent uPLc- QtoF analysis. uPLc analysis: the substances in the 6 μL ex- tracted sample were separated using a uPLc sys- tem (Acquity uPLc/XeVo G2 Q toF, waters) af- ter being loaded onto a high-resolution and high- 142 Ital. J. Food Sci., vol. 28 - 2016 performance uPLc beH c18 column (1.7 μm, 2.1 mm × 50 mm, waters). the column temper- ature was 50 °c and the flow rate was 0.3 mL/ min with solvent A (water + 0.1 % formic acid) and solvent b (acetonitrile + 0.1% formic acid). the elution program consist of 98:2 (A: b) for 1 min, increasing gradually to 100 % b by 16 min, then 100 % b held for 2 min, a ramp to 98:2 (A: b) within 10 s, and returned to 98:2 (A: b) for 2 min (re-equilibration of the column) before the loading of the next sample. Q-toF Ms conditions: the separated com- ponents from the uPLc were subsequently an- alyzed by quadrupole time of flight mass spec- trometry (Q-toF Ms) equipment (Acquity uPLc/ XeVo G2 Q toF, waters) operated using a neg- ative electrode for electrospray ionization. the settings were as following: data acquired in a full scan mode (mass: charge ratio (m/z) 50-1200) at a rate of 2 spectra/s; capillary and sampling cone voltages at 2500 and 35 V, respectively; de- solvation temperature at 350°c; desolvation gas flow at 720 L/h; cone gas flow at 50 L/h; source temperature at 105°c. All gas paths used nitro- gen and data were collected using Masslynx 4.1 data management software (waters, usA). the final data was expressed by the ID number, rel- ative m/z values, retention times and ion in- tensity. stAtIstIcAL AnALysIs this experiment was assigned two factors with two treatments and seven storage time levels.s. the mixed model procedure of sAs 9.0 (sAs Institute Inc.) was used to analyze the effects of garlic extracts and storage time. the following statistical model was used for analysis: y ik = μ + α i + β k + (αβ) ik + e ik where y ik is an observed value for tbArs, PV, -sH, pH, L*, a*, and b*, taken from sample re- ceiving treatment i at time k;μis the overall mean; α i is the fixed effect of treatment i; β k is the fixed effect of time k; (α β) ik is the interaction between treatment and storage time; and e ik is the resid- ual value. the variation tendency of pH and color, lipid oxidation with different storage times was an- alyzed by the contrast model of sAs 9.0. the means that were significantly different were analyzed using the t-test and different storage times were analyzed using Duncan’s multiple comparison tests. A level of P ≤ 0.05 was con- sidered significant and 0.05

1.5 were se- lected as different oxidative breakdown prod- ucts whose formulae were searched in metlin database. table 1 - the effects of garlic extracts and storage time on physical meat quality, lipid oxidation in raw ground beef during refrigerated storage. Items TBARS PV -SH pH L* a* b* Treatments C 22.41a 46.56a 6.62 5.86 51.22 13.50b 14.07b D 19.59b 30.22b 7.10 5.85 51.35 14.49a 14.59a SEM 0.6121 9.7158 0.3015 0.0027 0.2020 0.2086 0.1422 Times 1 10.25c 23.78b 7.16ab 5.91a 51.05b 18.05a 14.71a 3 13.50b 28.94b 8.14a 5.84b 50.13c 15.68b 13.52b 5 26.92a 42.11ab 7.23ab 5.84b 51.96a 15.44b 14.76a 7 24.20a 69.32a 7.12ab 5.83b 51.99a 13.98c 15.02a 10 26.07a 44.82ab 6.03bc 5.82c 51.01b 10.18d 13.55b 13 25.08a 21.39b 5.45c 5.91a 51.57ab 10.65d 14.43a SEM 0.6121 9.7158 0.3015 0.0027 0.2020 0.2086 0.1422 P A <0.001 0.0500 0.0636 0.0522 0.4248 <0.001 0.0001 P B L <0.001 0.5139 <.0001 0.1356 0.0123 <0.001 0.5835 Q <0.001 0.0085 0.0238 <.0001 0.0852 0.9871 0.8118 P AB 0.0383 0.9684 0.1200 0.0035 0.5571 0.0034 0.0152 Means in the same column with different superscripts are significantly different (P <0.05).C-control group, D-garlic extracts group. P A is the P value of treat- ments; P B is the P value of storage times; L stands for the linear effects of storage times; Q stands for the quadratic effects of storage times. P AB is the P value interaction between treatments and storage times. Ital. J. Food Sci., vol. 28 - 2016 143 resuLts The effects of garlic extracts and storage time on physical meat quality, lipid oxidation in raw ground beef during refrigerated storage According to table 1, L* and a* value pre- sented an increasing and decreasing line- ar change respectively (P b L<0.05), tbArs, PV, -sH, and pH had a quadratic change over stor- age time (P b Q<0.05). More details, tbArs val- ue significant increased in the first five storage days (P b <0.05), and then no significant chang- es in the residual storage time. PV and -sH val- ue were firstly increased and then decreased with the highest value at seventh day and third day respectively of storage time. on the contra- ry, pH was firstly decreased and then increased with the lowest value at tenth day of storage time, ranging from 5.82 to 5.91. However, stor- age time had neither linear nor quadratic effect on b* value in raw ground beef during refriger- ated storage. As shown in table 1, Adding garlic extracts could significant increased (P A ≤ 0.05) a* and b* values. the pH of D group had a decreasing ten- dency compared to c group (P A =0.0522). there was no significantly different on L* value be- tween treatments (P A =0.4248). tbArs and PV values of D group were significant lower (P A ≤ 0.05), and –sH tended to increase (P A =0.0636) than c group in raw ground beef during refrig- erated storage. because table 1 could not show the effects of garlic extracts on every storage time, the sig- nificant indicators a*, PV, and tbArs values (P A ≤ 0.05) influenced by garlic extracts were further analyzed on every storage time. Fig. 1 showed that a* value was significant increased following the addition of garlic extracts on days 3, 7 and 13 during refrigerated storage rela- tive to the control(P < 0.05). Adding garlic ex- tracts had a tendency to reduce the PV val- ues on days 5 and 13 (0.05