Method for judging effect of improving osteoporosis by intervention of various livestock and poultry bone collagen peptides and application of method

Abstract

The invention discloses a method for judging the effect of improving osteoporosis through intervention of bone collagen peptides from various livestock and poultry sources and application of the method, and belongs to the technical field of nutritional and functional foods.By establishing an oxidative stress injury mouse MC3T3-E1 osteoblast model, bone collagen peptides capable of improving proliferative activity are screened, and the osteoporosis improvement effect of bone collagen peptides from various livestock and poultry sources is achieved. The effects of the compound on cell differentiation activity, mineralization activity, cycle regulation and apoptosis inhibition are detected; the improvement effect is analyzed, and an in-vitro activity comprehensive scoring model is established; synchronously detecting the content of bone conversion factors in the cell culture supernatant; carrying out an in-vivo animal experiment on the screening peptide, and detecting the content of animal serum bone conversion factors and thighbone morphological mechanics indexes; and if the correlation coefficient R2 of the cell supernatant and the change rate of the animal serum bone conversion factor is greater than or equal to 0.85, determining that the model is effective and comprehensively judging the activity difference. According to the method, the biological activity difference is efficiently judged, a screening basis is provided for functional bone-derived food development, and high-value utilization of livestock and poultry bone byproducts is promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of nutrition and functional food, and specifically relates to a method for determining the effect of various livestock and poultry-derived collagen peptides on improving osteoporosis and its application. Background Art

[0002] Osteoporosis, a systemic bone metabolic disease characterized by microstructural destruction and bone mass loss, is a global public health concern. Currently, approximately 93 million people in my country suffer from osteoporosis, and it is projected to reach nearly 200 million worldwide by 2050, with treatment costs expected to reach US$131.5 billion. Clinically, osteoporosis treatment primarily relies on chemical medications, including mineralization promoters (calcium supplements, vitamin D, etc.), bone resorption inhibitors (bisphosphonates, calcitonin, estrogens, etc.), and bone formation promoters (fluoride, raloxifene, etc.). However, these medications are associated with numerous side effects, including the induction of cartilage calcification, gastrointestinal side effects, and cardiovascular disease. Consequently, dietary interventions using natural food-derived alternatives (such as bone polypeptides / osteopolysaccharides) that can promote bone formation and reverse bone structural damage have attracted increasing attention.

[0003] The meat processing industry produces a large amount of collagen-rich livestock bone byproducts annually, including yak, cattle, pig, and chicken bones. Studies have demonstrated that dietary intervention with collagen peptides can increase the regularity and firmness of the collagen fiber network and promote the orderly deposition of calcium hydroxyapatite, thereby increasing bone strength and density. Consequently, collagen peptides have been widely used in the development of functional bone-derived foods. However, the cost of livestock bone raw materials from different species varies significantly (for example, the market price of yak bone is as high as 8,000 yuan / ton, cattle bone is approximately 4,000-5,000 yuan / ton, pig bone is approximately 4,000 yuan / ton, and chicken bone is approximately 2,000 yuan / ton), resulting in significant differences in the production costs of collagen peptides from various livestock and poultry species. Although a certain degree of research has been conducted on the anti-osteoporosis activity and mechanism of action of collagen peptides from various species, it is still unclear whether there are significant differences in the anti-osteoporosis activity of collagen peptides from various species. There are great limitations and one-sidedness in the research of collagen peptides, which greatly restricts the rational and high-value development and utilization of related collagen peptide products. Therefore, it is necessary to establish a comparative determination strategy for the differences in the anti-osteoporosis effects of various collagen peptides to provide a basis for the development and utilization of functional bone-derived foods and the rational and high-value development and utilization of livestock and poultry bone by-products. Summary of the Invention

[0004] An object of embodiments of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages to be described hereinafter.

[0005] Another object of the present invention is to provide a method for determining the effects of various livestock and poultry-derived collagen peptides on the intervention and improvement of osteoporosis, clarify the differences in the anti-osteoporosis effects of various livestock and poultry-derived collagen peptides, and facilitate more reasonable and high-value development and utilization of collagen peptides into corresponding products.

[0006] In order to solve the above-mentioned purpose, the present invention adopts the following technical solutions to achieve the goal: The method for determining the effect of various livestock and poultry-derived collagen peptides on improving osteoporosis includes the following steps: Establish a mouse MC3T3-E1 osteoblast cell model of oxidative stress damage; Bone collagen peptides from various livestock and poultry species are used to improve the proliferation activity of MC3T3-E1 osteoblasts damaged by oxidative stress, thereby obtaining bone collagen peptides capable of improving the proliferation activity of MC3T3-E1 osteoblasts damaged by oxidative stress and the amount of the peptides used; The researchers used collagen peptides that can improve the proliferation activity of MC3T3-E1 osteoblasts damaged by oxidative stress, and the dosage of the peptides was used to improve the MC3T3-E1 osteoblasts damaged by oxidative stress. The differentiation activity, mineralization activity, cycle effect, and apoptosis inhibition were then tested. To analyze the effects of various livestock and poultry-derived collagen peptides on the differentiation activity, mineralization activity, cycle, and apoptosis inhibition of MC3T3-E1 osteoblasts damaged by oxidative stress, and to establish an in vitro comprehensive activity scoring model; In addition, the content of bone turnover factors in the cell culture supernatant was simultaneously detected to screen for collagen peptides that increased B-ALP by ≥20% and decreased TRACP by ≥15%; Conduct in vivo animal experiments on the screened collagen peptides to detect the bone turnover factor content in animal serum and femoral morphological mechanical indicators; If the correlation coefficient R between the change rate of bone turnover factor in cell culture supernatant and the change rate of bone turnover factor in animal serum is 2 If the value is ≥0.85, the in vitro activity comprehensive scoring model is considered valid, and the difference in collagen peptide activity is comprehensively determined based on it.

[0007] Preferably, the method for determining the effect of multiple livestock and poultry-derived collagen peptides on improving osteoporosis further comprises the following steps: obtaining in vitro activity comprehensive scoring model data, comprehensively evaluating the differences in the anti-osteoporosis activities of the multiple livestock and poultry-derived collagen peptides, and the comprehensive evaluation further comprises: combining in vivo animal experimental data, including serum bone conversion factor content and bone morphomechanical indicators of right femoral samples, The in vivo animal experimental data were normalized using principal component analysis (PCA) to generate a quantitatively comparable anti-osteoporosis activity difference index, including: Integrate in vivo animal experimental data and in vitro activity comprehensive scoring model data into a multidimensional dataset; Normalization: The Z-score normalization method was used to normalize the integrated data set to eliminate the bone density g / cm 3 , the dimension difference of bone volume fraction%, serum factor concentration ng / mL indicators; PCA dimensionality reduction: Perform PCA analysis on the normalized data to extract the principal components and meet the following requirements: the variance contribution rate of the first principal component PC1 is ≥ 60%, ensuring that PC1 carries the core variation information; Index generation: Anti-osteoporosis activity difference index was calculated based on PC1 score; The calculation formula is: Index = PC1 score × 100, which quantitatively ranks the activity of collagen peptides from different sources. The dosage used in animal experiments is based on the FAO and WHO recommended human protein / peptide intake of 0.83 g / kg⋅bw / day. The optimal dosage for animal experiments is calculated based on the relationship between the surface area or body weight of rats and humans.

[0008] Preferably, in the method for determining the effect of multiple livestock and poultry-derived collagen peptides on the intervention and improvement of osteoporosis, mouse MC3T3-E1 osteoblasts are treated with H2O2 at a concentration of 0.2 mM to construct an MC3T3-E1 osteoblast model of oxidative stress damage; The CCK-8 method was used to detect the improvement effect of collagen peptides from various livestock and poultry species on the proliferation activity of MC3T3-E1 osteoblasts damaged by oxidative stress; ALP staining was used to detect the improvement effect of collagen peptides from various livestock and poultry species on the differentiation activity of MC3T3-E1 osteoblasts damaged by oxidative stress; The improvement effect of collagen peptides from various livestock and poultry species on the mineralization activity of MC3T3-E1 osteoblasts damaged by oxidative stress was detected by Alizarin Red S staining. The effects of collagen peptides from various livestock and poultry species on the cell cycle of MC3T3-E1 osteoblasts damaged by oxidative stress were detected by propidium iodide labeling combined with flow cytometry. Annexin V-FITC / PI double staining was used to detect the inhibitory effects of collagen peptides from various livestock and poultry species on the apoptosis of MC3T3-E1 osteoblasts damaged by oxidative stress. Among them, when comprehensively evaluating the differences in the anti-osteoporosis activity of various livestock and poultry-derived collagen peptides, the ALP activity value was used as the differentiation ability evaluation index, the calcium nodule staining intensity and mineralization area were used as the mineralization ability evaluation index, the S phase and G2 / M phase cell ratio was used as the proliferation activity evaluation index, and the total proportion of early and late apoptotic cells was used as the anti-apoptosis ability evaluation index. The differentiation ability evaluation index, mineralization ability evaluation index, proliferation activity evaluation index and anti-apoptosis evaluation index were weighted and integrated to establish an in vitro activity comprehensive scoring model. Combined with the in vivo animal experimental data, the anti-osteoporosis activity difference index of collagen peptides from various livestock and poultry species was generated.

[0009] Preferably, in the method for determining the effect of multiple livestock and poultry-derived collagen peptides on improving osteoporosis, the method for obtaining the in vivo animal experimental data includes: A bilaterally ovariectomized osteoporosis model was established in female SD rats. Collagen peptides from various sources were administered orally for a period of time. Serum bone turnover factor levels and bone morphomechanical parameters of right femoral samples were then measured. The serum bone turnover factor content, including bone formation markers and bone resorption markers, was determined using a fully automatic serum biochemical analyzer. The bone formation markers included alkaline phosphatase, bone γ-carboxyglutamate-containing protein, and type I procollagen C-peptide. The bone resorption markers included tartrate-resistant acid phosphatase, serum type I collagen C-terminal peptide, and urine deoxypyridinoline. Micro-CT was used to measure the bone morphomechanical parameters of the right femoral specimens, including bone mineral density, bone volume fraction, trabecular number, and trabecular spacing.

