Effects Of Soy Protein Oxidative By Heating On Broilers And The Regulation By Vitamin E And Lipoic Acid

Protein quality is closely related to its digestibility, and poor protein may negatively affect animal health. Dietary proteins are often exposed to oxidants or oxidizing conditions during processing and storage. Therefore, the proteins are vulnerable to oxidative modification because of their abundance in foods and high rate constants for reaction. This study was conducted to evaluate the effects of storing and heating on protein oxidation of soybean meal, as well as the impacts of oxidative modification of soy protein isolate (SPI) after exposure to heat on the growth performance, redox state, immune function, and apoptosis of broilers and the regulation by vitamin E and lipoic acid. The study was composed of 6 trials as below:Trial 1 was conducted to study the effects of storing on protein oxidation of soybean meal, as well as the impacts of oxidative modification of soybean meal and SPI after exposure to heat.200 g fresh soybean meal was put into the zip-lock bag and stored at room temperature for different time (1,10,20,30, and 60 d); besides,20 g fresh soybean meal or SPI was put in an oven and heated at 100 ℃ for different time (0,1,2,4, and 8 h), then the contents of amino acid, protein carbonyl, and sulfhydryl of SPI and soybean meal were measured. The results showed that the content of threonine, tyrosine, lysine, and histidine of soybean meal and SPI decreased gradually with increased heating time. Compared with fresh soybean meal (control group), protein carbonyl of soybean meal was significantly increased by storage at room temperature or dry heat treatment for different time (P<0.05); protein sulfhydryl and total disulphide/sulphydryl groups of soybean meal decreased gradually with increased storage or heating time; 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 to unheated SPI.Trial 2 was conducted to evaluate the impacts of oxidative modification of SPI after exposure to heat on the growth performance, antioxidant function, intestinal morphology, and cell apoptosis of broilers. 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 unheated SPI or 1 of the 3 heat-treated SPI (heated for 1,4, and 8 h, respectively) for 21 days. The results showed that the body weight of broilers fed diets supplemented with SPI heated for 8 h were significantly lower than that of broilers fed diets supplemented with unheated SPI (P<0.05). Compared with unheated SPI, heat-treated SPI (heated for 8 h) increased the contents of ACTH and Cor in serum, H2O2 in mucosa, and MDA in serum and mucosa of broilers at 21 d (P<0.05); and the concentrations of GSH in serum and mucosa (at 21 d) were decreased by diets supplemented with heat-treated SPI (heated for 4 and 8 h) (P<0.05). Compared with unheated SPI, a significant increased was observed in the duodenal crypt depth (at 21 d), as well as caspase-3 activity and apoptotic index in mucosa of broilers fed the heat-treated SPI (heated for 8 h) (P<0.05); moreover, the intestinal V/C value and NOS activity in jejunal mucosa (at 21 d) of broilers were diminished by diets supplemented with heat-treated SPI (heated for 8 h) (P<0.05).Trial 3 was conducted to determine the effects of oxidative modification of SPI by heating on the organ weights, immunoglobulins, cytokines, and expression of genes related to inflammatory reaction in broilers. Experiment design was the same as that of trial 2. Compared with unheated SPI, heat-treated SPI (heated for 8 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 contents of IgG in serum and duodenal mucosa of broilers (at 14 d) were decreased by diets supplemented with heat-treated SPI (heated for 8 h) (P<0.01). Compared with unheated SPI, SPI heated for 8 h increased the level of TNF-a in mucosa (P=0.063), ICAM-1 in spleen and mucosa, and IFN-γ mRNA expression in mucosa of broiler at 21 d; besides, MPO activities in serum (at 14 d) and mucosa and mRNA expression of MPO in mucosa of broilers were increased by diets supplemented with that SPI (P<0.05).Trial 4 was conducted to evaluate the protective effects of vitamin E (VE) and a-lipoic acid (LA) on growth performance, serum hormones, and intestinal morphology of broilers fed diets supplemented with heat-treated SPI for a 21-d feeding trial. A total of 300 one-day-old chickens were allocated into 5 treatment groups with 6 replicates of 10 birds:1) Unheated SPI fed basal diet (Cont); 2) Heat-treated SPI (heat for 8 h) fed diet (HSPI); 3) HSPI fed diet plus 200 mg/kg VE (VE); 4) HSPI fed diet plus 200 mg/kg LA (LA); 5) HSPI fed diet both plus 200 mg/kg VE and LA (VE+LA). Compared with Cont, HSPI treatment (HSPI group) caused adverse effects on growth performance of broilers (P>0.05), significantly increased the levels of Cor, T3, and T4 in serum as well as jejunal crypt depth (14 d) of broilers (P<0.05), and diminished the duodenal villus height at 14 d and