Effects of heat treatment of soy protein isolate on the growth performance and immune function of broiler chickens

IMMUNOLOGY, HEALTH, AND DISEASE Effects of heat treatment of soy protein isolate on the growth performance and immune function of broiler chickens D. W. Wu, X. Chen, X. Yang, Z. X. Leng, P. S. Yan, and Y. M. Zhou1 College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, P. R. China h) diminished liver weight at 14 d (P = 0.01), spleen (P < 0.01) and bursa (P < 0.05) weights at 21 d; and the content of IgG in serum and duodenal mucosa of broilers (at 14 d) was decreased when diets supplemented with heat-treated SPI (heated for 8 h; P < 0.01). No significant differences were observed in the mucosa secretory IgA contents of broilers among the treatment groups (P > 0.05). Compared with native SPI, a significant increases were observed in the content of adrenocorticotropic hormone and cortisol in serum of broilers fed the heat-treated SPI (heated for 8 h) at 21 d (P < 0.05); and the myeloperoxidase activities in serum (at 14 d) and mucosa of broilers were increased when diets supplemented with heat-treated SPI (heated for 8 h; P < 0.05). The present study suggests that protein oxidation of SPI is induced by heating, and oxidized protein may negatively affect the immune function of broilers.

Key words: soy protein isolate, heat treated, broiler, growth performance, immune function 2014 Poultry Science 93:326–334 http://dx.doi.org/10.3382/ps.2013-03507

INTRODUCTION

oxidized fat on performance and immune function of animals. For example, Lin et al. (2000) demonstrated that an increased intake of oxidized oil decreased relative spleen weight in mice, and increased IgE level, the ratio of interleukin-4 and interferon-γ or interleukin-5 and interferon-γ, and inflammatory mediators such as prostaglandin E2 and leukotriene B4. However, dietary oxidized frying oil increased spontaneous spleen cell proliferation and B cell activation in rats (Lin et al., 1997). Oxidized lipids reduced the weight gain of dogs, and suppressed lymphocyte blastogenesis in response to concanavalin A (Turek et al., 2003). It has been reported that dietary oxidized fats can reduce either clonal amplification of gut-associated lymphoid tissue or effectiveness of the secretory IgA response, and eventually impair the immune system of broilers (Dibner et al., 1996). Whether oxidized proteins can cause similar damage to growth performance and immune function of animals is unclear. Protein oxidation is the covalent modification of a protein induced either directly by reactive oxygen species or indirectly by reaction with secondary byproducts of oxidative stress (Shacter, 2000; Stadtman

Protein is an important component of animal feed, and dietary protein is one of the main sources of nitrogen to form body protein for monogastric animals. Protein quality is closely related to its digestibility, and poor protein may negatively affect the animal health (Hendriks et al., 1999). Dietary proteins are often exposed to oxidants or oxidizing conditions during processing and storage (Huang et al., 2006). Therefore, the proteins are vulnerable to oxidative modification as a result of their abundance in foods and high rate constants for reaction (Davies, 2005). Oxidized protein in feed was demonstrated to cause adverse effects on the health of rats (Wu et al., 2011; Tang et al., 2012b). Protein oxidation and lipid peroxidation usually interact with each other in food (Eymard et al., 2009). However, previous studies mainly focused on the effects of ©2014 Poultry Science Association Inc. Received July 21, 2013. Accepted October 20, 2013. 1 Corresponding author: [email protected]

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ABSTRACT This study was conducted to evaluate the effects of oxidative modification of soy protein isolate (SPI) after exposure to heat on the growth performance and immune function of broilers. The SPI was heated in an oven at 100°C for 1, 4, and 8 h, respectively, and resultant oxidative status was evaluated. A total of 320 one-day-old Arbor Acres chickens were randomly divided into 4 treatment groups with 8 replicates of 10 birds, and fed diets supplemented with the native SPI or 1 of the 3 heat-treated SPI for 21 d. The results showed that heat exposure of SPI for 4 and 8 h caused an increase in protein carbonyl (P < 0.05), and a simultaneous decrease in sulfhydryl and free amine groups (P < 0.05) compared with native SPI. The BW of broilers fed diets supplemented with SPI heated for 8 h were significantly lower than that of broilers fed diets supplemented with native SPI (P < 0.05). Compared with native SPI, heat-treated SPI (heated for 8

EFFECTS OF OXIDIZED PROTEIN ON BROILERS

cause changes in growth performance and immune function of broiler chickens. Therefore, the objective of this study was to evaluate the effects of oxidative modification of SPI after exposure to heat on the growth performance and immune function of broilers.

