Comparison of Fatty Acids with Amino Acids Pro …...2.4 Quantification and quantitation of fatty acid in venison 2.4.1 Lipid extraction Muscle lipids were extracted following the

Comparison of Fatty Acids with Amino Acids Profiles of Wapiti Deer (Cervus elaphus) Muscle

Zhaohua Hou1*, Jing Luo2, Hui Zhao2, Bo Cui1, Bin Yu1

1. College of Food Science and Engineering, Qilu University of Technology, Ji’nan City, Shandong Province, P.R. China, 250353

2. Institute of Special Wild Economic Animals and Plants, Chinese Academy of Agricultural Sciences, Changchun City, Jilin Province, P.R. China, 130112

E-mail: [email protected]

Abstract: The fatty acids and amino acids compositions of different parts of wapiti deer (Cervus elaphus) muscle (loin, chops, shoulder and hindquarters) were analyzed in this study. In total, fifteen types of fatty acid were identified and quantified in fresh and freeze-dried samples, with monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) constituting approximately 47.82-59.04% (fresh venison) and 57.99-65.09% (freeze-dried venison) of all fatty acids, respectively. The highest levels of fatty acids were found in chops, followed by shoulder, hindquarters and loin. PUFAs/SFAs (saturated fatty acids) ratios in the fresh sample (0.30-0.41) were lower than those of freeze-dried sample (0.64-0.96). In addition, deer muscle were rich in total amino acids (TAAs) and essential amino acids (EAAs). The amino acid score (AAS), chemical score (CS) and the index of essential amino acids (EAAI) were calculated as correlation parameters of the biological value of the proteins. Overall, wapiti deer muscle contained good composition of fatty acids and amino acids which offer health benefits. Keywords: Wapiti deer (Cervus elaphus); deer muscle (venison); Fatty acids; Amino acids; Nutrional value; Gas chromatography-mass spectrometry (GC-MS). 1. Introduction Wapiti deer (Cervus elaphus) is one of the most important traditional and medicinal animal in many countries, which has great medicinal, visual and edible value. Deer is considered as the most recently domesticated species, and today deer farms provide an efficient use of land and also high-quality products [1]. Meats have an important role in human nutrition because of their nutritive value. The main health benefits associated with eating red meat (beef, veal, pork and lamb) relate to its nutritional composition. The value of meat is measured in terms of the major chemical components such as proteins and their composition, fats and their composition, vitamins, carbohydrates, minerals and fatty acids contents. Red meat contains high biological value protein, fatty acids, and important micronutrients, including iron, zinc and vitamin B12, all of which are essential for good health throughout life [2-4]. Deer muscle (venison) is often valued by consumers as an ecological and ethical alternative to the commercially produced beef, pork and chicken. In addition, venison is popular because of its unique flavor and nutritious compounds, such as high levels of fatty acids and amino acids, e.g. rich protein and low fat (21 and 1.2%, respectively) [1,5]. Especially, venison contains 50-80% less fat than traditional red meat [6]. Besides the fat content, the fatty acid composition of meat became a special interest to the consumers. Nowadays, there is increasing awareness to include higher proportions of unsaturated fatty acids in the diet that have favorable effect on human health [7]. Numerous authors have reported that venison has very low muscle fat contents consisting predominantly of structural lipid components (phospholipid and cholesterol) that have high proportions of polyunsaturated fatty acids. Venison has desirable polyunsaturated: saturated and n-6: n-3 fatty acid ratios [8]. Fatty acids not only enhance taste but also benefit human health by providing energy and essential compounds for the body. These include saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs). PUFAs possess positive pharmacological and physiological effects on human health, including reduced risk of coronary artery disease [9]. Cygan-Szczegielniak & Janicki (2011) [10] found that meat from roe deer does show in general a higher percentage proportion of SFAs and MUFAs, but lower of PUFAs than the meat from bucks. Nutritional analysis of fatty acids can be classified as qualitative and quantitative. Qualitative analysis of fatty acid produces data regarding the fatty acid composition in the form of

