The Mechanism Of Raising Bone Mineral Density Of Pigskin Gelatin Peptide And The Effect On Biomechanical Nature Of Bone

Calcium deficiency is easy to result in several kinds of disease,and children’s calcium deficiency will cause retarded growth and other diseases,while adults’calcium deficiency will lead to osteoporosis and fractures and so on.There are mainly two reasons for human body calcium deficiency:on the one hand,it is the lack of human body’s calcium absorption,on another hand,human body’s insufficient bone collagen content make the calcium which has been ingested and absorbed can not be fixed in the body.Therefore,it is important to supply both the mineral element calcium and bone collagen.At present,calcium preparations in the domestic market from the composition of calcium source are divided into two main categories which are inorganic calcium and organic acid calcium,and the absorption of these two calcium preparations are dependent on the stomach acidification and vitamin D induction.These two kinds of calcium preparations also have some defects,for instance,low bioavailability,a certain side effects and antagonism among body elements,etc.,therefore,to develop a new type of calcium supplement as soon as possible is very necessary.Collagen polypeptide biological calcium preparation can provide rich calcium for human body,and its absorption and utilization process does not rely on the induction of vitamin D,moreover,the rich polypeptide can promote the formation of bone collagen in human body.This paper by taking pigskin gelatin as raw materials studies the mechanism of pigskin collagen polypeptide on raising bone mineral density（BMD）as well as the effect of pigskin collagen polypeptide on bone biomechanical properties.1.The protein content in food grade pigskin gelatin was determined by the method of the Kjeldahl,and the protein content was90.75%.Amino acid composition of pigskin gelatin was analyzed by the automatic amino acid analyzer,and the pigskin gelatin was rich in a variety of essential amino acids.The results showed that the pigskin gelatin contained 16 kinds of amino acids.2.We chose 3 different typical kinds of protease to hydrolyze the pigskin gelatin under the optimal conditions,and the double enzyme system made up of pepsin and acid protease was used to optimize the hydrolysis process.The optimal hydrolysis process was A₂B₃C₁D₂,and the acid protease hydrolysis temperature was 50℃,and the hydrolysis solution p H was 6.0.The pepsin hydrolysis temperature was 45℃,and the hydrolysis solution p H was 3.0.And the hydrolysis degree of pigskin gelatin was 17.56%.SDS-PAGE of alkaline protease enzyme product,pepsin enzyme product,acid protease enzyme prodcut and double enzyme hydrolysis system enzyme product were carried out to find out that the relative ratio of molecular less than 5KDa in double enzyme hydrolysis system enzyme product was the highest.3.The combination between collagen polypeptide and calcium gluconate was studied by measuring chelate rate,and the chelate rate of collagen polypeptide chelated calcium（CP-Ca）could reach 78.3%.The structureofCP-Cawascharacterizedbytheultraviolet（UV）spectroscopy,infrared spectroscopy（IR）,X-ray diffraction（XRD）and scanning electron microscope（SEM）.The research confirmed the chelating reaction between calcium ions and collagen polypeptide,and the coordination reaction between calcium and carboxyl/amino group of collagen polypeptide occurred.4.Thecombination betweenL-Hydroxyproline（L-Hyp）and calcium gluconate was studied to prepare L-Hydroxyproline-Ca（L-Hyp-Ca）,and the chelate rate of L-Hyp-Ca was 79.5%.The composition and structure of L-Hyp-Ca were characterized by the UV,IR,SEM,XRD,elemental analysis and gravimetry.And the chelating reaction between Ca and carboxyl and imino groups in L-Hyp to form L-Hyp-Ca with a coordination ratio of 1:2,whose molecular formula was established as Ca（C₅H₈NO₃）₂.5.The physiological activity of CP-Ca and L-Hyp-Ca was investigated.CP-Ca and L-Hyp-Ca were able to remove DPPH·,·OH and O^2-·,implying their antioxidant activity.Calcium absorption and transport