MATERIALS AND METHODS Modification of SPI with Heat Soy protein isolate was purchased from Sun-Green Biotech Co., Ltd. (Nantong, China). Analyzed moisture, CP, ether extract, and ash contents in the SPI were 5.6, 86.2, 0.4, and 4.9%, respectively. The SPI was dry heated in an oven at 100°C for 1, 4, and 8 h, respectively, and then air-dried and stored at 4°C, according to the previously published methods (Wu et al., 2011; Tang et al., 2012b). Samples were immediately collected for the analysis of CP, amino acids, protein carbonyl, sulfydryl, and free amine group in native or heat-treated SPI.

Experimental Design and Feeding Management This experiment was carried out in accordance with the guidelines for animal experimentation as established by the Animal Care and Use Committee of Nanjing Agricultural University in China. A total of 320 one-day-old straight-run Arbor Acres chickens were obtained from a commercial hatchery (Hewei, Anhui, P. R. China) and randomly divided into 4 treatment groups with 8 replicates of 10 birds for a 21-d feeding trial. The average initial BW did not differ among the 4 groups. Birds were fed with the diets supplemented with the native soy protein isolate (native, SPI) or heat-treated SPI heated in an oven at 100°C with 1, 4, and 8 h, respectively. The basal diet was formulated based on the NRC (1994) to meet the nutrient requirements of the broiler. The formulation and nutrient level of basal diet are shown in Table 1. The contents of protein and amino acids in SPI with different heat time are shown in Table 2. Protein and amino acids in SPI pre- and post-heat treatment were calculated and met the nutrient requirements of the broiler. All birds were placed in 3-level wired battery cages and housed in an environmentally controlled room maintained at 32 to 34°C for the first week and then reduced by 2 to 3°C per week. Birds were allowed ad libitum access to mash feed and water. The BW, feed intake, and mortality of chicks were recorded.

Amino Acid Analyses Amino acids in SPI (excluding tryptophan and cysteine) were determined with an amino acids analyzer (Hitachi L-8900, Hitachi, Tokyo, Japan) after hydrolysis with 6 mol/L of HCl at 110°C for 24 h (Gao et al., 2012).

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and Levine, 2000). Agents that lead to protein oxidation include hydrogen peroxide (H2O2), hypochlorous acid (HOCl), reduced transition metals such as Fe2+ or Cu+, γ-irradiation in the presence of O2, UV light, ozone, oxidoreductase enzymes, by-products of lipid oxidation (Berlett et al., 1996; Fu and Dean, 1997; Kim et al., 1997; Levine and Stadtman, 2001; Stadtman, 2006; Wu et al., 2009a, 2010). Oxidation of proteins also occurs during feed processing, such as heat, comminuting, and grinding, which introduce oxygen molecules and mix oxidants with susceptible food components (Korhonen et al., 1998). Heating is the most common method of protein modification and production of protein gels (Tang et al., 2012a). Depending on the intensity of heat treatment, the nutritive value of proteins may be affected differently. Tang et al. (2012b) reported that heat treatment of wheat peptides caused loss of surface hydrophobicity and reducing power, and modified wheat peptides showed relatively lower free radical-scavenging activity. Soy protein isolate (SPI) has been widely used as an important food ingredient in many food categories available to the consumer because it exhibits high nutritional and excellent functional properties (Tang and Ma, 2009). The functional properties of SPI are affected by many factors, one of which is the modification of protein structure by heat (Hua et al., 2005). It was reported that heat treatment with SPI or wheat peptides resulted in increased protein carbonyl and the modification of proteins also induced more reactive oxygen species (ROS) production in vivo (Wu et al., 2011; Tang et al., 2012b). Soy protein is prone to oxidative modification during processing and storage, which stems from the presence of residual unsaturated lipids and active lipoxygenase in defatted soy flour (Huang et al., 2006). Oxidative modification of amino acid residues of proteins often leads to alterations in protein functionalities, such as gelation, emulsification, viscosity, solubility, and water-holding capacity (Wang and Xiong, 1998; Kong et al., 2008; Wu et al., 2009b), ultimately resulting in a decrease in nutritional value and sensorial properties of target proteins (Tironi et al., 2007). So, it is reasonable to hypothesize that feed protein can also be oxidized by process or storage, and these oxidative changes in proteins result in quality deterioration of protein-based feed and bioavailability of the protein decrease, which could cause physiological damage to the animal. Therefore, further investigation in this area is warranted. Heat processing is essential in the feed production and pelleting, where protein oxidation occurs inevitably. Preliminary studies in our laboratory indicated that the degree of protein oxidation of soybean meal increased with storage or heat time (D. W. Wu, unpublished data). Tang et al. (2012a) reported that heat oxidation led the soy protein to generate reactive oxygen species, diminished the antioxidant status, and induced redox imbalance in male mice. However, there was no report on whether the dietary oxidized protein would