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percentage of total fatty acids. Meanwhile, quantitative analysis is able to quantify the actual amount (weight) of each fatty acid that is present in the food [11]. Currently, these are limited qualitative fatty acid data on venison in different parts of the wapiti deer; meanwhile, no quantitative data are available especially as a means to utilize source of natural product for nutraceutical purposes. Proteins make up 80-90% of the organic matter in humans and animals are important for the growth and development. Protein quality is determined by the amino acids present [12]. Amino acids are essential nutrients for human growth and for physical functions such as physiology, biochemistry, and immunity. Proteins are constituted of about 20 types of amino acid, including eight essential amino acids (EAAs) [9, 13]. Since EAAs cannot be synthesized by mammals, it is necessary to obtain these constituents in food products. The remainder are synthesized and are said to be non-essential. A high quality protein diet contains all eight of the essential amino acids, in their correct proportion as required by the body [12, 14]. The processing and preparation of food for consumption considerably affects the constituents in the raw material, including amino acids [14] and fatty acids. Freeze-drying (lyophilization) is an effective method to extend the average lifespan of food, which is shown that it prevents the deterioration due to microbial growth or oxidations. Besides, freeze-dried meat can be stored for unlimited periods retaining the majority of their physical, chemical, biological and sensorial properties as in the fresh state. These freeze-dried products do not require cold chain and only have 10-15% of original weight that makes their storage, distribution and commercialization easy [15]. There is little data available on meat composition of the wapiti deer and its nutritional value in the human diet. For the first time, the present study was conducted to quantitatively and qualitatively evaluate the fatty acids and amino acids of fresh and freeze-dried wapiti deer muscle in four body parts (shoulder, hindquarters, loin and chops), which provide the basis for the research and utilization of venison. 2. Materials and methods 2.1 Meat samples The research included six wapiti deer (Cervus elaphus, female) from the Institute of Special Wild Economic Animals and Plants, Chinese Academy of Agricultural Sciences (Jilin, China). Wapiti deer (3-5 years old) muscle was transported from the supplier to the laboratory in less than 24h after slaughtering in a local slaughterhouse from Changchun city. Venison included four parts: shoulder, hindquarters, chops and loin. The venison (approximately 1000 g fresh weight) was transferred to the laboratory where visible fat and connective tissue was trimmed before the muscle was diced into 10cm×10cm×0.5cm portions. The venison was stored frozen at -80°C prior to fatty acids and amino acids analysis. This experiment was performed following the Animal Ethics Committee of the Institute of Special Wild Economic Animals and Plants, Chinese Academy of Agricultural Sciences. 2.2 Materials and standards A 37-component fatty acid methyl ester (FAME) mix was purchased from Nu-Chek Prep (Elysian, MN, USA). Chloroform, methanol, ethanol and n-hexane (chromatographic grade) were purchased from the Beijing reagent Company (Beijing, China). Anhydrous sodium sulphate, hydrochloric acid (HCl) and potassium hydroxide (KOH) (analytical grade) were acquired from the Tianjin Guangfu Chemical Reagent Company (Tianjin, China). Water utilized for preparation of standard and extraction solutions was deionized with a Milli-Q purification system (Millipore, Schwalbach/Ts, Germany).

2.3 Freeze-drying venison Venison was freeze-dried following the method described by [15, 16] with minor modifications. Preliminary experiments were done to define process conditions to be applied for venison lyophilization. Venison was diced into about 10cm×10cm×0.5cm portions, before these samples were placed in plastic bags into one layer and frozen in a chest freezer at -40°C for at least 12h. The samples were taken out from freezer and placed in an automatically controlled freeze-dryer (EYELA FDU-1100, Japan). Freeze-dryer was set at (-40°C) with a vacuum of 15.0±5 Pa and condensing plate temperature at -45°C. After completion of 30h freeze-drying, the deer muscle samples were ground using a grinder. The ground samples were placed in an airtight plastic container and stored at -80°C until used for compositional analysis. The weight yields of freeze-dried samples were calculated using the following expression: Yield (%)=Wf*100/Wo (1) Wf: weight of lyophilizated sample (g); Wo: weight of fresh sample (g).