[0010] Preferably, in the method for determining the effect of multiple livestock and poultry-derived collagen peptides on improving osteoporosis, The concentration of collagen peptide that can significantly improve the proliferation activity of MC3T3-E1 osteoblasts damaged by oxidative stress is determined by the following method: The CCK-8 method was used to detect the effects of various livestock and poultry collagen peptides at concentration gradients of 0.05, 0.1, 0.5, 1.0, and 5.0 mg / mL on cell proliferation activity; Screening for the lowest effective concentration that increases cell proliferation activity by ≥30% compared to the negative control group and has a statistically significant difference; When the minimum effective concentrations of various collagen peptides were different, 1.0 mg / mL was used as the unified test concentration for subsequent in vitro experiments.

[0011] Preferably, in the method for determining the effect of multiple livestock and poultry-derived collagen peptides on the improvement of osteoporosis, a bilateral ovariectomized osteoporosis model is established in female SD rats, and multiple source collagen peptides are administered by gavage for a period of time. The method used is: Three-month-old female SD rats underwent bilateral ovariectomy. After 4 weeks of postoperative recovery, they were given 1000 mg / kg of collagen peptides from various livestock and poultry species by oral gavage daily for 12 weeks.

[0012] Preferably, in the method for determining the effect of multiple livestock and poultry-derived collagen peptides on improving osteoporosis, In vivo animal experiments include the following steps: Establishment of a bilaterally ovariectomized osteoporosis model in female SD rats: 3-month-old female SD rats underwent bilateral ovariectomy. Serum estradiol levels and body weight recovery were monitored weekly for 4 weeks after surgery. The model was considered successfully established when serum estradiol levels were <15 pg / mL and body weight recovered to 95% ± 3% of the preoperative level. Determining the intervention dose of collagen peptides: The CCK-8 method was used to detect the improvement effect of each collagen peptide on the proliferation activity of MC3T3-E1 osteoblasts damaged by oxidative stress, and the minimum effective concentration (MEC) that increased cell activity by ≥30% compared with the negative control group and p < 0.05 was screened; According to the body surface area conversion formula, rat dose = human dose × 6.2, the MEC of each collagen peptide was converted into an equivalent oral dose; The patients were given an equivalent dose of collagen peptide by oral gavage daily, and serum bone-specific alkaline phosphatase B-ALP was measured every 2 weeks; If the B-ALP test value increases by ≥20% compared with the baseline for two consecutive times, the intervention will be terminated, with a minimum period of ≥8 weeks; otherwise, the intervention will be extended to 16 weeks.

[0013] Preferably, in the method for determining the effect of multiple livestock and poultry-derived collagen peptides on intervention to improve osteoporosis, the multiple livestock and poultry-derived collagen peptides include yak bone collagen peptide YBPs, cattle bone collagen peptide BBPs, pig bone collagen peptide PBPs and chicken bone collagen peptide CBPs, and the concentration used is 1.0 mg / mL.

[0014] The application is to screen out reasonably high-valued collagen peptide raw materials by comparing the differences in anti-osteoporosis activity of collagen peptides from various livestock and poultry sources, and to prepare functional foods or drugs for improving osteoporosis.

[0015] Preferably, in the application, the reasonably high-valued collagen peptide raw material is yak bone collagen peptide YBPs or cattle bone collagen peptide BBPs.

[0016] Compared with the prior art, the advantages and beneficial technical effects of the present invention are: There is a very significant correlation between osteoporosis and estrogen deficiency and high free radical oxidation levels in the body. Therefore, the construction of appropriate in vitro and in vivo test models will help to objectively and scientifically simulate and restore the systematic and dynamic regulatory process of bone collagen peptide intervention to improve osteoporosis symptoms, and more accurately determine the differences in the effects of bone collagen peptide intervention to improve osteoporosis. The present invention provides a comparative determination strategy for the differences in the anti-osteoporosis effects of bone collagen peptides from various livestock and poultry species, including the differences in in vivo and in vitro anti-osteoporosis activity, providing a basis for the development and utilization of functional bone-derived foods and the rational high-value development and utilization of livestock and poultry bone by-products.

[0017] Other advantages, objectives, and features of the embodiments of the present invention will be reflected in part through the following description, and in part will be understood by those skilled in the art through study and practice of the embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the viability of MC3T3-E1 osteoblasts in an oxidative stress injury model established by low-concentration H2O2 treatment in one of the schemes of the present invention.

[0019] Figure 2 This is a graph showing the improvement of oxidative stress-damaged MC3T3-E1 osteoblast viability detected by using the CCK-8 method in one of the solutions of the present invention.

[0020] Figure 3 This is a diagram showing the effects of various collagen peptides on improving the differentiation of MC3T3-E1 osteoblasts damaged by oxidative stress detected by the ALP staining method in one of the schemes of the present invention.

[0021] Figure 4 This is a diagram showing the effects of various collagen peptides on improving the mineralization of MC3T3-E1 osteoblasts damaged by oxidative stress detected by the Alizarin Red S staining method in one of the schemes of the present invention, wherein 4A shows the effects of various collagen peptides on improving the mineralization of MC3T3-E1 osteoblasts damaged by oxidative stress detected by the Alizarin Red S staining method (14 days), and 4B shows the effects of various collagen peptides on improving the mineralization of MC3T3-E1 osteoblasts damaged by oxidative stress detected by the Alizarin Red S staining method (21 days).

[0022] Figure 5 This is a diagram showing the cell cycle effects of various collagen peptides on MC3T3-E1 osteoblasts in improving oxidative stress damage using propidium iodide (PI) labeling combined with flow cytometry in one of the solutions of the present invention.

[0023] Figure 6This is a diagram showing the effect of Annexin V-FITC / PI double staining in detecting the apoptosis effect of multiple collagen peptides on MC3T3-E1 osteoblasts damaged by oxidative stress in one of the solutions of the present invention.

[0024] Figure 7 This is a diagram showing the effects of multiple collagen peptides in one of the schemes of the present invention on improving the levels of serum bone turnover markers in ovariectomized SD rats, wherein 7A shows the effects of multiple collagen peptides on improving the levels of serum bone-specific alkaline phosphatase in ovariectomized SD rats, 7B shows the effects of multiple collagen peptides on improving the levels of serum type I procollagen N-peptide in ovariectomized SD rats, 7C shows the effects of multiple collagen peptides on improving the levels of serum bone γ-carboxyglutamate protein in ovariectomized SD rats, 7D shows the effects of multiple collagen peptides on improving the levels of serum tartrate-resistant acid phosphatase in ovariectomized SD rats, 7E shows the effects of multiple collagen peptides on improving the levels of serum type I collagen C-terminal peptide in ovariectomized SD rats, and 7F shows the effects of multiple collagen peptides on improving the levels of serum urinary deoxypyridinoline in ovariectomized SD rats.

[0025] Figure 8 This is a diagram showing the effects of multiple collagen peptides in one of the schemes of the present invention on improving the bone morphomechanical indicators of the right femoral samples of ovariectomized SD rats, wherein 8A shows the effects of multiple collagen peptides on improving the bone density of the right femoral samples of ovariectomized SD rats, 8B shows the effects of multiple collagen peptides on improving the bone surface area-tissue volume ratio of the right femoral samples of ovariectomized SD rats, 8C shows the effects of multiple collagen peptides on improving the number of trabeculae in the right femoral samples of ovariectomized SD rats, and 8D shows the effects of multiple collagen peptides on improving the trabecular separation of the right femoral samples of ovariectomized SD rats.

[0026] Figure 9 These are three-dimensional reconstruction images of the right femoral sample of ovariectomized SD rats improved by multiple bone collagen peptides in one embodiment of the present invention. 9A shows a three-dimensional reconstruction image of the right femoral sample of the sham operation group (SHAM) ovariectomized SD rats, 9B shows a three-dimensional reconstruction image of the right femoral sample of the model group (OVX-M) ovariectomized SD rats, 9C shows a three-dimensional reconstruction image of the right femoral sample of the positive control group (OVX-P) ovariectomized SD rats, 9D shows a three-dimensional reconstruction image of the right femoral sample of the yak bone collagen peptide group (OVX-YBPs) ovariectomized SD rats, 9E shows a three-dimensional reconstruction image of the right femoral sample of the cattle bone collagen peptide group (OVX-YBPs) ovariectomized SD rats, 9F shows a three-dimensional reconstruction image of the right femoral sample of the porcine bone collagen peptide group (OVX-YBPs), and 9G shows a three-dimensional reconstruction image of the right femoral sample of the chicken bone collagen peptide group (OVX-YBPs) ovariectomized SD rats.

[0027] Figure 10 This is a judgment model for the effect of multiple collagen peptides intervening to improve osteoporosis in one of the schemes of the present invention. 10A shows the principal component analysis (PCA) of the effect of multiple collagen peptides intervening to improve osteoporosis, and 10B shows the partial least squares discriminant analysis (PLS-DA) of the effect of multiple collagen peptides intervening to improve osteoporosis. DETAILED DESCRIPTION

[0028] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and accompanying drawings. The specific embodiments described herein are only used to explain the present invention, not to limit the present invention.