jejunal V/C value (P<0.05). Compared with HSPI, supplementation of VE in diet significantly improved the BW, ADG, and ADFI (P<0.05), increased T4 contents in serum at 21 d and jejunal V/C value at 14 d (P<0.05), and reduced the jejunal crypt depth at 14 d (P<0.05). Dietary LA significantly reduced jejunal crypt depth at 14 d (P<0.05) as compared with HSPI. Combination of VE and LA in diet significantly improved the ADG, Cor contents in serum at 14 d, and intestinal V/C value (P<0.05), diminished jejunal crypt depth at 14 d (P<0.05), compared with HSPI.Trial 5 was conducted to evaluate the protective effects of vitamin E (VE) and a-lipoic acid (LA) on antioxidant function, Nrf2 signaling pathway, and intestinal apoptosis of broilers fed diets supplemented with heat-treated SPI. Experiment design was the same as that of trial 4. Compared with Cont, dietary HSPI significantly increased the concentrations of H2O2 and MDA in serum and liver (P<0.05), decreased the free radical scavenging capacity, CAT, SOD, and GSH-Px activity, and GSH concentrations in serum and tissues of broilers (P<0.05). Moreover, the Nrf2 signaling pathway and NF-κB in liver of birds were activated by HSPI treatment (P<0.05). Caspase-3 content and apoptotic index in mucosa of broilers in HSPI group were higher than in the Cont group (P<0.05). Compared with HSPI, supplementation of VE in diet significantly improved the free radical scavenging capacity in serum and tissues, liver CAT activity, and mucosa GSH content (P<0.05); reduced MDA content and inhibited the activation of Nrf2 and NF-κB in liver (P<0.05), and caspase-3 content and apoptotic index in mucosa were also decreased (P<0.05). Dietary LA significantly improved the hydroxyl radical scavenging capacity of liver, reduced MDA contents in serum and HO-1 protein expression in liver as compared with HSPI (P<0.05). Combination of VE and LA in diet significantly improved free radical scavenging capacity, SOD and GST activity, and GSH contents in serum and tissues (P<0.05); reduced MDA content and inhibited the activation of Nrf2 and NF-κB in liver (P<0.05) as well as caspase-3 contents and apoptotic index in mucosa (P<0.05), compared with HSPI.Trial 6 was conducted to study the protective effects of VE and LA on immune function of broilers fed diets supplemented with heat-treated SPI. Experiment design was the same as that of trial 4. Compared with Cont, dietary HSPI significantly increased the TNF-α and ICAM-1 contents and MPO mRNA expression in mucosa of broilers (P<0.05), diminished the mucosa NOS activity at 21 d (P<0.05). Compared with HSPI, supplementation of VE in diet significantly increased the IgG content in jejunal mucosa at 14 d (P<0.05), reduced the levels of TNF-a and ICAM-1 in mucosa and serum MPO activity at 21 d (P<0.05). Dietary LA significantly increased the IgG and SIgA contents and NOS activity in duodenal mucosa at 14 d (P<0.05), reduced ICAM-1 content and IFN-y mRNA expression in mucosa (P<0.05) as compared with HSPI. Combination of VE and LA in diet significantly improved the serum IgG content at 14 d and NOS activity in duodenal mucosa (P<0.05), reduced the ICAM-1 level and IFN-y mRNA expression in mucosa as well as serum MPO activity at 21 d, (P<0.05), compared with HSPI.It can be concluded as follows:(1) Protein oxidation of soybean meal increased gradually with the prolonged storage or heating time, dry heating can accelerate the oxidation of proteins in soy protein isolate.(2) Heat modification of proteins caused adverse effects on growth performance, and accelerated the metabolism in birds; dietary supplementation of 200 mg/kg VE can improve the growth performance of broilers.(3) Oxidized proteins caused adverse effects on the intestinal morphology of broilers by reducing villus height and increasing crypt depth; dietary VE or LA significantly improved the intestinal morphology of birds, the combined addition of VE and LA showed better results than when used alone.(4) Dietary supplementation of heat modification of proteins significantly decreased the antioxidant enzyme activity and free radical scavenging capacity, resulting in the accumulation of ROS in vivo, thereby activating Nrf2 signaling pathway and NF-κB; while diets supplemented with VE or LA improved the antioxidant function of broiler, thus attenuating the stimulation of ROS to Nrf2 pathway and NF-κB.(5) Heat modification of proteins caused significant negative impact on the immune function of broiler, increased the TNF-a, ICAM-1, and IFN-y levels and MPO activity, and reduced NOS activity in vivo, thereby increasing inflammation levels; dietary VE alone or in combination with LA improved the immunoglobulin contents and reduced inflammation levels in birds.(6) Caspase-3 can be activated by heat modification of proteins, and intestinal apoptosis index was also increased by oxidized protein; while diets supplemented with VE or LA significantly reduced the intestinal cell apoptosis.