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The Journal of Food Technology. Photon 106 (2014) 227-238 https://sites.google.com/site/photonfoundationorganization/home/the-journal-of-food-technology Original Research Article. ISJN: 3852-1875: Impact Index: 3.43

The Journal of Food Technology Ph ton

Evaluation of the addition of dry soybean sprouts on cooking yield and oxidative stability of enriched beef patties with soybeans oil

Romero Mara Cristinaa,b*, Garro Oscara,b, Romero Ana Maraa, Doval Mirtha Marinaa, Judis Mara Aliciaa

a Laboratorio de Industrias Alimentarias II, Universidad Nacional del Chaco Austral, Cte. Fernndez 755, (3700)

P.R. Senz Pea, Chaco, Argentina b CONICET, Consejo Nacional de Investigaciones Cientficas y Tcnicas, Argentina

The authors receive Thomas Edison Award-2014 in Food Technology for Inspiration and Knowledge Distribution among young research scholars.

Article history: Received: 29 January, 2014 Accepted: 02 February, 2014 Available online: 15 March, 2014

Keywords: Beef patties characteristics - healthier lipids - lipid oxidation - natural antioxidants - Glycine max L

Corresponding Author: Romero M.C. Professor Email: mara@uncaus.edu.ar Phone: 543644420137

Garro O. Professor Email: garro@uncaus.edu.ar Phone: 543644420137

Romero A.M. Professor Email: amr@uncaus.edu.ar Phone: 543644420137

Doval M.M. Professor Email: mdoval@uncaus.edu.ar Phone: 543644420137

Judis M.A Professor Email: judis@uncaus.edu.ar Phone: 543644420137

Abstract The improvement of fatty acid profile of beef patties through the replacement of pork back fat with soybean oil can be achieved, but it is necessary to use antioxidants, such as dry soybean sprouts, to maintain the oxidative stability. Besides, development of functional foods with soybean sprouts as food ingredient opens up new possibilities to their use as natural additive. This work was designed to investigate the effect of the addition of soybean sprouts on the quality characteristics of beef patties with partial replacement of pork back fat by soybean oil during frozen storage (90 days at 18 2 C). Results showed that addition of soybean sprouts improved protein and carbohydrate content; moisture and oil retention of cooked enriched patties were also enhanced. One percent (1%) of dry soybean sprouts (DSS) concentration was the most effective to retain healthier lipids and minimize the cooking loss. Besides, these products were microbiological stable and had a softer texture than those without soybean sprouts addition. This study indicates that proximate composition could be improved through incorporation of dry soybean sprouts, and it could be used to protect enriched beef patties from lipid oxidation without modifying their quality characteristics.

Citation: Romero M.C., Garro O., Romero A.M., Doval M.M., Judis M.A., 2014. Evaluation of the addition of dry soybean sprouts on cooking yield and oxidative stability of enriched beef patties with soybeans oil. The Journal of Food Technology. Photon 106, 227-238.

All Rights Reserved with Photon. Photon Ignitor: ISJN38521875D654215032014

1. Introduction

In recent years, the demand for fast food has been increasing rapidly. Since it is generally recognized that meat and meat products make an important contribution to nutrition, numerous efforts have been made to optimize their composition in order to help consumers adapt their diet to nutrient intake goals.

Burgers and patties are common meat products widely accepted by all population, and the possibility of inducing changes of composition to improve their nutritional value and their potential health-beneficial properties is among the advantages they present (Lpez-Lpez et al., 2010). Various ground beef products have been developed by replacing

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animal fat with vegetable oil so as to be more in line with health recommendations by reducing saturated fatty acids (SFA) and cholesterol, and increasing monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) (Dzudie et al., 2004; Hur et al., 2008; Lee et al., 2005). Therefore, in order to development functional meat products, liquid olive oil has been added to beef patties (Hur et al., 2008), also the addition of pre-emulsified soybean oil as replacement of pork back fat was studied (Muguerza et al., 2003); but this addition decrease the cooking yield and affect the oxidative stability of the enriched product.