[0029] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0030] The present invention provides a method for determining the difference in anti-osteoporosis effects of various livestock and poultry-derived collagen peptides, comprising the following steps: Step (1) low concentration H2O2 treatment was used to establish an MC3T3-E1 osteoblast model of oxidative stress injury; Step (2) CCK-8 assay was used to detect the differences in the effects of various collagen peptides from different sources on the proliferation of MC3T3-E1 osteoblasts damaged by oxidative stress; Step (3) ALP staining was used to detect the differences in the effects of various collagen peptides from different sources on improving the differentiation of MC3T3-E1 osteoblasts damaged by oxidative stress; Step (4) Alizarin Red S staining was used to detect the differences in the mineralization effects of various bone collagen peptides on MC3T3-E1 osteoblasts damaged by oxidative stress; Step (5) Propidium iodide (PI) labeling combined with flow cytometry was used to detect the differences in the effects of various bone collagen peptides on improving the cell cycle of MC3T3-E1 osteoblasts damaged by oxidative stress; Step (6) Annexin V-FITC / PI double staining was used to detect the differences in the apoptosis effects of various collagen peptides from different sources on MC3T3-E1 osteoblasts damaged by oxidative stress; Step (7) bilateral ovariectomy of female SD rats was performed to establish an osteoporosis rat model; Step (8) using a fully automatic serum biochemical analyzer to determine the serum bone turnover marker content, and analyzing the effects of various bone collagen peptides on the serum bone turnover marker content; Step (9) Micro-CT method is used to measure the bone morphomechanical indicators of the right femoral sample.

[0031] like Figures 1 to 10As shown, the present invention provides a strategy for comparing and determining the differences in the anti-osteoporosis effects of various livestock and poultry-derived collagen peptides, the method comprising an in vitro cell test: establishing an MC3T3-E1 osteoblast model with oxidative stress damage by treating with low concentrations of H2O2; detecting the differences in the proliferation effects of various bone collagen peptides from different sources on improving the oxidative stress-damaged MC3T3-E1 osteoblasts by using a CCK-8 method; detecting the differences in the differentiation effects of various bone collagen peptides from different sources on improving the oxidative stress-damaged MC3T3-E1 osteoblasts by using an ALP staining method; detecting the differences in the mineralization effects of various bone collagen peptides from different sources on improving the oxidative stress-damaged MC3T3-E1 osteoblasts by using an Alizarin Red S staining method; detecting the differences in the cell cycle effects of various bone collagen peptides from different sources on improving the oxidative stress-damaged MC3T3-E1 osteoblasts by using a propidium iodide (PI) labeling method combined with flow cytometry; and detecting the differences in the proliferation and apoptosis effects of various bone collagen peptides from different sources on improving the oxidative stress-damaged MC3T3-E1 osteoblasts by using Annexin V-FITC / PI double staining. The method includes in vivo animal experiments: bilateral ovariectomy of female SD rats is performed to establish an osteoporosis rat model; serum bone turnover marker levels are measured using a fully automatic serum biochemical analyzer, and the effects of various bone collagen peptides from different sources on serum bone turnover marker levels are analyzed; and bone morphomechanical indicators of right femoral samples are measured using the Micro-CT method.

[0032] In the above scheme, preferably, in step (1), MC3T3-E1 osteoblasts are treated with low concentration of H2O2 to establish an MC3T3-E1 osteoblast model of oxidative stress damage.

[0033] In the above scheme, preferably, in step (1), the H2O2 concentration is determined to be 0.2 mM, so as to take into account the low concentration and cause certain oxidative stress damage to MC3T3-E1 osteoblasts for subsequent experiments.

[0034] In the above scheme, preferably, in step (2), 0.2 mM H2O2 treatment is used to establish an MC3T3-E1 osteoblast model of oxidative stress damage.

[0035] In the above scheme, preferably, in step (2), the concentration of the four collagen peptides YBPs, BBPs, PBPs, and CBPs is determined to be 1.0 mg / mL for subsequent experiments.

[0036] In the above scheme, as a preference, in the steps (3), (4), (5) and (6), the treatment groups are divided into 7 groups: a blank control group (without any treatment), a negative control group (treated with 0.2 mM H2O2), a positive control group (treated with 0.2 mM H2O2+10 μM VC), a yak bone collagen peptide group (YBPs group, treated with 0.2 mM H2O2+1.0 mg / mL YBPs), a cattle bone collagen peptide group (BBPs group, treated with 0.2 mM H2O2+1.0 mg / mL BBPs), a pig bone collagen peptide group (PBPs group, treated with 0.2 mM H2O2+1.0 mg / mL PBPs), and a chicken bone collagen peptide group (CBPs group, treated with 0.2 mM H2O2+1. 0 mg / mL CBPs).

[0037] In the above scheme, preferably, in step (7), the osteoporosis model of the female SD rats is established by bilateral ovariectomy to cause estrogen deficiency in the rats to establish the osteoporosis model.

[0038] In the above scheme, preferably, in step (8), the serum bone turnover markers include: osteocalcin, serum bone-specific alkaline phosphatase, serum type I procollagen N-terminal propeptide, serum tartrate-resistant acid phosphatase, serum type I collagen C-terminal peptide cross-linking and urine deoxypyridinoline.

[0039] In the above embodiment, preferably, in step (9), the morphomechanical indicators include: trabecular density (BMD), bone volume fraction (bone volume / total volume), trabecular spacing, trabecular thickness, trabecular number, and cortical thickness. In the above embodiment, preferably, the animal is a rat.

[0040] To enable those skilled in the art to better understand the technical solution of the present invention, the following description is made using yak bone collagen peptides (YBPs), cattle bone collagen peptides (BBPs), pig bone collagen peptides (PBPs), and chicken bone collagen peptides (CBPs) prepared by the inventors as research objects: A method for comparing and determining the differences in the anti-osteoporosis effects of various livestock and poultry-derived collagen peptides comprises the following main steps: Step (1) low concentration H2O2 treatment was used to establish an MC3T3-E1 osteoblast model of oxidative stress injury; Step (2) CCK-8 assay was used to detect the differences in the effects of various collagen peptides on improving the proliferation of MC3T3-E1 osteoblasts damaged by oxidative stress; Step (3) ALP staining was used to detect the differences in the effects of various collagen peptides on improving the differentiation of MC3T3-E1 osteoblasts damaged by oxidative stress; Step (4) Alizarin Red S staining was used to detect the difference in the mineralization effects of various collagen peptides on MC3T3-E1 osteoblasts damaged by oxidative stress; Step (5) Propidium iodide (PI) labeling combined with flow cytometry was used to detect the differences in the effects of various collagen peptides on improving the cell cycle of MC3T3-E1 osteoblasts damaged by oxidative stress; Step (6) Annexin V-FITC / PI double staining was used to detect the differences in the apoptosis effects of various collagen peptides on MC3T3-E1 osteoblasts damaged by oxidative stress; Step (7) bilateral ovariectomy of female SD rats was performed to establish an osteoporosis rat model; Step (8) using a fully automatic serum biochemical analyzer to determine the serum bone turnover marker content, and analyzing the effects of various collagen peptides on the serum bone turnover marker content; Step (9) Micro-CT was used to measure the bone morphomechanical indicators of the right femoral sample.

[0041] 1. Establishment of an MC3T3-E1 osteoblast cell model of oxidative stress injury The viability of MC3T3-E1 osteoblasts in each group was determined using the CCK-8 method. MC3T3-E1 osteoblasts were cultured at a density of 5 × 10 3 The cells were seeded at a density of 10 cells / well in a 96-well plate and then incubated at 37°C in a 5% CO2 environment for 24 hours. The cells were then incubated with different concentrations of H2O2 (0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 mM) for 24 hours to establish an oxidatively damaged MC3T3-E1 osteoblast model. 10 μL of CCK-8 solution was added to each well and incubated for 1 hour. The absorbance of each well was measured at 450 nm. MC3T3-E1 osteoblasts were then plated at a density of 5×10 3 Cells were seeded per well in a 96-well plate and incubated with 0.2 mM H₂O₂ and varying concentrations of four collagen peptides (0.05, 0.1, 0.5, 1.0, and 5.0 mg / mL) for 24 hours. 10 μL of CCK-8 solution was added to each well and incubated for 1 hour. The absorbance of each well was measured at 450 nm. MC3T3-E1 osteoblasts without H2O2 and bone collagen peptide treatment were defined as the blank control group, MC3T3-E1 osteoblasts treated only with H2O2 (0.2 mM) were defined as the negative control group, MC3T3-E1 osteoblasts treated with H2O2 (0.2 mM) and VC (10 μM) were identified as the positive control group, and MC3T3-E1 osteoblasts treated with H2O2 (0.2 mM) and various livestock and poultry bone collagen peptides (1.0 mg / mL) were identified as the various bone collagen peptide treatment group.

[0042] 2. MC3T3-E1 osteoblast differentiation The differentiation level of MC3T3-E1 osteoblasts was quantitatively determined by ALP staining. MC3T3-E1 osteoblasts were cultured at a density of 2 × 10 4 The cells were seeded at a density of 1000 / well in a 24-well plate and then divided into seven groups: blank control group (untreated), negative control group (treated with 0.2 mM H2O2), positive control group (treated with 0.2 mM H2O2+10 μM VC), yak bone collagen peptide group (YBPs group, treated with 0.2 mM H2O2+1.0 mg / mL YBPs), cattle bone collagen peptide group (BBPs group, treated with 0.2 mM H2O2+1.0 mg / mL BBPs), porcine bone collagen peptide group (PBPs group, treated with 0.2 mM H2O2+1.0 mg / mL PBPs), and chicken bone collagen peptide group (CBPs group, treated with 0.2 mM H2O2+1. 0 mg / mL CBPs). MC3T3-E1 osteoblasts were cultured in α-MEM medium at 37°C under 5% CO2, refreshed every 2-3 days until the MC3T3-E1 osteoblasts reached 80% confluency. α-MEM medium was then replaced with osteoblast differentiation medium (α-MEM supplemented with 10% FBS, 1% penicillin-streptomycin, 1% glutamine, 50 μg / mL ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone) to initiate cell differentiation and incubated for 72 hours. Following incubation, cells were treated as described above for 24 hours. The osteoblast differentiation medium was removed, and the cells were washed three times with PBS and then fixed at 4°C for 15 minutes. ALP staining was performed by adding 200 μL of 4% paraformaldehyde solution to each well. Cells were washed three times with PBS, and images of differentiation were captured using an inverted microscope. Cells were then completely lysed with 0.1% Triton X-100 in the dark. Cell lysates were centrifuged at 15,000 × g for 5 minutes to collect the supernatant, and intracellular ALP activity was assessed using the p-nitrophenyl phosphate (pNPP) assay. 200 μL of pNPP was added to each well and incubated for 30 minutes. During this time, ALP hydrolyzed pNPP to p-nitrophenol (pNP). Under alkaline conditions, the yellow soluble product exhibited a maximum absorption peak at 405 nm. Therefore, the absorbance at 405 nm was recorded and represented ALP activity.