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2.4 Quantification and quantitation of fatty acid in venison 2.4.1 Lipid extraction Muscle lipids were extracted following the method described by [17-19] with minor modifications. The freeze-dried samples (about 1.0g) and fresh samples (about 2.0g) were minced and homogenized with 30mL of chloroform: methanol solution (2:1, v/v) in a glass bottle with screw-cap. After 3min of homogenization, the content of the bottle was filtered through filter papers to eliminate the solid residue, which consisted mostly of proteins, the filter paper was rinsed twice with 10mL solution. The filtrate was mixed thoroughly with 40mL of a 1 M KCl solution an left overnight at 4°C in order to obtain phase separation. The lower phase containing the lipids was collected and dried in a block heater for 30min at 50°C. The lipids content was determined gravimetrically. The extraction yields of lipids were calculated using the following formula: Yield (%) = Wl* 100/Ws (2) Wl: weight of lipids (mg); Ws: weight of fresh or freeze-dried sample (mg). 2.4.2 Methylation Two lipid extractions were performed for each sample [20-21]. Briefly, lipids were dissolved in 5mL hexane and 5mL of 1 M potassium hydroxide methanol solution and placed inside a 50mL screw capped test tube, then heated in a water-bath with the temperature adjusted to 50°C for 1h. Afterwards, hexane (5mL) and 5% HCl (10mL) were added and the solution was placed in a separation funnel, the top hexane phase, which contained the methylated fatty acids, was separated. The hexane solution obtained was made up to 10mL of the total volume with hexane, followed by dehydration over anhydrous sodium sulphate. 2.4.3 Gas chromatography- mass spectrometry (GC-MS) analysis Fatty acid profiles of deer muscle were determined by the method of Santos-Filho et al. (2005) [18]. Gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS) analysis were performed with a 7000B GC-MS Triple Quad system (Agilent technologies) equipped with a SPTM-2560 fused silica capillary column (100m×0.25mm×0.25μm; supelco, PA). The temperature program of the oven started at 140°C (hold time 5min) and was increased by 4°C/min to 200°C, then increased by 3°C/min to 220°C held for 26 min. Injection of 1.0μL was performed and helium was used as the carrier gas with a constant flow of 1.0 mL/min. Split mode was applied with a split ratio of 60:1. A solvent delay of 3 min was applied. The electron energy was 70eV and the temperature of the ion source was set at 230°C. The injector and detector temperatures were both set at 250°C. In the GC-MS full scan mode, m/z 50-500 was recorded. Fatty acids were identified by comparison of retention times and visual interpretation of the mass spectra to known standards (Fig. 2). Fatty acids are reported as mg/100g venison. Characterization and identification of FAMEs were performed in the scan mode. Quantification was done by selected ion monitoring (SIM) mode of the most intense fragments (saturated: m/z 74, mono-unsaturated: m/z 55, di-unsaturated: m/z 67, poly-unsaturated: m/z 79) (shown in Table 2). According to the equations [22], the atherogenic and thrombogenic indices (AI and TI, respectively) were calculated to measure the risk of venison to the incidence of coronary heart disease.

AI =

36

0:160:1440:12

nnPUFAMUFA (3)

T I = 633365.05.0

0:180:160:14

nnnPUFAnPUFAMUFA (4)

2.5 Amino acid analysis 2.5.1 Preparation of amino acid samples Amino acid analysis was carried out according to a method described by [9,14,23] with minor modifications. The content of amino acids was determined using an Hitachi L-8900 amino acid analyser (Hitachi Ltd., Japan). The analytical procedure applied was in accordance with the recommendations of the producer. About 30mg (dried weight) of each freeze-dried sample was placed into a 35mL hydrolysis tube, to which hydrochloric acid (6 M, 15 mL) was added. The tubes were then filled with the nitrogen gas for 1 minute and closed, followed by hydrolysis in a thermoelectric thermostat drying box (GZX-9140MBE; Shanghai Boxun Experimental Equipment Co., Ltd., China) at 110±1°C for 24h. After hydrolysis, the solution was transferred into a 50mL volumetric flask, and diluted with water to volume of 50mL. The liquid was filtered, and 1 mL of the resulting solution was dried in a vacuum drying oven (DZF-6051; Shanghai Jinghong Experimental Equipment Co., Ltd., China), then rehydrated using 2mL deionized water and dried again 2-3 times. Dried matter was dissolved in 0.02M hydrochloric acid buffer solution and filtered through 0.45μm filtration membrane to remove impurities. Twenty microliters of each sample was injected into the automatic amino acid analyser for analysis. The calculations were carried out according to the external standard. A two-channel visible detector with a 5 µL flow

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volume cuvette was operated at wavelengths of 440 and 570 nm. The amino acid composition in venison was expressed in g/100g dried weight. 2.5.2 Nutritional evaluation The nutritional evaluation was carried out according to the guidelines for the amino acid score and egg protein amino acid score issued by the Food and Agriculture Organization (FAO). The formulas for amino acid score (AAS), chemical score (CS), and essential amino acid index (EAAI) are as follows: AAS (%) = [amino acid content in venison (g/100 g protein)/amino acid content in FAO reference (g/100 g protein)] × 100, (5) CS (%) = [amino acid content in venison (g/100 g protein)/amino acid content in egg protein (g/100 g protein)]×100, (6)

EAAI= he

h

be

b

ae

an

100100100 (7)