Soy proteins are widely used in meat products for their nutritional value and functional properties in the form of flours, concentrates and isolates, since they increase water retention, fat retention, emulsion stability, nutritional content and cooking yield. Several researchers (Porcella et al., 2001; Chin et al., 2000; Feng et al., 2003) informed that soy protein isolates (SPI) can be used as additives in foods, especially in meat products, improving functional properties of the system such as water binding and textural properties. A further advantage of soy proteins is the antioxidant activity of some fractions of them (Pea-Ramos and Xiong, 2003; Romijn et al., 1991).

Germinated soybean sprouts are commercialized in our country as raw staple vegetables and used in soups and salads. It is particularly promising as meat ingredient due to its antioxidant compounds as vitamin C 475 mg/kg dry matter, phenolics compounds 391 mg/g dry sprouts, flavonoids content 184 mg/g quercetin equivalents of dry sprouts and superoxide dismutase (SOD) activity in crude extracts from dried soybean sprouts (DSS) was 3110 unit/g dry matters. In previous research, the effectiveness of different concentrations of DSS as antioxidant on cooked chicken patties stored at 6 1 C for 8 days was evaluated, showing a decrease of lipid oxidation in all cases, although they were only strongly effective to concentrations higher than 30 g/kg (Romero et al., 2008).

Even when soybean oil has been added to pork patties in liquid form (Jung and Joo, 2013) and the DSS have been investigated previously (Romero et al., 2008), there are no reports with the effect these products on the chemical composition and- or on technological properties during frozen storage of functional cooked meat products.

2. Objective of Research

Consequently, this work was designed (1) to evaluate the effects of the pork fat partial replacement by soybean oil and the addition dried soybean sprouts on the proximate composition and cooking properties of beef patties and (2) to investigate the effectiveness of DSS on reducing lipid oxidation of enriched cooked beef patties during frozen storage (90 days at 18 2 C). Also, texture and microbiology analyses were carried out over the samples that showed higher oil retention and acceptable oxidative stability

3. Materials and Methods

3.1 Study area The soybean (Glycine max L.) was obtained from soybean cultivars in the province of Chaco situated between the parallels 24 and 28 south latitude and between the meridians 58 and 63 west longitude, Argentina (America). This work was carried out between March of 2012 and July of 2013. This enriched beef patties with DSS and soybean oil provides both nutritional and phytochemical benefits of soybean to human population.

3.2 Materials Dry soybean sprouts were obtained from previously selected soybeans (Glycine max L. Merr, Nidera A8009RG, Chaco, Argentina) using as selection criteria hygienic condition, shape and size. The beans were soaked in water for one hour at 25 C and then germinated in darkness at 30 C in a controlled temperature chamber. Once the sprouts reached 3 cm of length approximately, they were separated from the beans and dehydrated at 30 C in a static drying chamber until 89% dry weight. Then dehydrated soybean sprouts were ground to a powder.

3.3 Patties preparation Enriched beef patties were prepared using fresh lean beef meat and pork back fat obtained from local markets, and soy oil containing SFA 16 g/100g, MUFA 35 g/100g, PUFA n6 60 g/100g and n3 6 g/100g (Aceitera General Deheza S.A., Crdoba, Argentina) that was used as fat partial replacer.

Six different formulations were elaborated as follows: one control sample without addition of antioxidants (C) with beef and 20% of pork back fat (similar to commercial products); and four enriched beef patties (EP) with beef, 10% of pork back fat, 10% of soybean oil and with addition of DSS as natural antioxidant (0%,

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0.5%, 1% and 2%), and 0.01% of butylated hydroxyanisole (BHA) as antioxidative positive control.

All formulations were shaped using Petri-dish (90 mm x 20 mm) to obtain 60 patties of approximately 100g each one. Half of the samples were cooked in an electric oven (200 C) until internal temperature reached 72 C. Cooking properties, proximate analysis, fatty acid composition, and texture profile were evaluated within 8 h after the samples were prepared.