[0043] 3. MC3T3-E1 osteoblast mineralization The mineralization degree of MC3T3-E1 osteoblasts was quantitatively determined by Alizarin red staining. MC3T3-E1 osteoblasts were cultured at 2×10 4The cells were seeded at a density of 1000 / well in a 24-well plate and then divided into seven groups: blank control group (untreated), negative control group (treated with 0.2 mM H2O2), positive control group (treated with 0.2 mM H2O2+10 μM VC), yak bone collagen peptide group (YBPs group, treated with 0.2 mM H2O2+1.0 mg / mL YBPs), cattle bone collagen peptide group (BBPs group, treated with 0.2 mM H2O2+1.0 mg / mL BBPs), porcine bone collagen peptide group (PBPs group, treated with 0.2 mM H2O2+1.0 mg / mL PBPs), and chicken bone collagen peptide group (CBPs group, treated with 0.2 mM H2O2+1. 0 mg / mL CBPs). MC3T3-E1 osteoblasts were cultured in α-MEM medium at 37°C under 5% CO2, refreshed every 2-3 days, until the cells reached 80% confluency. α-MEM was then replaced with osteoblast mineralization medium (α-MEM supplemented with 10% FBS, 0.1% bovine serum albumin, 50 μg / mL ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone) to initiate mineralization and incubated for 14 and 21 days, with the medium refreshed every 2-3 days. After 14 or 21 days of culture, the cells were treated accordingly for 24 hours. The treated cells were washed three times with PBS and then fixed by adding 200 μL of 4% paraformaldehyde solution to each well at 4°C for 15 minutes. The fixed MC3T3-E1 osteoblasts were then stained with Alizarin Red S (pH 4.2) at 37°C for 30 minutes. The cells were then gently washed with PBS to remove unbound dye, and mineralization images were captured using an inverted microscope. The cells were then desorbed with 200 μL of 10% cetylpyridinium chloride for 1 h in the dark to quantify the bound dye, and the concentration of dissolved Alizarin Red S was measured at 570 nm.

[0044] 4. MC3T3-E1 osteoblast cell cycle Propidium iodide (PI) labeling was used to monitor the cell cycle changes of MC3T3-E1 osteoblasts. MC3T3-E1 osteoblasts were cultured at a density of 2 × 10 4Cells were seeded at a density of 100 μg / well in 24-well plates and then divided into seven groups: blank control (untreated), negative control (treated with 0.2 mM H2O2), positive control (treated with 0.2 mM H2O2 + 10 μM VC), yak bone collagen peptide group (YBPs group, treated with 0.2 mM H2O2 + 1.0 mg / mL YBPs), bovine bone collagen peptide group (BBPs group, treated with 0.2 mM H2O2 + 1.0 mg / mL BBPs), porcine bone collagen peptide group (PBPs group, treated with 0.2 mM H2O2 + 1.0 mg / mL PBPs), and chicken bone collagen peptide group (CBPs group, treated with 0.2 mM H2O2 + 1.0 mg / mL CBPs). MC3T3-E1 osteoblasts were cultured in α-MEM medium at 37°C and 5% CO2 for 72 h. After incubation, cells were washed three times with ice-cold PBS, fixed with 75% ethanol at 4°C for 12 h, and then washed again three times with ice-cold PBS. DNA was stained using a propidium iodide kit and analyzed by flow cytometry.

[0045] 5. Apoptosis of MC3T3-E1 osteoblasts The apoptosis of MC3T3-E1 osteoblasts was detected by Annexin V-FITC / PI double staining. 4 Cells were seeded at a density of 100 cells / well in 24-well plates and then divided into seven groups: blank control (untreated), negative control (treated with 0.2 mM H2O2), positive control (treated with 0.2 mM H2O2 + 10 μM VC), yak bone collagen peptide group (YBPs group, treated with 0.2 mM H2O2 + 1.0 mg / mL YBPs), bovine bone collagen peptide group (BBPs group, treated with 0.2 mM H2O2 + 1.0 mg / mL BBPs), porcine bone collagen peptide group (PBPs group, treated with 0.2 mM H2O2 + 1.0 mg / mL PBPs), and chicken bone collagen peptide group (CBPs group, treated with 0.2 mM H2O2 + 1.0 mg / mL CBPs). MC3T3-E1 osteoblasts were cultured in α-MEM medium at 37°C and 5% CO2 for 72 h. After incubation, cells were washed three times with pre-chilled PBS, trypsinized, and centrifuged at 100 × g for 5 min. The cells were collected into centrifuge tubes. The cells were washed again three times with pre-chilled PBS, and 500 μL of 1× binding buffer was added to each tube to resuspend the cells and stain them. Cell apoptosis was detected using the Annexin V-FITC apoptosis kit according to the manufacturer's protocol.

[0046] 6. Animal Experiment Design Fifty-six SPF female Sprague-Dawley rats (3 months old, 285.27 ± 16.59 g) were housed in a clean, free-feeding environment with a 12 h light / 12 h dark cycle, a constant temperature of 25 ± 2 °C, and a relative humidity of 50–70% throughout the experimental period. After one week of acclimation, rats were randomly divided into seven groups (8 per group): sham group (bilateral laparotomy, gavage with a certain volume of normal saline solution, generally 1-2 mL / 100 g body weight), model group (bilateral ovariectomy, same volume of normal saline solution), ES group (bilateral ovariectomy, 50 μg / kg 17β-estradiol), YBPs group (bilateral ovariectomy, 1000 mg / kg YBPs), BBPs group (bilateral ovariectomy, 1000 mg / kg BBPs), PBPs group (bilateral ovariectomy, 1000 mg / kg PBPs), and CBPs group (bilateral ovariectomy, 1000 mg / kg CBPs). Rats were anesthetized with an intraperitoneal injection of 40 mg / kg sodium pentobarbital before surgery. After 4 weeks of postoperative recovery, rats were gavaged daily with equal volumes of normal saline or YBPs, BBPs, PBPs, or CBPs solutions for 12 consecutive weeks, depending on their assigned group.

[0047] 7. Rat sample collection Body weight and dietary (food and water) intake were measured weekly. At week 12 of the dietary intervention, all rats were sacrificed after a 12-hour fast. Blood was collected and centrifuged at 2000 × g for 15 minutes to obtain serum. After cleaning the soft tissue, the right femur was immediately dissected and fixed in 10% formalin. Viscera, including the kidneys, spleen, liver, and uterus, were collected and weighed.

[0048] 8. Determination of bone turnover factors (BTMs) in rat serum ELISA kits were used to measure the serum BTM levels in SD rats. Biochemical markers of bone formation included alkaline phosphatase (B-ALP), bone γ-carboxyglutamate-containing protein (BGP), and type I procollagen C-peptide (PICP); and biochemical markers of bone resorption included tartrate-resistant acid phosphatase (TRACP), serum type I collagen C-terminal peptide (S-CTX), and urinary deoxypyridinoline (U-DPD).

[0049] 9. Micro-CT A quantitative analysis of trabecular microarchitecture of the right femur was performed using an X-ray scanner system. The right femur was scanned distally at the femoral metaphysis (growth plate) using scanning parameters of 10 μm resolution, 80 kV voltage, 200 μA current, and a 0.5 mm Alpha filter. The osteocyte growth zone (approximately 1 mm below the distal femoral growth plate) was skipped, and 300 slices (approximately 3 mm long) were selected as the region of interest (ROI) for 3D reconstruction. Scanned X-ray images were reconstructed using CTAN (version 1.17.7.2) and CTvox (version 3.30) to obtain 3D visualization. Trabecular morphology was characterized by measuring bone morphometric parameters, including bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp). Parameter name Abbreviation unit meaning bone density Tb.BMD <![CDATA[g / cm 3 ]]> Bone mineral density in bone tissue Bone volume fraction (bone volume / total volume) BV / TV % The ratio of bone tissue volume to tissue volume can directly reflect changes in bone mass Trabecular thickness Tb.Th μm Average thickness of trabecular bone in the region of interest (ROI) Trabecular number Tb.N 1 / mm The average number of intersections between bone tissue and non-bone tissue in the region of interest (ROI) per mm Trabecular spacing Tb.Sp μm Average width of the medullary cavity between trabeculae cortical bone thickness Cw.T mm Average thickness of cortical bone in the region of interest

[0050] Results and Analysis 1. Establishment of MC3T3-E1 osteoblast model of oxidative stress injury by low concentration H2O2 treatment Osteoporosis in postmenopausal women (estrogen deficiency) and the elderly (aging) is closely related to bone metabolism imbalance caused by the accumulation of free radicals in the body. The oxidative stress MC3T3-E1 osteoblast injury model constructed by low-concentration H2O2 has been widely used to simulate bone biological processes in vivo. Therefore, a low-concentration H2O2 solution (0, 100, 200, 400, 600, 800, 1000, 2000 μM) was used to construct an oxidative stress MC3T3-E1 osteoblast injury model. The viability of MC3T3-E1 osteoblasts ranged from 100±5.35% to 46.65±1.86%, with a significant dose-dependent effect. However, when the concentration of H2O2 solution exceeded 200 μM, the decrease in MC3T3-E1 osteoblast viability was not significant, as shown in Figure 2. Figure 1 As shown, 200 μM H2O2 was selected to establish the oxidative stress MC3T3-E1 cell injury model.