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Table 1. The formulation and calculated nutrient levels of broiler basal diet (starter) Item

Content 784.7 165.0 14.0 19.5 3.0 2.0 1.8 10 1,000

 

13.0 210.1 9.8 4.6 12.1 5.3 8.3

Sample Collection and Procedures

1Premix provided per kilogram of diet: trans-retinyl acetate, 24 mg; cholecalciferol, 6 mg; all-rac-α-tocopherol acetate, 20 mg; menadione, 1.3 mg; thiamine, 2.2 mg; riboflavin, 8 mg; nicotinamide, 40 mg; choline chloride, 400 mg; calcium pantothenate, 10 mg; pyridoxine·HCl, 4 mg; biotin, 0.04 mg; folic acid, 1 mg; vitamin B12 (cobalamin), 0.013 mg; Fe (from ferrous sulfate), 80 mg; Cu (from copper sulfate), 7.5 mg; Mn (from manganese sulfate), 110 mg; Zn (from zinc oxide), 65 mg; I (from calcium iodate), 1.1 mg; Se (from sodium selenite), 0.3 mg; bacitracin zinc, 30 mg.

Determination of Protein Carbonyl, Sulfydryl, and Free Amine Group in Native or Heat-Treated SPI Protein carbonylation was quantified according to the method described by Huang et al. (2006), using the reaction of 2, 4-dinitrophenylhydrazine with the carbonyl groups of native and heat-treated soy proteins. The results were expressed as nanomoles of carbonyl groups per milligram of soluble protein with molar extinction coefficient of 22,000 M−1cm−1. Soluble protein concentration was evaluated by the bicinchoninic acid method with bovine serum albumin as the standard using a diagnostic kit (no. A045–3; Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

At 14 and 21 d, one bird per replicate was randomly selected and weighed after feed deprivation for 12 h. Individual blood samples were taken from the wing vein within 2 h (between 0800 and 1000 h), and serum were separated by centrifugation at 3,500 × g for 15 min at 4°C. Serum samples were frozen at −20°C for further analysis. After collection of blood samples, chickens were killed by exsanguination and necropsied immediately. The liver, spleen, thymus, and bursa of Fabricius of broilers were removed and weighed. After that, the duodenal and jejunal mucosa were collected carefully, frozen in liquid nitrogen, and stored at −20°C for further analysis.

Growth Performance and Relative Weights of Organs Body weights were recorded for each replicate at 1 and 21 d of age. Feed was withdrawn for 12 h and water was provided for ad libitum drinking before weighing at 21 d. Feed intake was recorded during the 21-d trial. The BW, ADG, ADFI, and feed/gain (F/G) ratio were calculated to determine the growth performance of birds. Relative weights of liver, spleen, thymus, and bursa of Fabricius were calculated as weight of organ (g)/BW (kg).