To evaluate oxidative stability, the cooked samples were packaged in oxygen permeable bags (2000 cm3/m2 day) using a packing machine (RAPI-VAC S-750 SERVIVAC S.R.L., Buenos Aires. Argentina) stored at -18 C and analyzed at 0, 15, 30, 60 and 90 days.

All chemical and physical analyses were carried out in duplicated for each formulation and heat treatment (raw or cooked).

3.4 Proximate composition Moisture, protein, fat and ash contents were analyzed according to AOAC (1999) methods. Total carbohydrates were quantified using anthrone Cleggs methods (1956) on an UV-Vis Beckman DU 640B (Fullerton, CA, USA) spectrophotometer. Sodium content was determined by a JENWAY PFP7 flame photometer (Burlington, NJ, USA) in pre-ashed and HCl acidified samples.

3.5 Determination of cooking properties Percent cooking yield was determined by calculating weight differences for samples before and after cooking as follows (Naveena, et al., 2006):

Fat retention percentage was calculated using the following equation (El-Magoli et al., 1996):

Moisture retention represents the percentage of moisture retained in the cooked product and was determined according to Alakali, et al. (2010):

Diameter and thickness of patties were measured with a Vernier caliper. Shrinkage percentagewas determined using the equations reported by Serdarolu (2006):

3.6 Fatty acids composition Total lipids were extracted by Bligh and Dyer (1959) method. Boron trifluoride/methanol was used for the preparation of fatty acid methyl esters (AOAC 1999) and then they were analyzed using GC Mass Spectrometer (Thermo Fisher Scientific, Austin, TX, USA) equipped with a 100% Cyanopropyl silicone capillary column (SPTM - 2340 60 m, 0.32 mm ID, film thickness 0.25 mm). The oven temperature was held at 140 C for 5 min and subsequently increased at 4 C/min to 220 C. Injector temperature was 250 C. Identification of fatty acid methyl esters was based on retention time of standard esters (Supelco 37 Components FAME Mixture, Bellefonte, PA) eluting from the capillary column. Peak areas were integrated using chromatography data software, and concentrations of each ester were calculated as a percentage of the total area of the chromatogram.

Atherogenic index (AI) and thrombogenic index (TI) were calculated according to Ulbricht and Southgate (1991).

3.7 Measurement of lipid oxidation To evaluate oxidative stability, the cooked samples were frozen at -18 C and analyzed at 0, 15, 30, 60 and 90 days. Samples with

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0.01% butylated hydroxyanisole (BHA) were used as positive reference in this assay.

Lipid oxidation of cooked beef patties was monitored by measuring conjugated dienes (CD) formation, peroxide value (PV) and thiobarbituric acid reactive substances (TBARS). Conjugated dienes were determined according to Aubourg (1998). Pre-weighed lipid samples were diluted with hexane and the absorbance was measured at 233 nm in a Beckman DU 640B spectrophotometer (Fullerton, CA). The hydroperoxides conjugated dienes concentration was expressed in milliliter per milligram of lipids. The results were calculated as:

Where Bis the absorbance reading, V denotes the volume (ml) of the solvent and w is the mass (mg) of sample measured. Determination of PV was conducted by the IDF method (Shanta and Decker, 1994). 0.01 g of samples was dissolved in 9.9 ml chloroform: methanol (70:30) solution and 0.05 ml of 30 % (w/v) ammonium thiocyanate were added and mixed. Precisely 5 min after the addition of 0.05 ml of ferrous chloride in 3.5 % v/v hydrochloride acid to the reaction mixture, the absorbance of the resulting red color was measured at 501 nm against solvent solution as blank. Results were expressed in meq O2 /kg of lipids. A modified thiobarbituric acid reactive substances (TBARS) method was used to evaluate the extent of lipid oxidation (Ahn and Kim, 1998). One hundred milligrams of lipids were taken, and the following reactives were sequentially added: 100 l (BHA 36 g/l) and 2 ml of TBA/trichloroacetic acid (TCA) solution (20 mM TBA in 150 g/l TCA). The mixture was heated in a 90 C water bath for 15 min and cooled at room temperature. Afterwards, two milliliters of chloroform were added and the mixture was centrifuged at 1,000 g for 15 min. The absorbance of the supernatant was measured at 532 nm in an UV-Vis Beckman DU 640B spectrophotometer against a blank containing 0.1 ml H2O and 2 ml TBA/TCA solution. Malonaldehyde (MAD) standard curves were prepared by 1,1,3,3-tetramethoxypropane and TBARS were expressed as mg/kg of MAD equivalents of lipids.

