Study on preparation of cattle bone collagen peptides-calcium chelate and its anti-osteoporosis activity

Cattle is one of the most important livestock resources globally. In 2019, the global production of beef was about 62.9 million tons, and collagen-rich bovine bones, skins, and tendons yield was up to 31.4 million tons. Protein content in cattle bone is 13.68–26.14 g/100 g (dry matter). In the EU, cattle bones have long been considered waste generated by the slaughterhouse industry and are typically disposed of in landfills or rendering plants. From economic, environmental protection, and human health perspectives, the high-value utilization of cattle bone resources is of great significance. Steam explosion has attracted attention as a method for extracting active ingredients from animal bone, due to its advantages of lower cost, lower energy usage, and environmental friendliness. Furthermore, the bioactivities of peptides-metal chelate, especially collagen peptides-calcium chelate (CPs-Ca), are poorly reported in vitro and in vivo. The objectives were: (1) to determinate the composition of cattle bone, (2) to extract collagen peptides using an eco-friendly steam explosion method, (3) to prepare collagen peptide-calcium chelate and analyze its structural characteristics and digestive stability in vitro, and (4) to evaluate its anti-osteoporotic activity through cell experiment (MC3T3-E1, mouse embryo osteoblast precursor cells) and rat animal experiment. First, 143 cattle bones were collected from eight provinces in China: Inner Mongolia, Shandong, Gansu, Henan, Shaanxi, Tibet, Sichuan, and Jilin. Sample species included yak and yellow cattle. The protein content, fat content, and elemental profile of cattle bone were determined. Sampling site included marrow bone, spine, and rib. Geographical origin and species did not show significant effect on protein content. Yak bone showed higher fat content that of yellow cattle bone. There were significant differences of protein content and fat content in different sampling sites. Meanwhile, to regulate the bone products market, the geographical origin and species of yellow cattle bone and yak bone can be discriminated by the bone elemental profiles. The combined use of multi-element analysis and Linear discriminant analysis was well-suited for the distinction of the geographic origin and species (yellow cattle vs. yak), with K-cross validation accuracies of 94.5% and 99.3%, respectively. Next, steam explosion technology was used to liquefy cattle bone for collagen peptides (CPs) extraction. The effects of experimental parameters (pressure and action time) on cattle bone liquefaction were investigated. The microstructural changes of the solid residue of the steam explosion treated bone were verified by scanning electron microscopy. Steam explosion indicated that with the increase of pressure and reaction time, the protein recovery rate and calcium/phosphorus release increased. Under 2.5 MPa-30 min, the protein recovery rate reached 60.7%. In addition, steam explosion significantly decreased the molecular weight determination of collagen peptides (P < 0.05). The results suggest that steam explosion can be used as a pretreatment method to liquefy cattle bone for environmentally friendly isolation of CPs. Cattle bone collagen peptides were applied to prepare CPs-Ca, and the preparation conditions (pH 7, temperature 60 ℃, time 60 min, peptides/CaCl2 5/1) were optimized by single factor test. The results of physical characteristics indicated that CPs-Ca was obviously different from CPs. The Fourier transform-infrared spectroscopy analysis demonstrated that chelating sites were carboxyl oxygen, hydroxyl oxygen, and amino nitrogen atom of CPs. The results of X-ray diffraction spectroscopy, scanning electron microscope, atomic force microscopy showed revealed alterations in the structure after calcium chelating. When calcium ions bind with peptides, they create links that lead to the organization of a crystal structure and the aggregation of the microstructure. According to the stability analysis, it was determined that CPs-Ca remains stable when subjected to thermal processing. The digestion experiment in vitro indicated that exposure to the gastric digestion environment may cause CPs-Ca to undergo partial dissociation. Those findings indicated that CPs-Ca has the potential function to improve calcium bioavailability as a calcium supplement. In addition, collagen peptides-calcium chelate prepared by ultrasound method (CPs-Ca-US) was successfully prepared and characterized, and the calcium-chelating ability reached 39.48 μg/mg. Compared with CPs-Ca prepared by hydrothermal method, the CPs-Ca-US did not show significant difference in structure characterizations. CPs-Ca-US showed a more stable property in gastric juice than former by digestion experiment in vitro. CPs-Ca-US could significantly promote the proliferation and mineralization of MC3T3-E1 cells (mouse embryo osteoblast precursor cells) and promote calcium absorption by interacting with calcium-sensing receptor, which regulates calcium and other metal ion homeostasis. CPs-Ca-US had a higher ability to promote the proliferation and mineralization of MC3T3-E1 cells (P > 0.05). Finally, the anti-osteoporosis bioactivity of CPs-Ca was studied in vivo (ovariectomized rat, a kind animal mode of osteoporosis). Our results indicated that dietary CPs-Ca may be effective in mitigating osteoporosis induced by estrogen deficiency, which could be due to changes in gut microbiota observed in fecal samples. For the osteoporosis rats, CPs-Ca dietary intervention increased the bone mineral density and enhanced the microstructure and changed the bone formation-related or bone resorption-related bone turnover markers levels in serum. CPs-Ca dietary intervention increased short-chain fatty acids contents in feces. In addition, CPs-Ca treatment increased the Bifidobacterium, Escherichia-Shigella, and Allobaculum, while decreased Firmicutes, and the Firmicutes/Bacteroidetes ratio at phylum level along with some specific gut microbiota community changed at genus level. Our study provided scientific evidence that CPs-Ca can be used as a functional food to treat osteoporosis.