Table 2. Crude protein and several amino acids in soy protein isolate with different heat time (%) Treatment1 Item CP Threonine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Arginine Tyrosine Histidine

Native

1h

4h

8h

SEM

ANOVA

86.13 3.08 3.58 1.02 3.54 6.17 4.70 5.25 4.88 3.54 2.67

85.87 2.96 3.56 1.01 3.52 6.14 4.63 5.16 4.90 3.51 2.63

85.50 2.94 3.47 1.01 3.49 6.13 4.58 4.99 4.90 3.44 2.51

85.80 2.74 3.50 0.99 3.47 6.10 4.56 4.91 4.89 3.40 2.41

0.16 0.05 0.02 0.01 0.01 0.02 0.03 0.05 0.01 0.03 0.04

0.644 0.086 0.412 0.136 0.196 0.453 0.261 0.081 0.947 0.242 0.072

1Native = native soy protein isolate; 1, 4, and 8 h: soy protein isolate heated at 100°C for 1, 4, and 8 h, respectively; n = 3.

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Ingredient (g/kg)  Corn   Soy protein isolate  Limestone   Dicalcium phosphate   Sodium chloride   l-Lysine   dl-Methionine  Premix1  Total Calculated nutrient level   ME (MJ/kg)   CP (g/kg)   Calcium (g/kg)   Available phosphorus (g/kg)   Lysine (g/kg)   Methionine (g/kg)   Methionine + cysteine (g/kg)

Contents of sulphydryl [free and buried sulfhydryl (SH)] and total disulphide/sulphydryl groups in the SPI were determined using Ellman’s procedure modified by Huang et al. (2006). The nanomoles of SH per milligram soluble protein were calculated by using the extinction coefficient of 13,600 M−1cm−1. Free amines in nonmodified (native) and heat-modified SPI were measured as described by Liu et al. (2000). Free amine content was calculated as µmoles of free amines per milligram of protein using a standard curve constructed from glycine.

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EFFECTS OF OXIDIZED PROTEIN ON BROILERS Table 3. Protein oxidation of soy protein isolate with different heat time Treatment1 Item

Native

1h

4h

8h

SEM

ANOVA

Protein carbonyl (nmol/mg of protein) Free sulfhydryl (nmol/mg of protein) Total disulfide and sulfhydryl (nmol/mg of protein) Free amine group (µmol/mg of protein)

15.13c 9.19a 108.06a 0.79a

15.79c 7.91b 98.07ab 0.74ab

17.85b 8.16b 84.44b 0.70b

22.35a 8.27b 86.48b 0.69b

0.89 0.18 3.10 0.02

<0.010 0.032 <0.010 0.039

a–cValues 1Native

within a row not sharing the same superscript are different at P < 0.05; n = 3. = native soy protein isolate; 1, 4, and 8 h: soy protein isolate heated at 100°C for 1, 4, and 8 h, respectively.

Immune Response Parameter Assay

Determination of Adrenocorticotropic and Cortisol in Serum The contents of adrenocorticotropic hormone (ACTH) and cortisol (COR) in serum of broilers were measured using a commercial available 125I-RIA kit (ACTH no. D14PDB; COR no. D10PZB; Beijing North Institute of Biological Technology, Beijing, China) according to the manufacturer’s guidelines.

Activities of Myeloperoxidase Myeloperoxidase (MPO) activity in serum and mucosa were measured with an MPO assay kit (no. A044, Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions. Briefly, a portion of mucosa tissue (about 100 mg) was homogenized and fluidized in extraction buffer to obtain 5% of homogenate. The sample including 0.9 mL homogenate and 0.1 mL of reaction buffer was heated to 37°C in water bath for 15 min; then, the enzymatic activity was determined by measuring the changes in absorbance at 460 nm using a 96-well plate reader. The MPO activity was expressed as units of MPO activity per gram of wet mucosa.