3.8 Microbiological analyses Ten grams of each sample were homogenized with 90 ml of 0.1% sterile peptone water, and appropriate serial dilutions were plated in duplicate on plate count agar (PCA) at 30 C

for 48 h for total bacterial count. Results were expressed as log number of colony forming units per g of sample (log cfu/g).

3.9 Texture profile analysis The cooked patties texture profile analysis (TPA) was subjected to a two-cycle compression test, as described by Pons and Fiszman (1996), using a texture analyzer (Stable Micro System TA-XT2i , Surrey, England), equipped with a load cell of 4.5 kg. Hardness, cohesiveness, gumminess, instantaneous springiness, delayed springiness and chewiness were obtained using the available computer software. Three different patties from each sample were analyzed.

4. Statistical Analyses

The results were expressed as mean standard deviation of the mean for the formulations under study. All statistical tests were performed using Statgraphics Plus for Windows software package. Data collected from proximate composition was analyzed by using 5x2 factorial categoricaldesign, being the analyzed factors: formulations (C; EP; EP 0.5% DSS; EP 1% DSS and EP2% DSS) and heat treatment (raw and cooked). Differences were considered significant at p

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Alimentary fiber 9.880.06 Ash 5.97 4.85E-05 Sodium 0.320.20

All values are mean standard deviation of three replicates

Table 2: Mean of fatty acid composition (% of fatty acids)

Fatty acid % Total Mean SD C14:0 0.460.004 C16:0 21.500.143 C16:1 1.550.239 C17:0 0.550.008 C18:0 5.230.036 C18:1n9 9.650.050 C18:2n6 37.600.149 C18:3n3 12.470.004 C20:0 1.480.003 C22:0 5.340.006 C22:2n6 4.140.156

All values are mean standard deviation of three replicates

5.2 Effect of soybean sprouts on proximate composition of beef patties with partial replacement of pork backfat by soybean oil The mean percent moisture, protein, fat, ash, carbohydrate contents and pH of raw and cooked beef patties are given in Tables 3 and 4.

Moisture analysis of all patties showed significant differences (p

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with the level of soybean sprouts added, due to its high carbohydrates and proteins contents, which improved the fat-absorption capacity (Ali et al., 2011).

Ashes analysis of all samples showed significant differences between heat treatment and formulation (p

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addition. Therefore, the impacts of added ingredients in the dimensional changes were evaluated. The results obtained in this case, showed that although pork fat was replaced and DSS were added to beef patties, significant changes in dimensional characteristics (diameter reduction, thickness and shrinkage were not observed (p>0.05).This is probably due to the binding and stabilizing properties of soybean buds, which maintain the meat particles together avoiding changes in the shape of the products (Choi et al., 2012).

5.4 Effect of soybean sprouts on fatty acids profile of cooked beef patties with partial replacement of pork fat by soybean oil Vegetable oils with a high content in PUFA such as corn, sunflower, cotton seed, and soybean oil, have been used to substitute the fat content in cooked meat burgers to increase the PUFA/SFA ratio as a consequence of a higher content in linoleic acid and -linolenic acid, which exerts a relevant influence on the nutritional quality of the products (Rodrguez-Carpena et al., 2012).

In this study, the replacement of pork fat by soybean oil in raw patties formulation increased n3 PUFA of short chain content from 1.65% to 4.55%, and PUFA from 23%

to 45% (Table 6 and Table 7). Moreover, as a consequence of replacement, SFA acids were reduced too; therefore the PUFA/SFA relationship increased around three times, and the n6/n3 ratio was maintained below 10.