Where n is the number of compared essential amino acids. The letters a-h denote the levels of essential amino acids (g/100 g protein) in the experiment, and ae-he denote the levels of amino acids in egg protein (g/100 g protein) [9]. 2.6 Statistical analysis Data were analyzed statistically using SPSS Statistical program (version 13.0 software, SPSS Inc. Chicago, USA). Each experiment was carried out in three replications. All data are expressed as the mean ± standard error. A value of P<0.05 was considered as statistically significant. 3. Results and discussion 3.1. Freeze-drying and lipids extraction results Weight yields of freeze-drying venison are obtained by the formula (1) and the results are shown in Table 1. The freeze-drying weight yields of the different muscle samples (chops, lion, shoulder and hindquarters) were 25.15±2.38%, 24.38±1.82%, 24.19±2.14% and 24.98±2.09%, respectively. The weight yield of freeze-dried chops is relative higher than the others, but there were no significant differences in four parts.

Table 1 Yields of freeze-drying and extraction of lipids in deer muscle

Sample Weight yield (%) of freeze-dried venison

Lipids of fresh venison Lipids of freeze-dried venison Weight (mg/g) Yield (%) Weight (mg/g) Yield (%)

Chops 25.15±2.38 51.65±4.17 2.54±0.25 94.25±8.69 9.13±0.64 Loin 24.38±1.82 35.65±0.35 1.75±0.31 54.72±12.58 5.36±1.25

Shoulder 24.19±2.14 30.10±3.68 1.89±0.32 52.35±5.02 5.56±1.28 Hindquarters 24.98±2.09 37.35±7.85 1.82±0.38 38.95±17.61 3.85±1.72

The contents and weight percentages of lipids in fresh and freeze-dried samples of deer muscle are calculated by the formula (2) and the results are shown in Table 1. In general, the amount of lipids from fresh samples are at the range of 30.10-51.65mg/g (1.75-2.54%), freeze-dried samples are 38.95- 94.25mg/g (3.85-9.13%). Results showed that there are significantly differences in four parts. Quantitatively, the freeze-dried samples generally contained lipids with significantly higher levels than that of the fresh samples. The lipid content of freeze-dried chops is up to 94.25mg/g, this shows that chops contained high level of crude fat. In contrast, the lipid content of the venison used in this experiment was relatively higher compared to the values found by Polak et al. (2008) [24], who reported values of 1.14-1.72% red deer meat. Deer have very low lipid concentrations in their muscles, with those present primarily being structural lipids, with little contribution from triacylglycerols [8]. Triumf et al. (2012) [25] found significantly lower levels of intramuscular fat (0.44-0.79%) in longissimus muscle of reindeer. The intramuscular fat content in wapiti deer meat (1.75-2.56%) in the present study is largely in agreement with the findings of Wiklund et al. (2001) [26], who reported 2.5-3.2% intramuscular fat in M. Longissimus of feral red deer. Similar results for the fat content in reindeer (Rangifer tarandus tarandus L) were reported by [26], while the values reported by [27] in the meat of the male fallow deer (Dama dama) from pasture and concentrate showed even lower values for fat content (0.42-0.69%).

3.2 Fatty acid analysis

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The contents and composition of fatty acid in four parts were summarized in Table 3 and 4. In this experiment, fifteen types of fatty acids were identified by GC-MS (Fig.1 and 2), the numbers of fatty acids quantified in fresh samples (chops, lion, shoulder, hindquarters) were 15, 14, 15, and 13, respectively, the numbers of fatty acids quantified in freeze-dired samples were 15. No significant differences of fatty composition was detected between freeze-dried and fresh samples. Among all fatty acids, 15 types of fatty acids were identified, including seven SFAs (lauric acid; myristic acid; pentadecanoic acid; palmitic acid; heptadecanoic acid; stearic acid; arachidic acid), four MUFAs (myristoleic acid; palmitoleic acid; oleic acid; cis-11-eicosenoic acid), four PUFAs (linoleic acid; linolenic acid; cis-8,11,14-eicosatrienoic acid; arachidonic acid).

Fig. 1 GC/MS chromatogram of a FAME standard mixture. The peaks are assigned in Table 2.