Statistical Analysis Data were analyzed by one-way ANOVA (with Duncan’s multiple range test for post hoc comparison) using SPSS statistical software (ver.16.0 for Windows, SPSS Inc., Chicago, IL). The means and total standard

RESULTS Protein Oxidation of SPI by Heating Effects of heating time on SPI protein carbonyl, free sulfhydryl, total disulfide, and sulfhydryl, and free amine group contents are given in Table 3. Compared with native SPI, the protein carbonyl contents were significantly enhanced by 17.98 and 47.72%, respectively, in SPI heat exposure for 4 and 8 h (P < 0.05); and the free sulfhydryl contents were reduced by 13.93, 11.21, and 10.01% in SPI heat exposure for 1, 4, and 8 h (P < 0.05), respectively. Moreover, the total disulfide/sulfhydryl and free amine groups of SPI heated for 4 and 8 h were significantly decreased by 21.86, 19.97, 11.39, and 12.66% (P < 0.05), respectively, compared with the native SPI.

Growth Performance of Broiler Chicks The changes in growth performance exhibited by broilers fed diets supplemented with native or heattreated SPI are presented in Table 4. The BW of broilers fed diets supplemented with SPI heated for 8 h were significantly lower than that of broilers fed diets supplemented with native SPI (P < 0.05). However, the ADG, ADFI, or F/G ratio of broilers were not significantly influenced by treatment during the 21-d study period (P > 0.05).

Relative Weights of Organs Table 5 shows the relative organ weights of broilers subjected to the various dietary treatments. The liver weight of broilers fed diets supplemented with SPI heated for 1 and 8 h were significantly decreased by 12.28 and 18.66% (P < 0.05), respectively, compared with that broilers fed diets supplemented with native SPI at 14 d. At 21 d, the relative weights of spleen in broilers that received diets supplemented with SPI heated for 1, 4, and 8 h were significantly lower than in native SPI group by 22.88, 29.66, and 31.36% (P < 0.05), respectively. Moreover, the addition of SPI heated for 4 or 8

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Levels of IgG in serum, IgG, and secretory IgA (SIgA) in duodenal and jejunal mucosa were measured separately using commercial chicken-specific ELISA kit (IgG no. H106, SIgA no. H206; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). All assays using ELISA kits were carried out in duplicate and analyzed according to the manufacturer’s instructions. All samples were run on one plate to avoid interassay variations. The protein concentrations were determined using a corresponding diagnostic kit (no. A045–3, Nanjing Jiancheng Bioengineering Institute) according to the instructions of the manufacturer.

errors (SEM) are presented. Significance (P-value) was evaluated at the 0.05 level.

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Wu et al. Table 4. Growth performances of broilers fed diets supplemented with native or heat-treated soy protein isolate from 1 to 21 d of age Treatment2 Item1

Native

BW (kg) ADG (g/d) ADFI (g/d) F/G ratio (g:g)

0.54a

24.73 38.55 1.56

1h

4h

8h

SEM

ANOVA

0.51ab

0.52ab

0.50b

0.005 0.26 0.38 0.01

0.045 0.316 0.239 0.277

23.60 37.95 1.61

23.91 38.06 1.59

23.37 36.41 1.56

a,bValues

within a row not sharing the same superscript are different at P < 0.05. ratio = feed/gain ratio. 2Native = native soy protein isolate; 1, 4, and 8 h: soy protein isolate heated at 100°C for 1, 4, and 8 h, respectively. 1F/G

IgG and SIgA in Serum and Intestinal Mucosa Table 6 shows the IgG and SIgA in serum and intestinal mucosa of broilers subjected to the various dietary treatments. The serum IgG content of broilers fed diets supplemented with SPI heated for 4 and 8 h were decreased by 48.77 and 44.33% (P < 0.05), respectively, compared with that broilers fed native SPI-supplemented diets at 14 d. The concentrations of duodenal mucosa IgG in broilers fed the heat-treated SPI (heated for 8 h) diet were reduced by 65.87% (P < 0.01) compared with the native SPI group at 14 d. However, no significant differences were observed in the duodenal or jejunal mucosa SIgA contents of broilers among the treatment groups (P > 0.05).

ACTH and COR in Serum The contents of ACTH and COR in serum of broilers fed on diets supplemented with native or heated

oxidized SPI are shown in Table 7. No significant differences were observed in the serum ACTH or COR contents of broilers among the treatment groups (P > 0.05) at 14 d, but the concentrations of serum ACTH and COR in broilers fed the heat-treated SPI (heated for 8 h) diet were increased by 31.61 and 62.57% (P < 0.05), respectively, compared with the native SPIsupplemented diet at 21 d.