The improvement in raw products fatty acid composition was affected by the cooking process, decreasing 21% and 18% the linoleic acid content and the alpha-linolenic content, respectively. This loss could be explained by the lower melting point of polyunsaturated fatty acids and the poor retention capacity of the meat matrix used. However, in cooked patties when DSS was added the loss of these fatty acids were minimized, further improving the PUFA/SFA relationship, and maintaining n6/n3 ratio below 10 (Figure 1 A and B).

Products obtained in this ways, were healthier due to the higher contents of unsaturated acids and essential fatty acids. This is in accordance with Lpez-Lpez et al. (2011), who reported a significant increase of the PUFA/SFA ratio in some formulations of patties with olive oil and Wakame seaweed added. Salcedo-Sandoval et al. (2013) informed that the n6/n3 ratio remained near 10 in meat products with reduced fat content (pork back fat replaced by emulsified oil-in-konjac matrix).

Table 6: Fatty acid profile of raw patties formulated with different concentrations of dry soybean sprouts (DSS) in g/100g dry product

Fatty acid Raw Samples C EP0% DSS EP 0.5 % DSS EP 1% DSS EP 2% DSS

(14:0) 1.230.01c 0.620.00a 0.610.01a 0.730.03b 0.720.01b (16:0) 21.490.15d 15.300.14a 15.090.16a 16.120.03c 15.620.03b (16:1) 1.900.21b 0.960.01a 0.930.02a 1.110.11a 0.990.01a (17:0) 0.530.17b 0.300.03a 0.290.01a 0.340.01ab 0.340.00ab (17:1) 0.480.21d 0.230.03b - 0.280.01c 0.230.03b (18:0) 11.770.37b 7.870.08a 7.880.06a 8.290.21a 8.290.17a (18:1)t n9 0.410.11c - - 0.200.06b 0.260.11bc (18:1)c n9 39.230.20d 29.720.08b 29.080.07a 31.130.03c 29.530.08b (18:2)c n6 12.420.35a 37.090.07d 36.770.01d 33.240.21b 35.930.04c (20:0) - 0.260.02bc 0.260.01bd 0.220.02b 0.300.01c (18:3) n3 1.650.35a 4.550.20c 4.550.28b 4.100.04b 4.470.10b (18:2) CLA 8.900.45e 3.110.01c 4.600.07b 4.230.07b 3.320.03a SFA 35.010.71d 24.350.28ab 24.120.25a 25.700.30c 25.260.23bc MUFA 42.020.74d 30.910.17ab 30.010.01a 32.730.04c 31.010.05b PUFA 22.961.16a 44.750.37cd 45.870.30d 41.570.33b 43.720.17c

Results are presented as means standard deviation. Means in the same row with different letters are significantly differentamong formulations (P

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containing only animal fat presented greater values than frankfurters with healthier oil replacement. Furthermore, DSS addition did not affect these indices in both raw and cooked patties. This behavior was similar to

the results found by Lpez-Lpez et al. (2011) when seaweeds were added to beef patties whose pork fat was partially or totally replaced by olive oil in water emulsion.

Figure 1: Fatty acid profile of cooked meat products, (A) without soybean oil and (B) with soybean oil

SI: C 19:0, Internal Standard; RT: 17.373 C14:0; RT: 20.965 C16:0; RT: 22.087 C16:1; RT: 22.670 C17:0; RT: 23.744 C17:1; RT: 24.483 C18:0; RT: 25.192 C18:1 n9 t; RT: 25.498 C18:1 n9 c; RT: 26.947 C18:2 n6; RT: 28.673 C18:3 n3; RT: 30.112 CLA C18:2 n6

Table 7: Fatty acid profile of cooked patties formulated with different concentrations of dry soybean sprouts (DSS) in g/100g dry product