(a) fresh chops (b) freeze-dried chops

(c) fresh hindquarters (d) freeze-dried hindquarters

(e) fresh shoulder (f) freeze-dried shoulder

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(g) fresh loin (h) freeze-dried loin

Fig. 2 GC/MS chromatograms of fatty acids in wapiti deer muscle

The weight of freeze-dried samples is significant higher than fresh samples, however, the weight percentages is similar in both the fresh and freeze-dried samples. The results showed that freeze drying could protect the quality of venison. Oleic acid, Stearic acid, palmitic acid, and linoleic acid were dominant with approximately 10 % of overall contents, respectively. The weight percentages of MUFAs of venison varied from 26.71-42.39%; the weight percentages of PUFAs varied from 14.05-34.37%. The present showed lower PUFA and higher MUFA values for venison; this is not in agreement with the findings of Polak et al. (2008) [24], who indicated a lower PUFA and higher MUFA content for red deer meat. Nowadays, there is increasing awareness to include higher proportions of unsaturated fatty acids in the diet that have favorable effect on human health. The recommended ratio of polyunsaturated and saturated fatty acids (PUFAs/SFAs) in the human diet should be above 0.4. The PUFA/SFA ratio in the diet is considered to be more important than the total fat content (Ugarković, & Ugarković, 2013). In the present study, the ratio of PUFAs to SFAs in fresh sample (0.30-0.41) were lower than those of freeze-dried sample (0.64-0.96). Accordingly, the P/S ratio of freeze-dried venison (0.64-0.96) is more favorable for the human diet compared to beef (0.17-0.18). Other studies have indicated similar, less favorable P/S ratios for beef and lamb when compared with the data in the literature for deer meat [28-29].

Table 2 Positional and geometrical FA isomers, retention times (RT), fragment ions used for

quantification and nominal masses of the FAMEs. The number refers to the chromatogram shown in Fig.1.

Nr. Fatty acid RT m/z mass 1 Lauric acid methyl ester(C12:0) 16.789 74 214 2 Myristic acid methyl ester (C14:0) 20.048 74 242

3 Myristoleic acid methyl ester (C14:1) c9 21.564 55 240 4 Pentadecanoic acid methyl ester(C15:0) 21.748 74 256 5 Palmitic acid methyl ester (C16:0) 23.458 74 270 6 Palmitoleic acid methyl ester(C16:1) c9 24.766 55 268 7 Heptadecanoic acid methyl ester(C17:0) 25.149 74 284 8 Stearic acid methyl ester (C18:0) 26.817 74 298 9 Oleic acid methyl ester (C18:1n9c) c9 27.987 55 296

10 Linoleic acid methyl ester (C18:2n6c) c9,c12 29.768 67 294 11 Arachidic acid methyl ester (C20:0) 30.251 74 326 12 Cis-11-Eicosenoic acid methyl ester (C20:1) c11 31.549 55 324 13 Linolenic acid methyl ester (C18:3n3) c9,c12,c15 31.966 79 292 14 Cis-8,11,14-Eicosatrienoic acid methyl ester

(C20:3n6) c8,c11,c14 35.314 79 320

15 Arachidonic acid methyl ester (C20:4n6) c5,c8,c11,c14 36.437 79 318

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Table 3 GC-MS analysis results of fatty acid in venison Nr. fatty acid (mg FA/100g venison) fresh shoulder freeze-dried shoulder fresh hindquarters freeze-dried hindquarters

contents ratios % contents ratios % contents ratios % contents ratios % 1 Lauric acid methyl ester(C12:0) 1.72±1.68 0.19±0.06 2.09±1.06 0.15±0.01 0.69±0.72 0.13±0.03 1.03±0.08 0.10±0.01

2 Myristic acid methyl ester (C14:0) 48.00±9.79 5.18±1.98 60.17±31.07 4.17±0.36 22.63±6.65 3.85±1.57 30.18±2.29 2.88±0.29 3 Myristoleic acid methyl ester