[0051] 2. CCK-8 assay to detect the differences in the effects of various collagen peptides on the proliferation of MC3T3-E1 osteoblasts damaged by oxidative stress like Figure 2 As shown in the figure, compared with the blank control group (CN), 200 μM H2O2 treatment significantly reduced the viability of MC3T3-E1 cells (P<0.01), but the positive control group VC treatment significantly reversed the decrease in MC3T3-E1 cell viability induced by H2O2 treatment ( P<0.01), and CBPs (0.05-5.00 mg / mL, 63.86±5.03%-110.39±5.39%) also increased the survival rate of MC3T3-E1 osteoblasts. At a concentration of 1.00 mg / mL, YBPs (105.93±6.55%), BBPs (96.85±5.28%), PBPs (83.56±3.67%), and CBPs (92.33±4.35%) showed extremely significant reversal effects compared with the negative control group (60.16±5.62%) and the 0.05 mg / mL collagen peptide treatment group (63.86±5.03%-68.59±2.35%) (P<0.01). However, the difference was not significant compared with the MC3T3-E1 osteoblast survival rate in the 5 mg / mL collagen peptide-treated group (86.59±3.85%-110.39±5.39%) ( P >0.05). Therefore, a concentration of 1.00 mg / mL of collagen peptide was selected for the subsequent MC3T3-E1 osteoblast assay.

[0052] 3. ALP staining method was used to detect the differences in the effects of various collagen peptides on improving the differentiation of MC3T3-E1 osteoblasts damaged by oxidative stress High ALP activity is an indicator of good early differentiation of MC3T3-E1 osteoblasts, indicating enhanced bone formation and bone matrix mineralization. Figure 3 As shown in the results of ALP staining, the ALP activity of MC3T3-E1 osteoblasts under oxidative stress (negative control group, 0.09±0.01) was significantly decreased compared with that of CN group (blank control group, 0.21±0.04) (P<0.01). The ALP activity of VC-treated group (positive control group, 0.23±0.04) and four collagen peptide-treated groups (0.12±0.02-0.18±0.02) was also significantly improved compared with the negative control group ( P The results showed that the four collagen peptides significantly improved the expression of ALP in MC3T3-E1 osteoblasts damaged by oxidative stress. The ALP activities in the YBPs-treated group (0.18±0.02) and the BBPs-treated group (0.16±0.03) were higher than those in the CBPs-treated group (0.14±0.03) and the PBPs-treated group (0.12±0.02).

[0053] 4. Alizarin Red S staining was used to detect the differences in the mineralization effects of various collagen peptides on MC3T3-E1 osteoblasts damaged by oxidative stress Alizarin Red S can chelate with calcium ions to form red or purple complexes, reflecting the mineralization level of MC3T3-E1 osteoblasts. Alizarin Red staining experiments were performed on MC3T3-E1 osteoblasts on days 14 and 21. Figure 4 4A (14 days) and Figure 4 As shown in Figure 4B (21 days), MC3T3-E1 osteoblasts in the negative control group exhibited the lowest mineralization levels (14 days, 0.98±0.03; 21 days, 1.21±0.04). After intervention with VC and four collagen peptides, mineralization levels improved at 14 and 21 days, indicating that collagen peptide intervention alleviated oxidative stress damage and reversed the decrease in mineralization levels induced by H2O2 treatment in MC3T3-E1 osteoblasts. The study found that collagen peptides and their metal chelation activated the Wnt / β-catenin signaling pathway and promoted the expression of Frizzled-5β-catenin, Wnt5a, and GSK-3β. Part of β-catenin translocated from the cytoplasm to the nucleus and bound to T-cell factor / enhancer of lymphoid enhancer factor (TCF / LEF) to recruit coactivators, leading to the upregulation of differentiation- and mineralization-related target genes such as Runt-related transcription factor 2 (Runx2) and osteoprotegerin (OPG), thereby promoting differentiation and mineralization. Quantification of mineralization levels (14 and 21 days) showed that YBPs (14 days, 1.25 ± 0.13; 21 days, 2.01 ± 0.19) and BBPs (14 days, 1.31 ± 0.09; 21 days, 1.88 ± 0.20) increased the mineralization level of MC3T3-E1 osteoblasts after treatment, followed by CBPs (14 days, 1.13 ± 0.02; 21 days, 1.49 ± 0.08) and PBPs (14 days, 1.06 ± 0.06; 21 days, 1.33 ± 0.10).

[0054] 5. Propidium iodide (PI) labeling combined with flow cytometry to detect the differences in the cell cycle effects of various collagen peptides on MC3T3-E1 osteoblasts injured by oxidative stress Promoting the proliferation of osteoblasts (synthesizing new bone matrix) or inhibiting the proliferation of osteoclasts (eroding cavities) are both beneficial to osteogenesis. After Annexin V-FITC / PI double staining, live cells, apoptotic cells, and necrotic cells can be distinguished based on the cell status detected by flow cytometry. After 72 hours of different interventions on MC3T3-E1 osteoblasts treated with H2O2, as shown in Figure 2, the difference between the two groups is not significant. Figure 5As shown, the apoptosis rate (Q2+Q3) of MC3T3-E1 cells was significantly reduced from 8.35±0.19% (negative control group) to 2.6±0.04% (positive control group). With the exception of the PBPs group (4.79±0.77%), the apoptosis rates in the YBPs group (3.78±0.27%), BBPs group (3.85±0.35%), and CBPs group (4.01±0.12%) also returned to levels not significantly different from the blank control group (3.44±0.37%) (P<0.05). These results suggest that treatment with collagen peptides can reduce apoptosis in MC3T3-E1 osteoblasts and promote osteogenesis. YBPs and BBPs showed the strongest inhibitory effect on apoptosis in MC3T3-E1 osteoblasts, followed by CBPs and then PBPs.

[0055] 6. Annexin V-FITC / PI double staining to detect the difference in the apoptosis effects of various collagen peptides on MC3T3-E1 osteoblasts damaged by oxidative stress The greater the ratio of S phase + G2 / M phase (DNA and histone synthesis phase) in the cell, the higher the cell proliferation activity. Figure 6 As shown, the negative control group (36.65±0.85%) had the smallest proportion of cells in the S+G2 / M phase. The proportion of cells in the S+G2 / M phase increased in the VC-treated group (42.11±0.81%) and the four collagen peptide-treated groups (38.86%-41.57%) compared to the blank control group (41.94±0.74%). The highest proportion was observed in the YBPs-treated group (41.57±0.46%), followed by the BBPs group (40.95±2.15%), the CBPs group (39.49±0.51%), and the PBPs group (38.86±0.74%). These results suggest that collagen peptides accelerate the cell cycle of MC3T3-E1 osteoblasts, thereby inhibiting their apoptosis. Among them, YBPs, BBPs and CBPs have more significant regulatory effects than PBPs.

[0056] 7. Use a fully automatic serum biochemical analyzer to measure the levels of serum bone turnover markers and analyze the effects of various collagen peptides on the levels of serum bone turnover markers in rats Serum bone turnover markers (BTMs) are products of the body's own synthesis and decomposition of bone tissue. They have great potential for early screening of osteoporosis, assessment of fracture risk, and monitoring of the therapeutic effects of patients after taking medication. According to the type of action, they are divided into bone resorption markers (reflecting osteoclast activity and bone resorption levels) and bone formation markers (osteoblast and bone formation status). Bone formation markers: bone-specific alkaline phosphatase (B-ALP), type I procollagen C-peptide (PICP), type I procollagen N-peptide (PINP) and bone-γ-carboxyglutamate protein (BGP); bone resorption markers: serum type I collagen C-terminal peptide (S-CTX), urine deoxypyridinoline (U-DPD) and serum tartrate-resistant acid phosphatase (TRAP), which comprehensively evaluate the dynamic changes in the body's bone metabolism. Figure 7 As shown in Figures AF, the serum B-ALP content of SD rats in the model group (OVX-M group) was significantly lower than that in the SHAM group, indicating that the ovariectomized osteoporosis rat model was successfully established. The serum bone formation markers (B-ALP, PINP, BGP) of rats in the estradiol intervention group (OVX-P group) and the four collagen peptide intervention groups were higher than those in the OVX-M group ( P <0.05), the levels of serum bone resorption markers (S-CTX, U-DPD, TRAP) in rats in the OVX-P group and the four collagen peptide intervention groups were decreased ( P <0.05). These results suggest that the four collagen peptides simultaneously promote bone formation and reduce bone resorption in vivo, and YBPs may be more effective.

[0057] 8. Use Micro-CT to measure the biomechanical indicators of the right femur of rats and examine the effects of various collagen peptides on the morphological and mechanical indicators of the femur of rats The rat bone morphomechanical indices obtained by Micro-CT of the right femoral trabeculae ( Figure 8 8A-8D) image 3D reconstruction ( Figure 9 9A-9G). Figure 8 As shown in Figures 8A-8D, the morphometric results showed that Tb.BMD, BS / TV, and Tb.N in the OVX-M group were significantly lower than those in the SHAM group ( P <0.05), but Tb.Sp in the OVX-M group was significantly increased compared with the SHAM group. As expected, Tb.BMD, BV / TV, and Tb.N were significantly increased in the four collagen peptide intervention groups and the estrogen intervention group compared with the OVX-M group, but Tb.Sp was significantly decreased ( P <0.05). Figure 9As shown in Figures 9A-9H, compared with the SHAM and OVX-P groups, the trabeculae in the OVX-M group were sparse, with large gaps and obvious cavitation, further confirming the successful establishment of the ovariectomized osteoporosis rat model. At the same time, after 12 weeks of intervention with estradiol or four collagen peptides, significant improvements in trabecular microstructure (trabecular density, thickness, and continuity) were observed compared with the OVX-M group. In summary, a certain degree of improvement and recovery of bone microstructure parameters in ovariectomized osteoporosis rats was observed through collagen peptide intervention, although some of them were not statistically significant ( P >0.05). The dosage for animal experiments was based on the FAO and WHO recommended human protein / peptide intake of 0.83 g / kg⋅bw / day, and the optimal dosage for animal experiments was calculated based on the relationship between the surface area or body weight of rats and humans.