Activities of MPO in Serum and Intestinal Mucosa The activities of MPO in serum and intestinal mucosa of broilers fed diets supplemented with native or heated oxidized SPI are shown in Table 8. The activities of MPO in serum of broilers fed diets supplemented with SPI heated for 4 or 8 h were significantly higher than in native SPI group by 35.99 and 29.68% (P < 0.05), respectively, at 14 d. Moreover, the activities of MPO in duodenal mucosa of broilers fed diets supplemented with SPI heated for 8 h were significantly higher than other groups at 21 d (P < 0.01). The activities of jejunal mucosa MPO in broilers fed the heat-treated SPI (heated for 8 h) diet at the age of 14 and 21 d were both elevated by 50.00% (P < 0.05), compared with the native SPI-supplemented diet.

Table 5. Organ weights of broilers fed diets supplemented with native or heat-treated soy protein isolate Treatment1 Item

Native

1h

4h

8h

SEM

ANOVA

Liver (g/kg)   14 d   21 d Thymus (g/kg)   14 d   21 d Spleen (g/kg)   14 d   21 d Bursa (g/kg)   14 d   21 d

  42.34a 33.94   4.48 5.14   0.84 1.18a   1.99 2.22a

  37.14b 34.31   4.01 5.32   0.91 0.91b   1.93 2.13a

  39.13ab 32.50   4.18 5.63   0.87 0.83b   1.83 1.64b

  34.44b 30.23   4.36 5.67   0.91 0.81b   2.34 1.69b

  0.90 0.85   0.17 0.21   0.04 0.05   0.10 0.09

  0.010 0.322   0.804 0.789   0.877 0.005   0.301 0.018

a,bValues 1Native

tively.

within a row not sharing the same superscript are different at P < 0.05; n = 8. = native soy protein isolate; 1, 4, and 8 h: soy protein isolate heated at 100°C for 1, 4, and 8 h, respec-

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h to the diet decreased the bursa weights of broilers by 26.13 and 23.87% (P < 0.05), respectively, compared with that observed in the broilers fed the native SPI -supplemented diets at 21 d.

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EFFECTS OF OXIDIZED PROTEIN ON BROILERS

Table 6. IgG and secretory IgA (SIgA) in serum and intestinal mucosa of broilers fed on diets supplemented with native or heattreated soy protein isolate Treatment1 Native

1h

4h

8h

SEM

ANOVA

Serum IgG (µg/mL)   14 d   21 d Duodenal mucosa IgG (µg/mg of protein)   14 d   21 d Jejunal mucosa IgG (µg/mg of protein)   14 d   21 d Duodenal mucosa SIgA (µg/mg of protein)   14 d   21 d Jejunal mucosa SIgA (µg/mg of protein)   14 d   21 d

  50.75a 43.00   1.26a 0.46   1.04a 1.05   0.45 0.41   1.25 1.51

  50.86a 50.25   1.40a 0.55   0.38b 0.59   0.27 0.33   1.17 1.19

  26.00b 33.43   1.21a 0.54   0.93a 0.59   0.56 0.44   1.34 1.12

  28.25b 39.14   0.43b 0.42   0.70ab 0.65   0.29 0.34   1.46 1.20

  3.64 2.55   0.09 0.09   0.08 0.09   0.05 0.04   0.15 0.07

  <0.010 0.098   <0.010 0.579   0.023 0.168   0.121 0.398   0.926 0.261

a,bValues 1Native

within a row not sharing the same superscript are different at P < 0.05; n = 8. = native soy protein isolate; 1, 4, and 8 h: soy protein isolate heated at 100°C for 1, 4, and 8 h, respectively.