(C14:1) 12.99±4.69 1.48±0.66 17.12±9.44 1.18±0.18 8.54±1.75 1.61±0.33 9.47±0.75 0.90±0.07

4 Pentadecanoic acid methyl ester(C15:0)

6.69±6.05 0.76±0.18 9.89±5.45 0.66±0.09 3.47±3.92 0.61±0.21 5.34±0.88 0.51±0.09

5 Palmitic acid methyl ester (C16:0) 133.89±42.44 16.43±1.46 216.68±99.24 15.31±0.49 77.10±27.46 14.68±2.59

142.29±13.87 13.55±0.93

6 Palmitoleic acid methyl ester(C16:1) 82.11±22.10 9.66±2.86 115.69±59.01 8.03±0.63 61.00±59.50 11.82±1.68

79.78±7.48 7.59±0.42

7 Heptadecanoic acid methyl ester(C17:0)

6.96±6.26 0.79±0.20 10.92±6.11 0.73±0.10 3.91±4.56 0.67±0.26 6.44±0.89 0.61±0.08

8 Stearic acid methyl ester (C18:0) 167.06±42.95 21.49±3.35 299.56±143.61 20.80±1.68 87.55±4.80 17.04±2.34

202.27±24.47 19.25±1.80

9 Oleic acid methyl ester (C18:1n9c) 205.15±80.74 24.09±4.07 310.56±172.24 20.72±3.52 131.84±35.00 24.79±4.99

200.65±19.37 19.12±1.59

10 Linoleic acid methyl ester (C18:2n6c)

85.51±19.23 14.99±6.75 277.22±99.15 20.72±4.19 60.29±9.39 17.47±8.69

269.40±42.58 25.53±2.35

11 Arachidic acid methyl ester (C20:0) 0.84±0.74 0.10±0.03 1.50±0.89 0.10±0.02 0.99±0.09 0.30±0.16 0.85±0.18 0.08±0.02 12 cis-11-Eicosenoic acid methyl ester

(C20:1) 0.98±0.52 0.12±0.06 1.80±0.14 0.45±0.36 1.55±0.41 0.15±0.03

13 Linolenic acid methyl ester (C18:3n3)

1.79±1.06 0.21±0.04 6.86±5.61 0.54±0.54 1.71±0.24 0.42±0.31 5.81±5.71 0.58±0.63

14 Cis-8,11,14-Eicosatrienoic acid methyl ester (C20:3n6)

1.08±0.58 0.18±0.15 4.87±2.47 0.35±0.09 0.61±0.10 0.15±0.11 4.49±1.01 0.42±0.06

15 Arachidonic acid methyl ester (C20:4n6)

20.59±5.59 4.39±3.58 97.05±58.99 6.55±1.33 17.19±9.14 6.34±4.88 93.12±38.98 8.74±3.27

Total Content (mg/100g) 775.36±109.80 100 1430.18±283.94 100 479.32±62.59 100 1052.67±105.97 100 SFA 365.16 47.09 600.81 42.01 196.34 40.96 388.40 36.89 MUFA 301.23 38.85 443.37 31.00 203.18 42.39 291.45 27.69 PUFA 108.97 14.05 386.00 26.99 79.80 16.65 372.82 35.42 PUFA/SFA 0.30 0.64 0.41 0.96 Atherogenic index (AI) 2.54 2.03 1.65 1.31 Thrombogenic index (TI) 1.66 1.33 1.28 0.71

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Table 4 GC-MS analysis results of fatty acid in venison

Nr. fatty acid (mg FA/100g venison) fresh chops freeze-dried chops fresh loin freeze-dried loin contents ratios % contents ratios % contents ratios % contents ratios %

1 Lauric acid methyl ester(C12:0) 1.62±2.36 0.15±0.07 2.09±0.29 0.12±0.01 1.72±0.90 0.16±0.02 1.35±0.86 0.13±0.03 2 Myristic acid methyl ester (C14:0) 39.01±55.83 3.65±1.42 71.93±12.51 4.39±0.21 48.00±14.63 4.49±0.71 38.91±12.21 3.75±0.63 3 Myristoleic acid methyl ester (C14:1) 6.65±6.68 1.03±0.26 33.22±5.75 2.03±0.10 12.99±2.96 0.99±0.88 10.38±5.29 1.02±0.17 4 Pentadecanoic acid methyl

ester(C15:0) 9.56±14.96 0.71±0.57 8.16±1.98 0.50±0.15 6.69±3.75 0.94±0.23 5.64±2.99 0.50±0.21

5 Palmitic acid methyl ester (C16:0) 121.49±18.41 13.56±1.88 240.44±46.17 14.63±0.35 133.89±71.21 12.29±0.66 148.89±72.66 14.61±1.39 6 Palmitoleic acid methyl ester(C16:1) 51.20±52.81 7.69±1.75 232.10±45.04 14.11±0.40 82.11±27.43 8.21±5.52 77.80±41.44 7.56±0.99 7 Heptadecanoic acid methyl

ester(C17:0) 11.22±7.83 0.79±0.71 8.87±1.31 0.54±0.04 6.96±5.86 1.14±0.30 6.16±5.29 0.55±0.22

8 Stearic acid methyl ester (C18:0) 239.31±51.37 21.15±10.17 243.69±36.49 14.94±1.11 167.06±44.03 28.65±8.19 192.25±54.26 18.13±3.63 9 Oleic acid methyl ester (C18:1n9c) 197.53±26.54 19.33±6.39 381.67±66.32 23.29±0.98 205.15±99.11 25.03±1.33 180.15±49.31 16.19±5.80