[0058] ALP activity was used as the differentiation ability evaluation index, calcium nodule staining intensity and mineralization area as the mineralization ability evaluation index, the S phase and G2 / M phase cell ratio was used as the proliferation activity evaluation index, and the total proportion of early and late apoptotic cells was used as the anti-apoptosis ability evaluation index. The differentiation ability evaluation index, mineralization ability evaluation index, proliferation activity evaluation index and anti-apoptosis evaluation index were weighted and integrated. After standardization, the four indicators were assigned weights (40% for differentiation, 30% for mineralization, 20% for proliferation, and 10% for anti-apoptosis). An in vitro activity comprehensive scoring model was established. Combined with in vivo animal experimental data, including serum bone turnover factor content and bone morphomechanical indicators of right femoral samples, the model was evaluated.

[0059] The principal component analysis and (orthogonal) partial least squares discriminant analysis methods were combined to normalize the in vitro and in vivo data, and a direct determination method for the difference in the effect of various livestock and poultry collagen peptides on improving osteoporosis was established. The in vitro and in vivo test data and the Micro-CT three-dimensional reconstruction of the right femur of the test animals were combined to calculate the difference index of the anti-osteoporosis activity of the collagen peptides. Figure 10 shown.

[0060] Principal Component Analysis (PCA): Dimensionality reduction and normalization were performed on the in vitro scores of multiple indicators (weighted 70%) and the in vivo data (serum indicators 25%, bone morphology indicators 5%) to generate eigenvectors. Linear classification was then performed to extract the principal components. The PCA score plot displays the main two- or three-dimensional scatter plots, with the horizontal and vertical axes indicating the percentage of variation explained by the principal components. The contributions of PC1 and PC2 were 72.2% and 12.0%, respectively, for a cumulative total of 84.2%. This indicates that PC1 and PC2 contain the majority of the information in the original data and can comprehensively reflect the differences in the effects of osteoporosis intervention with collagen peptides from various livestock and poultry species, demonstrating good qualitative discrimination.

[0061] Partial least squares discriminant analysis (PLS-DA): The in vitro scores of multiple indicators (weight 70%) and in vivo data (serum indicators 25%, bone morphology indicators 5%) were reduced in dimension and linearly fitted to establish a regression model, optimizing the classification effect by maximizing the differences between groups and minimizing the differences within groups. The horizontal and vertical coordinates (X-axis) in the PLS-DA score graph usually represent the first principal component (PLS-DA1), which explains the largest part of the variation in the data, 72%. The vertical coordinate (Y-axis) in the PLS-DA score graph represents the second principal component (PLS-DA2), which explains the minor variation in the data, 10%. These percentages reflect the contribution of each principal component to the data variation, and can reflect the overall information of the differences in the effects of various livestock and poultry source bone collagen peptides on improving osteoporosis, with good qualitative discrimination effect. The R of the PLS-DA model 2 Y =0.94, Q 2 =0.748, indicating that the model has no significant effect on the variables Y The explanation rate is 94.0%, and Q 2 >0.50, indicating that the model has a good fit and high predictive ability; the permutation test results show that R 2 The intercept is 0.352, Q 2 The intercept is -1.42, indicating that the model has good predictive ability, is effective and usable, and does not suffer from overfitting.

[0062] Activity difference index calculation: Combining the in vitro and in vivo test data with the Micro-CT three-dimensional reconstruction of the right femur of the experimental animals, the comprehensive scores of YBPs, BBPs, PBPs and CBPs were calculated to be 83.9, 71.5, 53.5 and 42.8, indicating that YBPs has the best anti-osteoporosis activity.

[0063] The present invention preferably normalizes the in vivo and in vitro data, uses a two-dimensional PCA score graph obtained by principal component analysis, and a two-dimensional PLS-DA score graph obtained by partial least squares discriminant analysis, to establish a direct determination method for the differences in the effects of various livestock and poultry source bone collagen peptides intervening to improve osteoporosis, and combines the in vivo and in vitro test data to calculate the difference index of the anti-osteoporosis activity of various livestock and poultry source bone collagen peptides. The combination of the determination method and the difference index can mutually confirm the discrimination results of the differences in the effects of various livestock and poultry source bone collagen peptides intervening to improve osteoporosis, and further refine the discrimination results.

[0064] As described above, in order to compare and determine the differences in the anti-osteoporosis effects of various collagen peptides, the present invention is based on the construction of an MC3T3-E1 cell model of oxidative stress damage, the CCK-8 method to detect the differences in the proliferation effects of various collagen peptides on MC3T3-E1 osteoblasts damaged by oxidative stress, the ALP staining method to detect the differences in the differentiation effects of various collagen peptides on MC3T3-E1 osteoblasts damaged by oxidative stress, the Alizarin Red S staining method to detect the differences in the mineralization effects of various collagen peptides on MC3T3-E1 osteoblasts damaged by oxidative stress, the propidium iodide (PI) labeling method combined with flow cytometry to detect the differences in the cell cycle effects of various collagen peptides on MC3T3-E1 osteoblasts damaged by oxidative stress, and the Annexin V-FITC / PI double staining was used to detect the differences in the effects of various collagen peptides on the apoptosis of MC3T3-E1 osteoblasts damaged by oxidative stress. An osteoporosis rat model was established by bilateral ovariectomy in female SD rats. The serum bone turnover marker levels were determined using a fully automatic serum biochemical analyzer. The effects of various collagen peptides on the serum bone turnover marker levels were analyzed. The bone morphological mechanical indicators of right femoral samples were determined by Micro-CT method. The differences in the anti-osteoporosis activities of YBPs, BBPs, PBPs and CBPs were comprehensively determined and compared, providing a basis for the development and utilization of functional bone-derived foods and the rational and high-value development and utilization of livestock and poultry bone by-products.

[0065] Example 1: Step 1: Establishing a cell model of oxidative stress Mouse MC3T3-E1 osteoblasts were treated with 0.2 mM H2O2 for 24 hours to establish an injury model. Step 2: In vitro activity and supernatant factor detection The damaged cells were intervened with yak bone collagen peptide, cattle bone collagen peptide, pig bone collagen peptide, and chicken bone collagen peptide (concentration 1.0 mg / mL). Detect cell proliferation, differentiation, mineralization activity and apoptosis inhibition; The cell culture supernatant was collected simultaneously, and the levels of bone formation marker B-ALP and bone resorption marker TRACP were detected by ELISA. Step 3: In vitro pre-screening of qualified collagen peptides Screening of collagen peptides that increase supernatant B-ALP by ≥20% and decrease TRACP by ≥15%. result: Yak collagen peptide: B-ALP↑28%, TRACP↓18%, finally reaching the standard Cattle collagen peptide: B-ALP↑22%, TRACP↓16%, finally reaching the standard Porcine collagen peptide: B-ALP↑15%, TRACP↓10%, ultimately failed to meet the standard (continued only as a control) Chicken collagen peptide: B-ALP↑8%, TRACP↓5%, ultimately failed to meet the standard Step 4: Animal experiment verification Rat experiments were conducted on collagen peptides that met the standards (yak and cattle): The ovariectomized SD rat model of osteoporosis was established; Collagen peptide was administered orally (1000 mg / kg, 12 weeks); The levels of B-ALP and TRACP in animal serum and femoral bone mineral density were detected.

[0066] Step 5: In vitro-in vivo correlation validation Calculate the correlation coefficient of the bone turnover factor change rate between in vitro supernatant and animal serum: Yak collagen peptide: R 2 =0.92 (B-ALP), R 2 =0.89 (TRACP) result>0.85, valid.

[0067] Cattle collagen peptide: R 2 =0.87 (B-ALP), R 2 =0.84 (TRACP) result>0.85, valid.

[0068] Conclusion: The in vitro model can accurately predict the in vivo effect, and yak bone collagen peptide has the best anti-osteoporosis activity.

[0069] Comparative Example 1 1. Relying solely on in vitro cell experiments Step 1: Establish an oxidative stress cell model Mouse MC3T3-E1 osteoblasts were treated with H2O2 for 24 hours to establish an injury model.

[0070] Step 2: Detect cell proliferation and differentiation activity The damaged cells were intervened with yak bone collagen peptides (YBPs), bovine bone collagen peptides (BBPs), porcine bone collagen peptides (PBPs) and chicken bone collagen peptides (CBPs) (concentration 1.0 mg / mL).

[0071] The cell proliferation activity was detected by CCK-8 assay, and the cell differentiation activity was detected by ALP staining.

[0072] Step 3: Directly sort the effect of collagen peptides The effects of collagen peptides were ranked according to cell proliferation activity (CCK-8 results) and differentiation activity (ALP activity): Proliferative activity: pig bone collagen peptide (83.56%) > chicken bone collagen peptide (92.33%) > cattle bone collagen peptide (96.85%) > yak bone collagen peptide (105.93%) Differentiation activity ranking: pig bone collagen peptide (ALP activity value 0.12) < chicken bone collagen peptide (0.14) < cattle bone collagen peptide (0.16) < yak bone collagen peptide (0.18) Comprehensive ranking: Pig bone collagen peptide has the best activity (because of its higher proliferation activity and lower cost than yak and cattle bone collagen peptides).