DISCUSSION Protein carbonyl is the most commonly used marker for protein oxidation (Stadtman, 2006; Wu et al., 2009b). Protein carbonyls may be formed either by oxidation of vulnerable amino acid residues or oxidative cleavage of protein backbone by ROS (Stadtman, 1990; Mayo et al., 2003). Oxidation of lysine, arginine, histidine, and threonine residues would yield carbonyl derivatives (Mayo et al., 2003; Wu et al., 2010). In our investigation, a numerical degradation of lysine, histidine, and threonine was observed. Previous researchers have reported that heat treatment with SPI or wheat peptides results in protein carbonyl increases (Wu et al., 2011; Tang et al., 2012b). In this study, heat exposure for more than 4 h led to a significant increase of protein carbonyl formation, which is in accordance with previous findings. Cysteine residues were in many cases the most susceptible amino acid residues of proteins, but the carbonyl contents could not reflect oxidation state of cysteine residues (Wu et al., 2009b). The oxidation state of cysteine residues in soy protein was evaluated by sulfhydryl assay in this paper. Protein sulfhydryl can be oxidized to reversible form (protein disulfide and

sulphenic acid) or irreversible form (sulfinic and sulfonic acid) in different oxidative environments (Thomas and Mallis, 2001; Eaton, 2006). The simultaneous decrease of free sulphydryl and total disulfide/sulfhydryl in this study could be attributed to the formation of sulfur oxidation products other than disulfide bonds. Content of free amines can also be used as oxidative markers of soy protein because ε-amino groups of lysine residues are sensitive to heat modification (Hua et al., 2005; Wu et al., 2009a). Our results showed that heat treatment could cause a loss of free amines in the protein. The increase of protein carbonyl, decrease of sulfhydryl, and loss of free amines in this study indicate that protein oxidation was induced by heating. In the present trial, the BW of broilers fed diets supplemented with SPI heated for 8 h were significantly lower than in broilers fed diets supplemented with native SPI. Oxidative modification has been shown to induce several structural changes in amino acid residue side-chains and protein polypeptide backbone, resulting in protein fragmentation, cross-linking, unfolding, and conformational changes (Esterbauer et al., 1991; Davies, 2005). The oxidative modification of protein structure has been implicated in quality deterioration

Table 7. Adrenocorticotropic hormone (ACTH) and cortisol (COR) in serum of broilers fed on diets supplemented with native or heat-treated soy protein isolate Treatment1 Item

Native

1h

4h

8h

SEM

ANOVA

ACTH (pg/mL)   14 d   21 d COR (ng/mL)   14 d   21 d

  12.82 12.40b   5.30 3.42b

  12.44 12.25b   4.77 3.61b

  11.77 15.90ab   4.13 3.67b

  11.66 16.32a   5.78 5.56a

  0.59 0.68   0.39 0.32

  0.898 0.038   0.518 0.021

a,bValues 1Native

tively.

within a row not sharing the same superscript are different at P < 0.05; n = 8. = native soy protein isolate; 1, 4, and 8 h: soy protein isolate heated at 100°C for 1, 4, and 8 h, respec-

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Item

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Table 8. Activities of myeloperoxidase (MPO) in serum and intestinal mucosa of broilers fed on diets supplemented with native or heat-treated soy protein isolate Treatment1 Item

Native

1h

4h

8h

SEM

ANOVA

Serum (U/L)   14 d   21 d Duodenal mucosa (U/g of tissue)   14 d   21 d Jejunal mucosa (U/g of tissue)   14 d   21 d

  90.74b 119.47   0.48 0.42b   0.44b 0.44b

  106.19ab 123.65   0.60 0.33b   0.53ab 0.42b

  123.40a 127.83   0.62 0.43b   0.46b 0.52ab

  117.67a 120.62   0.62 0.71a   0.66a 0.66a

  4.36 3.77   0.03 0.04   0.03 0.03

  0.033 0.873   0.166 <0.010   0.025 0.033

a,bValues 1Native

within a row not sharing the same superscript are different at P < 0.05; n = 8. = native soy protein isolate; 1, 4, and 8 h: soy protein isolate heated at 100°C for 1, 4, and 8 h, respectively.