10 Linoleic acid methyl ester (C18:2n6c) 97.67±63.14 20.16±10.49 294.43±74.80 17.73±0.80 85.51±48.23 10.69±0.93 245.94±46.85 26.21±6.68 11 Arachidic acid methyl ester (C20:0) 4.39±1.38 0.82±0.77 1.08±0.19 0.07±0.01 0.84±0.65 0.13±0.05 0.81±0.76 0.07±0.03 12 cis-11-Eicosenoic acid methyl ester

(C20:1) 2.98±1.13 0.53±0.48 2.27±0.54 0.17±0.02 0.98±0.27 0.12±0.04 2.03±0.13 0.20±0.08

13 Linolenic acid methyl ester (C18:3n3) 5.81±0.66 1.23±1.29 3.48±0.73 0.21±0.02 1.79±1.58 0.25±0.07 2.72±1.72 0.23±0.01

14 Cis-8,11,14-Eicosatrienoic acid methyl ester (C20:3n6)

1.24±0.33 0.24±0.23 4.95±1.62 0.29±0.04 1.08±0.21 0.08±0.06 4.36±0.97 0.47±0.15

15 Arachidonic acid methyl ester (C20:4n6)

27.89±12.06 9.89±8.42 122.35±61.62 7.06±2.24 20.59±9.94 1.89±0.71 94.89±46.11 10.54±6.12

Total Content (mg/100g) 817.57±95.16 100 1650.73±349.69 100 775.36±106.32 100 1012.28±140.05

100

SFA 426.20 52.13 576.26 34.91 365.16 47.09 395.01 38.92 MUFA 258.36 31.60 649.26 39.33 301.23 38.85 270.36 26.71 PUFA 132.61 16.22 425.31 25.76 108.97 14.05 347.91 34.37 PUFA/SFA 0.31 0.74 0.30 0.88 Atherogenic index (AI) 2.31 1.77 2.54 1.63 Thrombogenic index (TI) 1.90 1.02 1.66 1.20

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3.3 Amino acids analysis

The amino acid compositions and content of venison were analyzed, and seventeen amino acids were identified and quantified (Table 5). Moreover, the amino acid score (AAS), chemical score (CS), and essential amino acid index (EAAI) were calculated and presented in Table 6.

Table 5 Amino acid composition in venison (g/100g venison)

Amino acid shoulder hindquarters loin chops

Alanine (Asp) 5.35±0.74 6.84±0.97 6.06±1.17 5.83±0.33 Threonine (Thr)A 2.78±0.61 3.38±0.64 3.04±0.28 2.94±0.16 Serine (Ser) 2.32±0.76 2.81±0.56 2.51±0.65 2.46±0.21 Glutamic acid (Glu) 9.18±1.06 10.98±1.21 9.87±0.72 9.60±1.01 Glycine (Gly) 2.32±0.31 2.77±0.29 2.50±0.57 2.49±0.47 Aspartic acid (Ala) 3.39±0.55 3.98±0.39 3.64±0.71 3.56±0.99 Cystine (Cys)A 0.63±0.35 0.59±0.15 0.64±0.03 0.58±0.11 Valine (Val)A 3.01±1.01 3.49±0.22 3.29±0.68 3.16±0.97 Methionine (Met)A 1.83±0.21 1.87±0.15 1.83±0.21 1.83±0.29 Isoleucine (Ile)A 2.86±0.99 3.30±0.82 3.05±0.62 2.94±0.74 Leucine (Leu)A 4.99±0.23 5.71±0.23 5.26±0.13 5.18±0.97 Tyrosine (Tyr)A 2.14±0.55 2.33±0.83 2.19±0.28 2.27±0.44 Phenylalanine (Phe)A 2.57±0.46 2.86±0.66 2.66±0.46 2.62±0.49 Lysine (Lys)A 5.60±1.46 6.19±1.23 5.82±1.18 5.67±1.01 Histidine (His)A 2.15±0.17 2.56±0.14 2.48±0.85 2.23±0.81 Arginine (Arg) 3.96±0.37 4.35±0.91 4.07±0.73 4.04±0.66 Proline (Pro) 1.58±0.23 2.28±0.96 1.94±0.91 1.84±0.33 Total essential amino acids (EAAs) 28.56 32.28 30.26 29.64 Total non-essential amino acids (NEAA)

28.10 34.01 30.59 29.82

Total amino acids (TAA) 56.66 66.29 60.85 59.46 EAA/TAA 50.41% 48.70% 49.73% 49.85%

A Essential amino acid (EAA) It is considered that the composition of amino acid content, which may be one of the factors indicative of the quality of venison, is greatly variable with respect to different parts. In our study, venison contained 56.66-66.29g total amino acids in 100g venison; Essential amino acids constituted 49.73-50.41% of total amino acids. The dominant amino acids were glutamic acid (9.18-10.98% of the total content of amino acids), lysine (5.60-6.19%), alanine (5.35-6.84%) and leucine (4.99-5.71%). Quantitative changes in the composition of amino acids can depend on different parts.