Fatty acid Cooked Samples C EP EP DSS 0.5% EP DSS 1% EP DSS 2% (14:0) 1.300.51b 0.780.08ab 0.750.01ab 0.710.06ab 0.650.06a (16:0) 21.691.75b 16.700.21a 16.480.13a 16.520.35a 15.750.14a (16:1) 1.970.42b 1.260.01a 1.120.00a 1.120.11a 0.950.04a (17:0) 0.540.11b 0.360.01a 0.330,07a 0.330.04a 0.310.03a (17:1) 0.500.16b 0.330.06ab 0.260.01a 0.250.01a 0.170.08a (18:0) 11.771.53b 8.410.07a 8.680.13a 8.460.04a 8.210.10a (18:1)t n9 0.380.16a 0.230.01a 0.180.14a 0.220.13a 0.330.10a (18:1)c n9 39.961.27d 32.900.35c 31.080,13ab 31.260.38b 29.490.20a (18:2)c n6 12.002.87a 29.160.35b 33.390.20c 33.460.14c 33.960.04c (20:0) NDa 0.180.01b 0.220.02bc 0.200.01bc 0.270.03c (18:3) n3 1.740.33a 3.700.30b 4.140.18b 4.150.06b 4.070.08b (18:2) CLA 8.211.03c 5.980.03b 3.360.04a 3.320.11a 5.840.16b SFA 35.250.19c 26.420.17b 26.460.14b 26.220.20b 25.190.33a MUFA 42.802.01c 34.720.61b 32.640.28ab 32.850.64ab 30.940.42a PUFA 21.954.93a 38.841.34b 40.860.32b 40.930.31b 43.890.27b n3 1.740.33a 3.700.30b 4.110.08b 4.150.06b 4.070.08b n6 20.213.9a 35.141.05b 36.750.24bc 36.780.25bc 39.820.19c n9 39.961.27d 32.910.35c 31.080.13ab 31.260.38b 29.490.20a PUFA/SFA 0.620.05a 1.470.03b 1.540.01bc 1.560.02c 1.740.03d n6/n3 11.610.07c 9.520.48ab 8.940.13a 8.860.06a 9.780.16b AI 0.410.02c 0.270.00b 0.270.00ab 0.260.01ab 0.250.00a TI 0.940.00d 0.560.01c 0.550.01bc 0.540.00b 0.520.00a

Results are presented as means standard deviation. Means in the same row with different letters are significantly differentamong formulations (p

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due to the fact that the heating process leads to a dramatic increase of oxidative reactions of lipids in meat, which cause a warmed-over-flavour (WOF) (Bastida et al., 2009).

The formation of Conjugated Dienes (CD) occurs parallel to the production of hydroperoxides (measured as PV) and take place at the early stages of lipid oxidation (Frankel, 1996); later, these compounds decompose into secondary products such as aldehydes and ketones. The increase of PV in cooked samples may result from catalysis of intracellular compounds due to the destruction of self-structures by NaCl and processing (Roginsky and Barsukova, 2001). TBARS values represent the content of secondary lipid oxidation products mainly aldehydes (or carbonyls), which contribute to off-flavors in oxidized meat and meat products.

Moreover, the oxidative stability decreases more when the lipid composition of these products was improved with polyunsaturated fatty acids; thus, antioxidants are usually used to prevent lipid oxidation. Earlier, DSS had shown to possess a moderate antioxidant effect in other matrixes, therefore to verify if DSS added to enriched meat patties exert this effect, conjugated dienes, hydroperoxides and aldehydes formed during frozen storage were continuously monitored. These parameters (CD, PV and TBARS) were determined on cooked meat patties and are presented in

Table 8. Using the CD determination as lipid oxidation indicator, DSS concentrations did not show antioxidant activity until day 30, although the CD formation at 60 and 90 days was lower than control for 0.5% and 1% of this additive (p

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5.6 Texture profile analysis (TPA) of beef patties with partial replacement of pork fat by soybean oil Table 9 shows the effects of replacing fat content with soybean oil and soybean sprouts as natural antioxidants on the textural properties of selected cooked beef patties. Significant changes in hardness,

cohesiveness, chewiness and delayed elasticity were recorded. The hardness of cooked meat patties significantly decreased (p

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