Effects of Long-Term Protein Restriction on Meat Quality, Muscle

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Effects of long-term protein restriction on meat quality, muscle amino acids, and amino acid transporters in pigs Jie Yin, Yuying Li, Xiaotong Zhu, Gang Liu, Xingguo Huang, Rejun Fang, tiejun li, and Yulong Yin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02746 • Publication Date (Web): 02 Oct 2017 Downloaded from http://pubs.acs.org on October 2, 2017

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Journal of Agricultural and Food Chemistry

20-18-16% CP Meat quality

17-15-13% CP

14-12-10% CP

ACS Paragon Plus Environment


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Effects of long-term protein restriction on meat quality, muscle amino acids, and amino acid transporters in pigs

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Jie Yin1,2, Yuying Li1,2, Xiaotong Zhu1, Hui Han1,2, Wenkai Ren1,2, Shuai Chen1,2, Peng Bin1,2, Gang Liu1*, Xingguo Huang3,4, Rejun Fang3,4, Bin Wang5, Kai Wang6, Liping Sun6, Tiejun Li1,4*, Yulong Yin1,4*

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Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences; National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production; Hunan Provincial Engineering Research Center for Healthy Livestock and Poultry Production; Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Changsha, Hunan 410125, P. R. China; 2 University of Chinese Academy of Sciences, Beijing 100039, China; 3 Department of Animal Science, Hunan Agriculture University, Changsha, Hunan 410125, China 4 Hunan Co-Innovation Center of Animal Production Safety, Changsha, Hunan 410128, China 5 School of Food, Jiangsu Food & Pharmaceutical Science College, Higher Education Park in Huaian, Jiangsu Province, P. R. China 223005 6 Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093, China

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Jie Yin and Yuying Li contributed equally to this study.

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*Corresponding authors:

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Gang Liu, [email protected];

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Tiejun Li, [email protected];

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Yulong Yin, [email protected]

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ABSTRACT: This study aimed to investigate the long-term effects of protein restriction from piglets to finishing pigs for 16 weeks on meat quality, muscle amino acids, and amino acid transporters. 39 piglets were randomly divided into three groups: a control (20-18-16% crude protein, CP) and two protein restricted group (17-15-13% CP and 14-12-10% CP). The results showed that severe protein restriction (14-12-10% CP) inhibited feed intake and body weight, while moderate protein restriction (17-15-13% CP) had little effect on growth performance in pigs. Meat quality (i.e. pH, color traits, marbling, water-holding capacity, and shearing force) were tested and the results exhibited that 14-12-10% CP treatment markedly improved muscle marbling score and increased yellowness (b*). pH value (45min) was significant higher in 17-15-13% CP group than that in other groups. In addition, protein restriction reduced muscle histone, arginine, valine, and isoleucine abundances and enhanced glycine andlysine concentrations compared with the control group, while the RT-PCR results showed that protein restriction downregulated amino acids transporters. Mechanistic target of rapamycin (mTOR) signaling pathway was inactivated in moderate protein restricted group (17-15-13% CP), while severe protein restriction with dietary 14-12-10% CP markedly enhanced mTOR phosphorylation. In conclusion, long-term protein restriction affected meat quality and muscle amino acid metabolism in pigs, which might be associated with mTOR signaling pathway.

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KEYWORDS: Protein restriction, meat quality, amino acids, pig

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INTRODUCTION

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Protein restriction is an effective strategy for preventing diseases and promoting health span via regulating nutrient-signaling pathways in human and animals (1). In piglets challenged with an enterotoxigenic strain of Escherichia coli, dietary a low-protein diet reduces indices of protein fermentation and the incidence of post-weaning diarrhea in weaned pigs (2). In adult pigs, protein restriction alters the composition of gut microbiota and improves intestinal barrier function (3). Our previous reports have suggested that dietary protein restriction involves in lipid, protein, and energy metabolism in skeletal muscle and the mechanism is mechanistic target of rapamycin (mTOR) signaling pathway-dependent (4-6). Proteomic profiling of skeletal muscles identified 132 differentially expressed proteins in response to protein restriction in piglets and these proteins were mapped in various nutritional metabolic pathways, including lipid, carbohydrate, and amino acids (7). Thus, we anticipated that muscle also serves as a major target in response to protein restriction in pigs and nutritional metabolism in muscle is highly associated with meat quality. Meat quality has exerted a crucial role in human evolution and is an important component of a healthy and well-balanced diet due to its nutritional richness. Meanwhile, protein restriction has been reported to exhibit a beneficial role in metabolic regulation, which may be further associated with muscle metabolism and



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meat quality. However, current references are limited to understand to the effect of protein restriction on meat quality in pigs. In this study, long-term effect of protein restriction from piglets to finishing pigs for 16 weeks was investigated and the results showed that protein restriction affected meat quality and muscle amino acid metabolism.

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MATERIALS AND METHODS

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Animals and groups

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39 male piglets (Landrace×Large White) with average body weight (11.34+0.70kg) were randomly divided into three groups: a control group in which piglets received a basal diet according to the NRC 1998 and two protein restricted groups (Table 1). The experiment lasts for 16 weeks and assigns into three periods: piglet (5 weeks), growing pig (5 weeks), and finishing pig (16 weeks). At week 10 and 16, 6-7 pigs were randomly sacrificed for sample collection. Each animal was fed in independent pens (same size) for 3 times per day and free to drinking water.

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Growth performance

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Feed intake in each pen was recorded daily and average feed intake in every week was calculated. Body weight were weighed at 0, 5, 10, and 16 weeks. Carcass length and backfat thickness were tested in finishing pigs to evaluate carcass traits.