[0073] 2. Ignoring key verification steps Bone turnover factors (such as B-ALP and TRACP) in the cell supernatant were not detected, so it was not possible to pre-screen the active ingredients through in vitro indicators.

[0074] No animal experiments were conducted to verify the results, no osteoporosis rat model was established, and no bone turnover factors (such as B-ALP, TRACP) or femoral bone density were detected in animal serum.

[0075] 3. Misjudging the risk Comparative Example 1 Conclusion: The proliferation activity of porcine bone collagen peptides (PBPs) (83.56%) is higher than that of chicken bone collagen peptides (92.33%), and the cost is lower, so the development of porcine bone collagen peptide products is given priority.

[0076] Key comparison notes 1. Defects of Comparative Example 1: The effects of collagen peptides were ranked based solely on in vitro cell activity (proliferation and differentiation), without pre-screening the active ingredients through supernatant bone turnover factors (B-ALP, TRACP).

[0077] Failure to conduct animal experiments to verify the claim led to the misjudgment that pig bone collagen peptide is superior to chicken bone collagen peptide (in fact, chicken bone collagen peptide has better activity in vivo).

[0078] 2. Rigor of Example 1: Pre-screening of supernatant factors: By detecting the changes in B-ALP and TRACP in the cell culture supernatant (porcine collagen peptide: B-ALP↑15%, TRACP↓10%), invalid collagen peptides (porcine collagen peptide does not meet the standards) are excluded, and only animal experiments are conducted on potential collagen peptides (yak, cattle).

[0079] In vivo-in vitro correlation validation: Yak collagen peptide: R2 =0.92 (B-ALP), R 2 =0.89 (TRACP)>0.85, effective; Cattle collagen peptide: R 2 =0.87 (B-ALP), R 2 =0.84 (TRACP) >0.85, effective.

[0080] This ensures that in vitro models reliably predict in vivo effects.

[0081] 3. Conclusion: Comparative Example 1: Due to the neglect of pre-screening and verification steps, low-efficiency raw materials (such as porcine collagen peptide) may be mistakenly selected, which wastes resources and has poor development effects.

[0082] Example 1 Through scientific multi-step verification (in vitro pre-screening + animal experiment + correlation analysis), it is accurately determined that the yak bone collagen peptide has the best anti-osteoporosis activity, providing a reliable basis for the development of functional foods.

[0083] According to another embodiment of the present invention, after completing in vitro screening and animal validation, the comprehensive evaluation phase begins. Four serum bone conversion factors (including bone formation factors such as alkaline phosphatase and bone γ-carboxyglutamate protein, and bone resorption factors such as tartrate-resistant acid phosphatase) obtained from animal experiments and three femoral morphomechanical indicators (bone density, trabecular number, and trabecular spacing) are integrated into a 7-dimensional data set. The score output by the in vitro cell activity comprehensive scoring model is simultaneously incorporated to form a multi-source data matrix of "in vitro score + in vivo indicator." This matrix is ​​subjected to Z-score normalization: for bone density units of g / cm 3 , serum factor unit ng / mL and other different dimensions, subtract the mean item by item and divide by the standard deviation to eliminate the difference in numerical scale. Perform principal component analysis on the standardized data, and require that the variance contribution rate of the first principal component PC1 exceeds 60% (72.2% in actual implementation) to ensure that the core biological variation is concentrated in PC1. Based on the PC1 score, a 0-100 anti-osteoporosis activity difference index is generated linearly. For example, yak bone collagen peptide scored 83.9 points, cattle bone collagen peptide scored 71.5 points, and pig bone collagen peptide scored 53.5 points. This index directly quantifies the activity ranking of different bone collagen peptides, guiding the selection of high-activity raw materials such as yak bone collagen peptides for the development of high-value products at the thousand-yuan / ton level.

[0084] Existing technologies use a single-dimensional osteoporosis assessment strategy: first, only the levels of bone alkaline phosphatase and tartrate-resistant acid phosphatase in rat serum are detected, and the bone metabolic balance is judged by the ratio of the two. This method ignores changes in bone microstructure and cannot reflect substantial improvements in bone strength. Second, only femoral bone density and the number of trabeculae are measured by Micro-CT. Although it can characterize the degree of bone loss, it is not sensitive to early metabolic imbalances. More importantly, the data of the two methods cannot be integrated - the serum index unit is ng / mL, and the bone density unit is g / cm 3 The lack of standardized treatments leads to incomparable results. Consequently, traditional approaches often lead to misjudgments: for example, pig bone collagen peptides were prioritized for development due to their low cost, but their actual bone density improvement rate was only 5.3%, far lower than the 12.7% achieved with yak bone collagen peptides. This invention overcomes limitations through three technological innovations: first, mandatory Z-score normalization eliminates dimensionality barriers for seven indicators, including serum factors and bone density; second, a PC1 contribution threshold of ≥60% is set to ensure extraction of core variants (actually reaching 72.2%); and finally, a quantifiable activity index is generated. This index, as demonstrated in the examples, clearly demonstrates that yak collagen peptides are 56% more active than porcine collagen peptides, technically resolving the industry debate over whether high-priced collagen peptides are worth developing and providing an irreplaceable decision-making basis for the differentiated development of livestock and poultry bone by-products.

[0085] Technical effect verification: In an experiment involving 56 osteoporotic rats, this method yielded key evidence: PCA analysis extracted PC1, which contributed 72.2%, PC2, which contributed 12.0%, and a cumulative contribution of 84.2%, far exceeding the 60% threshold, demonstrating the model's reliability. The yak collagen peptide group achieved an activity index of 83.9 points, corresponding to significant biological effects: serum bone morphogenetic factor increased by 28.1%, femoral bone mineral density increased by 12.7%, and trabecular number recovered to 91.3% of that in the sham-operated group. In contrast, the porcine collagen peptide group achieved a score of only 53.5, demonstrating a 5.3% improvement in bone mineral density and no reduction in trabecular spacing.

[0086] This implementation establishes a new objective evaluation standard for osteoporosis improvement through standardized integration of multi-source data, mandatory constraints on principal component contribution rates, and the generation of a percentage-based activity index. Compared to traditional single-dimensional methods, this breakthrough lies in three aspects: addressing the incomparability between serum indicators and bone structure data; extracting true biological signals through a high PC1 contribution of 72.2%; and resolving disputes over raw material screening with a quantitative conclusion of 83.9 > 53.5.

[0087] According to another embodiment of the present invention, when determining the most suitable amount of collagen peptide, a gradient concentration experiment is first performed: yak collagen peptide, cattle collagen peptide, pig collagen peptide, and chicken collagen peptide are respectively prepared into five concentration gradient solutions of 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 5.0 mg / mL. The CCK-8 method is used to detect the effect of each concentration on the proliferation activity of MC3T3-E1 osteoblasts damaged by oxidative stress, and cells treated with only 0.2mM hydrogen peroxide are used as a negative control group (the cell activity baseline value is set at 60.16%). The screening criteria require that the cell proliferation activity of the collagen peptide-treated group is increased by at least 30% compared with the negative control group, and the p value is confirmed to be less than 0.05 by t-test. For example, yak bone collagen peptide reached 96.85% activity at a concentration of 0.5 mg / mL, a 60.5% increase compared to the negative control, meeting the minimum effective concentration (MINC) requirement. In contrast, chicken bone collagen peptide exhibited only 68.59% activity at 0.5 mg / mL, falling short of the threshold and requiring an increase to 1.0 mg / mL to reach 92.33%. When MINCs varied among different collagen peptides, a uniform 1.0 mg / mL concentration was used for subsequent differentiation activity assays: cells were cultured in osteogenic differentiation medium containing 50 μg / mL ascorbic acid and 10 mM β-glycerophosphate for 72 hours, and alkaline phosphatase activity was measured by ALP staining. Mineralization activity was assayed by quantifying calcium nodule area 21 days after staining with Alizarin Red S. This strategy allowed for a 50% reduction in raw material during the proliferation phase (MINC of 0.5 mg / mL) while ensuring that all collagen peptides achieved the same baseline activity during differentiation experiments.

[0088] Existing technology uniformly uses a 1.0 mg / mL assay for all collagen peptides. This protocol obscures the optimal activity of porcine collagen peptide at 0.8 mg / mL (experimental value 98.7% vs. 1.0 mg / mL value 83.56%), falsely reducing its potency by 15.4%. It also leaves the high activity of yak collagen peptide at 0.5 mg / mL (96.85%) unutilized, resulting in a waste of raw materials.

[0089] The present invention uses a 30% activity increase as a rigid standard, combined with a statistical difference of p<0.05, to accurately capture the effective concentration critical points of yak bone collagen peptide at 0.5 mg / mL and chicken bone collagen peptide at 1.0 mg / mL. The proliferation experiment uses the lowest concentration specific to the species, and the subsequent differentiation / mineralization experiments are unified at 1.0 mg / mL. In a test involving four types of bone collagen peptides, this scheme makes the ALP activity value of yak bone collagen peptide reach 0.18 at a uniform concentration, and that of pig bone collagen peptide is 0.12, with a difference rate of 33.3%; if the yak bone collagen peptide is forced to use 0.5 mg / mL to detect ALP, its activity rises to 0.21, but the cross-species comparison is invalid.

[0090] Although the activity of chicken collagen peptide at 0.1 mg / mL was 63.86%, which was higher than that of the negative control, the increase was only 6.2% and had no statistical significance, so it was correctly excluded from the effective concentration range.

[0091] According to another embodiment of the present invention, healthy female SD rats aged 3 months were selected and bilateral ovariectomy was performed. Key physiological indicators were monitored weekly for 4 consecutive weeks after surgery: serum estradiol levels were measured by blood sampling from the tail vein, and the level was required to be stable below 15 pg / mL; body weight recovery was simultaneously recorded, and it was required to reach the range of 95% to 103% of the preoperative body weight. Only rats that met the standards for both indicators entered the subsequent experiments; those that did not meet the standards were excluded. This step ensures that the osteoporosis model has a high degree of consistency.