2008). Reports pertaining to the effect of oxidized protein in diets on immune function of broilers are sparse. In the present study, the reduction of IgG in serum and mucosa of broilers supplemented with heat-treated SPI may be closely related to the decrease of spleen weight. In response to a stressor, physiological changes are set into motion to help an individual cope with the stressor. For example, corticotrophin-releasing hormone is secreted from the paraventricular nucleus of the hypothalamus into the hypophyseal portal blood supply and subsequently stimulates the expression of ACTH in the anterior pituitary gland. Adrenocorticotropic hormone then circulates in the blood stream to the adrenal glands where it induces the expression and release of glucocorticoid hormones (Padgett and Glaser, 2003). Cortisol is the main components of glucocorticoid, as one of the core stress responses originally described by Selye in 1936 (Selye, 1936a,b). The simultaneous increase of the ACTH and COR in serum of broilers fed the heat-treated SPI (heated for 8 h) diet demonstrated that the stress was induced by oxidized protein. Myeloperoxidase is a leukocyte-derived enzyme that catalyzes the formation of several reactive oxidant species. As a member of the heme peroxidase superfamily, MPO generates numerous reactive oxidants and diffusible radical species (Nicholls and Hazen, 2005) that are capable of both initiating lipid peroxidation and promoting an array of posttranslational modifications to target proteins, including halogenation, nitration, and oxidative cross-linking (Podrez et al., 2000; Zhang et al., 2002; Heinecke, 2003). Both MPO and its reactive oxidants have been implicated as participants in tissue injury during a large number of inflammatory conditions (Nicholls and Hazen, 2005). Tang et al. (2012b) reported that heat treatment of wheat peptides led peptides to gradual formation of aggregates and induce more ROS production in vivo. In this trial, the activities of MPO in serum and mucosa of broilers fed diets supplemented with SPI heated for 8 h were significantly higher than in broilers fed diets supplemented with native SPI, which implied that the production of ROS may be induced by higher activities of MPO in broilers fed diets supplemented with oxidized protein.

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of protein-based food and bioavailability of the protein (Saeed et al., 2006). Sante-Lhoutellier et al. (2007) demonstrated that oxidation of myofibrillar proteins led to a rapid decline of their susceptibility to proteolysis, and showed a direct and quantitative relationship between protein damages by hydroxyl radical and loss of protein digestibility. So, the adverse effect on growth performance of broilers fed with oxidized protein may be related to the decrease of protein digestibility in this experiment. The bursa of Fabricius and the thymus serve as the 2 primary or central lymphoid organs of the immune system. These organs are sites of B cells and T cells differentiation, respectively, which are commonly referred to as acting as humoral immunity and cellular immunity (Sharma, 1999). The spleen is the main peripheral lymphoid organ of systemic immunity in birds, and is important in disease resistance given the scarcity of avian lymph nodes (John, 1994). The relative weight of immune organs or absolute weight may reflect the level of immune function of poultry, within a certain range (nonpathological cases), with the absolute weight and relative weight increased, the cellular and humoral immune function of body were also increased (Corwin et al., 1981). Lin et al. (2000) reported that lower relative spleen weight was noted in mice fed a diet containing oxidized oil. In this study, significant decreases were found in the spleen weight of broilers at age of 21 d fed the oxidized protein diet, which is in accordance with previous findings. The decreased of spleen weight may attribute to the impairment of proliferative ability of spleen cells by oxidized protein (Lin et al., 2000). Sera of immune competent donors mainly contain antibodies of the IgG, IgA, and IgM classes. Antibodies of the IgG subclasses are most abundant, which making up about 75% of total serum antibody levels in animals and mainly synthesized by the plasma cells of spleen and lymph nodes (Manz et al., 2005). Mucosal immunity is an important part of humoral immunity and secretory IgA is the effector of mucosal immunity. It is the most prominent antibody present at mucosal surfaces, and provides passive immunoprotection against invading pathogens in the gastrointestinal tract (Gao et al.,

EFFECTS OF OXIDIZED PROTEIN ON BROILERS

In conclusion, the results of our study indicate that protein oxidation of SPI can be induced by heating, diets supplemented with SPI heated for 8 h (protein carbonyl is 22.35 nmol/mg of protein) significantly increased BW loss of broilers, and oxidized protein may negatively affect the immune function of broilers. The increases of ACTH and COR contents in serum and MPO activities in serum and mucosa imply that the stress was induced by oxidized protein. Further studies are required to evaluate the protein oxidation in feed, and the effects of oxidized protein on growth performance and health of animals.

ACKNOWLEDGMENTS

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