| Calcium is one of the most abundant elements in the body.Calcium plays an important physiological function in the transmission of nerve impulse,blood coagulation,excitability of neuromuscle and maintenance of cell membrane function.Calcium deficiency can lead to a range of diseases,such as osteoporosis and hyperplasia in middle-aged and elderly people and menopausal women,and rickets in children.At present,there are problems such as poor absorption and utilization rate of calcium supplements in the market.Therefore,the absorption and utilization of calcium has attracted much attention.On the basis of seeking rich natural calcium sources,it is extremely urgent to develop a calcium supplement with high bioavailability.Porcine bone is rich in calcium,protein,amino acids and other nutrients,but these components exist in the form of conjugated state,difficult to be directly absorbed by the human body.Through microbial fermentation can make porcine bone nutrients released,more conducive to the absorption and utilization of the body.In this study,the physicochemical and nutritional components of porcine bone enzymes were determined and the bioavailability of calcium in porcine bone enzymes in vivo was studied.The main research contents and results were as follows:1.Determination of physicochemical components of porcine bone enzymes:It mainly included the determination of pH value of porcine bone enzymes;the total acid content was determined by pH meter potentiometric titration;HPLC-Q-Orbitrap-HRMS was used for qualitative and quantitative analysis of organic acids and methodology verification.The results showed that the pH value of the broth was4.09 and the total acid content of the broth was 0.826 g/100m L;a method for organic acid detection by high performance liquid chromatography-high resolution mass spectrometry was established,the results of methodology investigation showed that the regression curve had good linear relationship,and the method had good recovery rate,precision,repeatability and stability,only one organic acid,succinic acid,was detected in porcine bone enzymes,and its content was 1.388 g/100m L.2.Determination of nutritional components of porcine bone enzymes:It mainly included the determination of soluble protein content by coomassie bright blue method;the content of amino acid nitrogen was determined by acidity meter;HPLC-Q-Orbitrap-HRMS were used for qualitative and quantitative analysis of free amino acids and methodology verification;the content of free calcium ion was determined by EDTA titration;and the antioxidant capacity of porcine bone enzymes were evaluated in vitro.The results showed that the soluble protein content of porcine bone enzymes was0.4437 g/100m L.The content of amino acid nitrogen was 0.3681 g/100m L.A method for amino acid detection by high performance liquid chromatography-high resolution mass spectrometry was established.The results of methodology investigation showed that the regression curve had good linear relationship,and the method had good recovery rate,precision,repeatability and stability.The results showed that there were 13 kinds of amino acids in porcine bone enzymes,the content of amino acids ranged from 0.0104 mg/100m L to 1.1304 mg/100m L,including 6 kinds of essential amino acids,1 kind of semi-essential amino acid and 6 kinds of non-essential amino acids,among which the content of Ala was the highest（1.1304 mg/100m L）.The content of free calcium ion in porcine bone enzymes was 0.4091 g/100m L.Porcine bone enzymes has certain scavenging ability on DPPH free radical and hydroxyl free radical and reducing ability on Fe3+.3.Study on calcium bioavaibility of porcine bone enzymes:A calcium deficient rat model was established in four-week-old male SD rats.The calcium deficient rats were given calcium carbonate and porcine bone enzymes with the same calcium content by intragastric administration,respectively.The effects of porcine bone enzymes on calcium absorption and utilization of calcium deficient rats were evaluated by weight growth rate,calcium apparent absorption rate,serum indexes,femur weight and bone calcium content of the rats.The results of animal experiments showed that compared with inorganic calcium carbonate,porcine bone enzymes could significantly promote the weight gain of rats,significantly improve the absorption and utilization of calcium in calcium-deficient rats,restore the serum calcium,phosphorus and alkaline phosphatase to normal levels in calcium-deficient rats,and significantly increase the weight of femur and bone calcium content in calcium-deficient rats.The results showed that under the condition of similar calcium intake,the composite organic calcium formed by the combination of polypeptide and amino acid in porcine bone enzymes has better bioavailability than inorganic calcium.Porcine bone enzymes can maintain the normal development of rats,maintain the stability of the internal environment and the quality of bone calcium in rats.In summary,this study comprehensively determined the physicochemical and nutritional components of porcine bone enzymes,which can provide data basis for further development and application of porcine bone enzymes.The calcium in porcine bone enzymes has high bioavailability in rats and is expected to be a new calcium supplement product. |
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