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Meat quality

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Samples of the longissimus thoracis (LT) muscle were taken from the area of the last thoracic vertebrae to analyze the quality of carcasses. Meat acidity was measured 45 min and 24 h after slaughter with a pH meter (Matthaus pH Star, Germany).

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Meat color traits (lightness L*, redness a*, and yellowness b*) were measured by reflectance spectrophotometer Minolta CR-410 (Kinica Minolta Sensing Inc., Osaka, Japan);

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Marbling scores (ranging from 0 to 3 with 0 = devoid and 3 = overly abundant) were subjectively evaluated.

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Water-holding capacity were tested using pressing loss (PL). Briefly, approximately 20 g samples were weighed before being covered with gauze and filter papers and placed into Tenove Meat-1 (Beijing, China) for 5 min, then reweighed. The difference between the initial and final weights was used to calculate water-holding capacity: PL=(Initial weight – Final weight)/Initial weight * 100%.

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Muscle shearing force was determined via GR-150 Warner-Bratzler Shear Machine


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(Shakopee, USA). Briefly, steaks were cooked by placing in a water bath to reach an internal temperature of 70 °C. After cooling, a minimum of five meat pieces, 1 × 1 × 1 cm (height × width × length) parallel to the muscle fiber direction were sheared in the Warner-Bratzler cell. The maximal force recorded during shearing (at rupture time) was used as the shear force value and expressed in Newtons. Averages were calculated to determine the shear force value per sample.

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Blood biochemical parameters

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At week 5, 10, 15, blood samples were harvested from anterior vena cava and serum were separated after centrifugation at 300 rpm for 10 min under 4 °C. Cobas c-311 coulter chemistry analyzer was used to test serum biochemical parameters, including blood urea nitrogen (BUN) (04460715190), triglyceride (TG) (20767107322), cholesterol (CHOL) (03039773190), low density lipoprotein (LDL) (03038866322), high density lipoprotein (HDL) (04399803190), and NH3L (20766682322).

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Muscle amino acids

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0.5 g muscle samples were homogenized with 5 ml 0.01M hydrochloric acid and centrifuged at 5000 rpm for 5 min, then 0.5 ml supernatants were mixed with 8 % salicylsulfonic acid for one night under 4 °C. The mixtures were further centrifuged at 12000 rpm for 10 min for twice. The final supernatants were used for amino acids analysis using High-speed Amino Acid Analyzer L-8900 (Japan).

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Real-time quantitative (RT-PCR)

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Total RNA from muscle samples was isolated from liquid nitrogen frozen and ground tissues with TRIZOL regent (Invitrogen, USA) and then treated with DNase I (Invitrogen, USA) (8). The reverse transcription was conducted at 37°C for 15 min, 95°C 5 sec. Primers used in this study were designed via Primer 5.0 according to mouse and pig gene sequence (Table 2). β-actin was chosen as the house-keeping gene to normalize target gene levels. The PCR cycling condition was 36 cycles at 94°C for 40 sec, 60 °C for 30 sec and 72°C for 35 sec. The relative expression was expressed as a ratio of the target gene to the control gene using the formula 2-(∆∆Ct), where ∆∆Ct=(CtTarget-Ctβ-actin)treatment-(CtTarget-Ctβ-actin)control (9, 10). Relative expression was normalized and expressed as a ratio to the expression in the control group.

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Western blot

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Total proteins from muscle samples were extracted using protein extraction reagents (Thermo Fisher Scientific Inc., Waltham, MA, USA) (11) and 30 ug proteins were separated by a reducing SDS-PAGE electrophoresis. Then the proteins were transferred onto a PVDF membrane (Millipore, MA, USA) and blocked with 5% non-fat milk in Tris-Tween buffered saline buffer for 1.5 hour. Then the membranes



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were incubated with primary antibodies: β-actin (60008-1, 1:5000, Proteintech), mTOR (20657-1-AP, 1:500, Proteintech), p-mTOR (ab137133, 1:2000, Abcam), AMPK (#2603, 1:1000, CST), p-AMPK (#2535, 1:1000, CST), CAT1 (sc-66824, 1:500, Santa), CAT2 (sc-87036, 1:100, Santa), EAAT1 (22515-1-AP, 1 µg/ml, Proteintech), and EAAT2 (ab41751, 1：500, Abcam). The HRP-conjugated secondary antibodies were subsequently incubated for 2 hours at room temperature. Then the membrane developed the blots using Alpha Imager 2200 software (Alpha Innotech Corporation, CA, USA). We digitally quantified the resultant signals and normalized the data to GAPDH abundance.

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Statistical Analysis

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All data were performed by using the one-way analysis of variance (ANOVA) to test homogeneity of variances via Levene’s test and followed with student’s T test (IBM SPSS 23 software) (12, 13). Data are expressed as the mean ± SEN. Values in the same row with different superscripts are significant (P < 0.05).

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RESULTS

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Effect of protein restriction on growth performance in pigs

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From week 5 to 16, severe protein restriction with dietary 14-12-10% CP significantly reduced body weight in piglets, growing, and finishing pigs (P0.05). Daily feed intake was recorded and the results showed that severe protein restriction with dietary 14-12-10% CP markedly inhibited feed intake from week 1 to 13 (P0.05), indicating a potential of protein restriction to improve meat quality.

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In finishing pigs, 14-12-10% CP diet tended to reduce backfat thickness (16.81±2.73) compared with the control group (23.42±2.17) (P>0.05) (Table 3), which may be associated with the reduced body weight. Middle protein restriction with dietary 17-15-13% CP significantly decreased meat pH (45 min) compared with the severe protein restriction and control groups (P

Effects of Long-Term Protein Restriction on Meat Quality, Muscle

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