The ratios of EAAs/TAA did not differ significantly among four parts. Comprehensively, hindquarters were rich in EAAs and had higher nutrient levels. The total content of essential amino acids for all investigated venison (48.70 to 50.41%) was higher than the standard (33.90%). The comparison of wapiti deer muscle with goose muscles has shown similar amino acid content for goose muscles [30].

We compared the levels in four parts of venison to calculate the amino acid score (AAS), chemical score (CS), and essential amino acid index (EAAI) (Table 6). The results for AAS and CS indicated that the highest scores were found for loin, followed in decreasing order by shoulder, hindquarters, and chops. We can observe that the CS of the His was highest in all parts, reaching a maximum value of 240.12 in loin, in comparison with other amino acids. Compared with the score of each sample, among the essential amino acids, the amino acid limiting the quality of protein was valine, cystine with methionine. Four amino acids (His, lys, Phe and Thr) had high scores, which could be used as amino acid supplements for other food lacking these components. The EAAI index was the highest for shoulder, followed by loin and chops, whereas the lowest value was obtained for the hinderquarters.

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Table 6 Comparison of AAS, CS, and EAAI in four parts of venison

Sample shoulder hindquarters loin chops

AAS of essential amino acids

Thr 122.85 127.25 124.65 124.13

Val 106.36 105.48 108.22 106.76

Met Cys 123.91 105.74 116.31 116.14

Ile 126.1 124.55 125.38 123.95

Leu 125.89 123.04 123.56 124.83

Phe Tyr 138.55 130.62 132.85 137.62

Lys 179.67 169.87 173.69 174.05

His 378.8 386.9 408.2 375.8

Total 1302.13 1273.45 1312.86 1283.28

CS of essential amino acids

Thr 104.55 108.29 106.09 105.64

Val 80.58 79.91 81.98 80.88

Met Cys 76.09 64.93 71.42 71.32

Ile 93.41 92.26 92.87 91.81

Leu 102.47 100.15 100.57 101.6

Phe Tyr 89.39 84.27 85.71 88.78

Lys 141.26 133.47 136.47 136.76

His 222.82 227.59 240.12 221.06

Total 910.57 890.87 915.23 897.85

Indexes of samples

AAS 106.36 105.48 108.22 106.76

CS 76.09 64.93 71.42 71.32

EAAI 107.06 103.62 106.29 105.42

The amino acid score (AAS) and chemical score (CS) of essential amino acids (EAAs) were calculated using Eqs. (5) and (6), respectively. The EAA with the minimal AAS (or CS) could be described as the first limiting amino acid (FLAA). The AAS (or CS) of the EAA in a certain sample, shown in parentheses (or square brackets), was equal to the AAS (or CS) of the same sample. The essential amino acid index (EAAI) was calculated from the CS of EAAs by Eq. (7)

4. Conclusions In our study, fifteen types of fatty acid were identified and quantified in fresh and freeze-dried samples, with monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) constituting approximately 47.82-59.04% (fresh venison) and 57.99-65.09% (freeze-dried venison) of all fatty acids, respectively. The highest levels of fatty acids were found in chops, followed by hindquarters, fresh shoulder and loin. Oleic acid, Stearic acid, palmitic acid, and linoleic acid were dominant with approximately 10 % of overall contents, respectively. PUFAs/SFAs (saturated fatty acids) ratios in fresh sample (0.30-0.41) were lower than those of freeze-dried sample (0.64-0.96). According to FAO/WHO recommendations, amino acid was abundant in venison (especially hindquarters). Loin and shoulder are higher than average scores, indicating their better nutritive value. Taken together, our results illustrate that wapiti deer muscle can provide a highly nutritious food rich in fatty acids and amino acids which offer health benefits and may be used for nutraceutical purposes in the future.

Acknowledgements The present study was supported by the National Science and Technology Supporting Program (No. 2013BAD16B09-04) from Ministry of Science and Technology, the Special Fund of Agricultural Technology

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