Optimization of the Preparation Process and Ameliorative Efficacy in Osteoporotic Rats of Peptide-Calcium Chelates from Skipjack Tuna (Katsuwonus pelamis) Meat

Abstract

This study aimed to establish the preparation process of peptide-calcium chelates (TMP-Ca) using skipjack tuna meat and investigate the function and mechanism of TMP-Ca in an osteoporosis model of rats. The results indicated that trypsin is more suitable for preparing the Ca-chelating hydrolysates of tuna meat, and the optimal hydrolysis conditions were derived as follows: digestion time 4 h, material-liquid ratio 1:10, and enzyme dose 3%. The conditions for chelating Ca with tuna meat hydrolysate were optimized to be chelation time 50 min, temperature 50 °C, pH 8.0, and a peptide-Ca ratio 1:10. The prepared hydrolysate was subjected to ultrafiltration, and the fraction (TMP) (MW <1 kDa) showed the highest Ca chelation rate (51.27 ± 1.42%) and was made into the peptide-Ca chelates (TMP-Ca). In osteoporotic rats, TMP-Ca significantly improved the decrease in ovarian indexes caused by retinoic acid. It also elevated serum Ca, phosphorus, and bone turnover indexes, increased the number of bone trabeculae, and improved bone microstructure. In addition, we confirmed that TMP-Ca could regulate the OPG/TRAF6 pathway to reduce osteoclast differentiation, inhibit bone resorption, and promote bone formation. Therefore, TMP-Ca could significantly ameliorate osteoporosis, and this study provides a functional component for the preparation of healthcare products using skipjack tuna meat to treat osteoporosis.

Keywords: meat; osteoporosis; peptide–calcium chelates; skipjack tuna (Katsuwonus pelamis).

Figure 1

Effect of different proteases (A), hydrolysis time (B), enzyme dose (C), and material–liquid ratio (D) on Ca chelation rate of tuna meat hydrolysates. All values are means ± SD (n = 3). ^a–d No significant difference between the same letters (p > 0.05).

Figure 2

Effect of chelation pH (A), time (B), temperature (C), and material to Ca ratio (D) on the Ca chelation rate of the tuna meat hydrolysates. All the values are means ± SD (n = 3). ^a–e No significant difference between the same letters (p > 0.05).

Figure 3

The Ca chelation rates of the five peptide fractions from the tuna meat hydrolysate. All the values are means ± SD (n = 3). ^a–e No significant difference between the same letters (p > 0.05).

Figure 4

Effect of TMP-Ca chelates on body weight of model rats. Blank control: 0.9% saline; model: 0.9% saline + retinoic acid; positive control (alendronate): 5 mg alendronate/kg + retinoic acid; low dose of TMP-Ca: 400 mg/kg + retinoic acid; medium dose of TMP-Ca: 600 mg/kg + retinoic acid; high dose of TMP-Ca: 800 mg/kg + retinoic acid.

Figure 5

Effect of TMP-Ca chelates on rat blood biochemical indicators. (A) Serum calcium; (B) serum phosphorus; (C) alkaline phosphatase; (D) acid phosphatase; (E) osteocalcin. All values are means ± SD (n = 8). ^*** p < 0.001 vs. blank group; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 vs. model group.

Figure 6

Effect of TMP-Ca chelates on wet weight (A) and dry weight (B) of femur and tibia in retinoic acid-induced rats. All values are means ± SD (n = 8). ^** p < 0.01 and ^*** p < 0.001 vs. blank group; ^# p < 0.05, ^## p < 0.01, vs. model group.

Figure 7

Effect of TMP-Ca chelates on the length (A) and diameter (B) of the femur and tibia in the retinoic acid-induced rats. All the values are means ± SD (n = 8). ^*** p < 0.001 vs. blank group; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 vs. model group.

Figure 8

Effect of TMP-Ca chelates on the bone calcium (A) and phosphorus (B) contents of the femur and tibia in the retinoic acid-induced rats. All the values are means ± SD (n = 8). ^** p < 0.01 and ^*** p < 0.001 vs. blank group; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 vs. model group.

Figure 9

Effect of TMP-Ca chelates on histomorphology of rat femoral head using Masson staining method. Alendronate (5 mg/kg) was used as a positive control. Yellow circles indicate bone trabeculae and epiphyses.

Figure 10

Morphological images of cross-section of femoral epiphysis using H&E staining method. Yellow circles indicate bone trabeculae and epiphyses.

Figure 11

Effect of TMP-Ca chelates on the protein expression of the OPG/TRAF6 signaling pathway. (A) The protein expression of OPG; (B) the protein expression of TRAF6. All the values are means ± SD (n = 8). ^*** p < 0.001 and ^** p < 0.01 vs. blank group; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 vs. model group.

Figure 12

Mechanism of TMP-Ca chelates to ameliorate retinoic acid-induced osteoporosis of rats. The black arrow in the figure indicates the rising or falling trend of the sample group.