test showed that CP-Ca and L-Hyp-Ca exhibited promoting effects on calcium transport rate of Caco-2 cells.Under the simulated stomach environment,part of the Ca²⁺in the chelated complex were released to free ions.However,under the simulated intestinal tract environment,the increase of p H led to the further chelation between Ca ions and peptide.The Ca could be absorbed in the form of free ions or chelated complex,which improved its digestibility.Cell survival test showed that:CP-Ca and L-Hyp-Ca had no poisonous effect on Caco-2cells,and they were safe.6.Theeffectsofpigskingelatin（PG）,pigskingelatin hydrolysate（PGH）,collagen polypeptide（CP）on the raising of bone mineral density（BMD）were investigated by female SD rats.The body weight and length of the rats were recorded weekly.3 days calcium metabolization experiment was carried out in the fourth week to estimateapparentabsorption.Serumcalcium（S-Ca）,serum phosphorus（S-P）,andalkalinephosphatese（ALP）,bonemineral density（BMD）and bone calcium content（BMC）were measured.The results showed that PG,PGH,CP-1（M>5KDa）and CP-2（M<5KDa）could increase the body weight of rats,and increase the growth of the rats’bone,and decrease the activity of ALP,and increase rats’calcium apparent absorption rate,and they were playing a role of increasing calcium absorption in rats.The group of CP-2+Ca CO₃ more obviously decreased the ALP activity of rats and reduced it by 40.37%when comparing with the control group.CP-2 had the effect of promoting rats’bone growth and development,and its effect was more prominent than other groups.7.This paper studied the effect of collagen hyhrolysate calcium on the increasing of rats’BMD with weaned female SD rats as models.During the experiment,we weekly measured these rats’body weight and body length,performed 3 days of calcium metabolism test in the fourth week after intragastric administration,and measured the levels of S-Ca,S-P,ALP,BMD and BMC of the rats,and made SEM analysis on rats’femur.The results showed that the middle dose group CP-Ca,high dose group CP-Ca and high dose group CP+Ca CO₃ could significantly facilitaterats’weightdevelopment（p<0.05）,decreaseALP activity（p<0.05）and increase BMD（p<0.05）.The calcium apparent absorption rate of group CP-Ca and group CP+Ca CO₃ were respectively higher than that of group Ca CO₃.Compared to control groups,the high dose group CP-Ca increased rats’weight and length by 99.92%,decreased ALP activity by 48.66%,increased BMD by 15.06%and increased rats’bone calcium content by 18.47%,so CP-Ca plays an active role in promoting rats’physical development,bone growth and bone calcium deposition.SEM results showed that CP-Ca and CP+Ca CO₃ could prevent osteoporosis caused by long-term insufficient calcium intake in rats.The analysis of rats’femur biomechanics properties found that the high dose group CP-Ca improved rats’bone structure mechanics characteristic parameters such as the femoral stiffness,maximum load,absorbed energy,maximum stress and elastic modulus by 14.27%,8.17%,17.91%,14.43%and 12.10%respectively.When the rats had intake CP and Ca²⁺,it could increase the mechanical strength of the bone.8.After calcium intake,the rats mainly absorbed it through intestinal tract,and a small amount of calcium was absorbed through stomach,and there was still part of（35%）unabsorbed calcium excreted directly in the form of feces.The absorbed calcium entered the rats’blood and intercellular fluid through active transport and passive diffusion.Part of calcium was then delivered to the soft tissue and cells of the body,part of calcium was transported to bone tissues and deposited in these tissues,and part of it（25%）was excreted by kidney metabolism and glomerular filtration.Through the analysis of calcium transport pathways in rats,it was found that the target of calcium absorption in rats was the intestinal tissue,and the target of calcium transport medium in rats was mainly rats’blood.What’s more,the rats’calcium deposition target was mainly rats’bone tissue,and there were also a small amount of calcium deposition in rats’heart,liver,spleen,brain and other organizations.