[0092] The minimum effective concentration of each collagen peptide was then determined through in vitro experiments: MC3T3-E1 osteoblasts were exposed to 0.2 mM hydrogen peroxide solution for 24 hours to construct an oxidative stress injury model.

[0093] The damaged cells were intervened with gradient concentration solutions of four collagen peptides from yak, cattle, pig and chicken (0.05, 0.1, 0.5, 1.0, 5.0 mg / mL).

[0094] The proliferation activity was detected using the CCK-8 method. The screening criteria were that the cell activity increased by at least 30% compared with the injury control group and the difference was statistically significant (p<0.05).

[0095] Example: Yak bone collagen peptide achieved 96.85% cell viability at 0.5 mg / mL (60.16% in the injured group), so its minimum effective concentration is 0.5 mg / mL. Porcine bone collagen peptide required 1.0 mg / mL to reach 83.56%. Converting the minimum effective concentration to an in vivo dose: Based on the principle of cross-species conversion based on body surface area (rat dose = human equivalent dose × 6.2), using yak bone collagen peptide as an example: The reference dose for humans is 500 mg, corresponding to the minimum effective concentration, and the oral dose for rats is 500×6.2=3100 mg / kg.

[0096] After verifying safety through preliminary experiments, the practical dosage of yak bone collagen peptide was finally adjusted to 620 mg / kg, and the dosage of pig bone collagen peptide was increased to 1240 mg / kg due to its lower activity.

[0097] Dynamic intervention cycle adjustment: personalized dose of collagen peptide solution is administered by gavage daily.

[0098] After the intervention began, blood was collected from the tail vein every 2 weeks, and the serum bone-specific alkaline phosphatase (B-ALP) level was measured by enzyme-linked immunosorbent assay.

[0099] Termination criteria: If the B-ALP test value increases by ≥20% compared with the baseline for two consecutive times, the intervention will be terminated (minimum period ≥8 weeks).

[0100] Extension criteria: If the criteria are not met, the program will be extended to 16 weeks.

[0101] Example: In the yak collagen peptide group, B-ALP increased by 25% compared to baseline at week 8 and by 32% at week 10, meeting the termination criteria (end of week 10).

[0102] The pig collagen peptide group only improved by 15% by the 14th week, and the experiment needed to be extended to 16 weeks to complete.

[0103] Comprehensive evaluation of effects: After the intervention, morphological indicators such as femoral bone mineral density and trabecular number were measured. Combined with serum bone turnover factor data, the anti-osteoporosis activity difference index was generated through principal component analysis to quantitatively evaluate the efficacy of different collagen peptides.

[0104] In existing protocols, all collagen peptides are administered orally at a dose of 1000 mg / kg for 12 weeks. Intervention begins only four weeks after oophorectomy, without verifying estrogen levels or weight recovery rates. Relying on data from a single time point makes it impossible to adapt to the speed of action of different collagen peptides.

[0105] This protocol features innovative breakthroughs: 1. Precision Dosing: Existing technologies use a uniform 1000 mg / kg dose of yak collagen peptide, resulting in an overdose (620 mg / kg is actually sufficient), while porcine collagen peptide dosages are insufficient (requiring 1240 mg / kg). This protocol, through in vitro screening for the lowest effective concentration and body surface area conversion, reduces the yak collagen peptide dosage by 38% and scientifically improves the porcine collagen peptide dosage. 2. Improved Model Reliability: Existing technologies can cause data deviation in approximately 30% of rats due to residual estrogen or poor recovery. This protocol eliminates unqualified individuals through dynamic monitoring, ensuring model stability. 3. Optimized Cycle Flexibility: Existing technologies show efficacy in yak collagen peptides by week 8, but this effect is still extended to 12 weeks. Porcine collagen peptides peak at 16 weeks, but are mistakenly considered ineffective due to the fixed cycle. This protocol dynamically adjusts the cycle based on bone turnover markers, shortening the yak collagen peptide experiment to 10 weeks and extending the porcine collagen peptide experiment to 16 weeks to fully release its activity.

[0106] All experiments in the present invention were repeated multiple times to obtain stable experimental results. Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and embodiment. They can be applied to various fields suitable for the embodiments of the present invention. For those skilled in the art, further modifications can be easily realized. Therefore, without departing from the general concept defined by the claims and their equivalents, the embodiments of the present invention are not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for determining the effect of various livestock and poultry-derived collagen peptides on osteoporosis, characterized in that: The steps include: Establish a mouse MC3T3-E1 osteoblast cell model of oxidative stress damage; Bone collagen peptides from various livestock and poultry species are used to improve the proliferation activity of MC3T3-E1 osteoblasts damaged by oxidative stress, thereby obtaining bone collagen peptides capable of improving the proliferation activity of MC3T3-E1 osteoblasts damaged by oxidative stress and the amount of the peptides used; The researchers used collagen peptides that can improve the proliferation activity of MC3T3-E1 osteoblasts damaged by oxidative stress, and the dosage of the peptides was used to improve the MC3T3-E1 osteoblasts damaged by oxidative stress. The differentiation activity, mineralization activity, cycle effect, and apoptosis inhibition were then tested. To analyze the effects of various livestock and poultry-derived collagen peptides on the differentiation activity, mineralization activity, cycle, and apoptosis inhibition of MC3T3-E1 osteoblasts damaged by oxidative stress, and to establish an in vitro comprehensive activity scoring model; In addition, the content of bone turnover factors in the cell culture supernatant was simultaneously detected to screen for collagen peptides that increased B-ALP by ≥20% and decreased TRACP by ≥15%; Conduct in vivo animal experiments on the screened collagen peptides to detect the bone turnover factor content in animal serum and femoral morphological mechanical indicators; If the correlation coefficient R between the change rate of bone turnover factor in cell culture supernatant and the change rate of bone turnover factor in animal serum is 2 If the value is ≥0.85, the in vitro activity comprehensive scoring model is considered valid, and the difference in collagen peptide activity is comprehensively determined based on it.

2. The method for determining the effect of multiple livestock and poultry-derived collagen peptides on osteoporosis according to claim 1, wherein: The following steps are also included: Obtain in vitro activity comprehensive scoring model data to comprehensively evaluate the anti-osteoporosis activity differences of various livestock and poultry-derived collagen peptides. The comprehensive evaluation also includes: combining in vivo animal experimental data, including serum bone conversion factor content and bone morphomechanical indicators of right femoral samples, The in vivo animal experimental data were normalized using principal component analysis (PCA) to generate a quantitatively comparable anti-osteoporosis activity difference index, including: Integrate in vivo animal experimental data and in vitro activity comprehensive scoring model data into a multidimensional dataset; Normalization: The Z-score normalization method was used to normalize the integrated data set to eliminate the bone density g / cm 3 , the dimension difference of bone volume fraction%, serum factor concentration ng / mL indicators; PCA dimensionality reduction: Perform PCA analysis on the normalized data to extract the principal components and meet the following requirements: the variance contribution rate of the first principal component PC1 is ≥ 60%, ensuring that PC1 carries the core variation information; Index generation: calculation of anti-osteoporosis activity difference index based on PC1 score Calculation formula: Index = PC1 score × 100, to achieve quantitative ranking of the activity of collagen peptides from different sources.

3. The method for determining the effect of multiple livestock and poultry-derived collagen peptides on osteoporosis according to claim 1, wherein: Mouse MC3T3-E1 osteoblasts were treated with 0.2 mM H2O2 to establish an MC3T3-E1 osteoblast model of oxidative stress injury. The CCK-8 method was used to detect the improvement effect of collagen peptides from various livestock and poultry species on the proliferation activity of MC3T3-E1 osteoblasts damaged by oxidative stress; ALP staining was used to detect the improvement effect of collagen peptides from various livestock and poultry species on the differentiation activity of MC3T3-E1 osteoblasts damaged by oxidative stress; The improvement effect of collagen peptides from various livestock and poultry species on the mineralization activity of MC3T3-E1 osteoblasts damaged by oxidative stress was detected by Alizarin Red S staining. The effects of collagen peptides from various livestock and poultry species on the cell cycle of MC3T3-E1 osteoblasts damaged by oxidative stress were detected by propidium iodide labeling combined with flow cytometry. Annexin V-FITC / PI double staining was used to detect the inhibitory effects of collagen peptides from various livestock and poultry species on the apoptosis of MC3T3-E1 osteoblasts damaged by oxidative stress. Among them, when comprehensively evaluating the differences in the anti-osteoporosis activity of various livestock and poultry-derived collagen peptides, the ALP activity value was used as the differentiation ability evaluation index, the calcium nodule staining intensity and mineralization area were used as the mineralization ability evaluation index, the S phase and G2 / M phase cell ratio was used as the proliferation activity evaluation index, and the total proportion of early and late apoptotic cells was used as the anti-apoptosis ability evaluation index. The differentiation ability evaluation index, mineralization ability evaluation index, proliferation activity evaluation index and anti-apoptosis evaluation index were weighted and integrated to establish an in vitro activity comprehensive scoring model. Combined with the in vivo animal experimental data, the anti-osteoporosis activity difference index of collagen peptides from various livestock and poultry species was generated.

4. The method for determining the effect of multiple livestock and poultry-derived collagen peptides on osteoporosis according to claim 1, wherein: Methods for obtaining in vivo animal experimental data include: A bilaterally ovariectomized osteoporosis model was established in female SD rats. Collagen peptides from various sources were administered orally for a period of time. Serum bone turnover factor levels and bone morphomechanical parameters of right femoral samples were then measured. The serum bone turnover factor content, including bone formation markers and bone resorption markers, was determined using a fully automatic serum biochemical analyzer. The bone formation markers included alkaline phosphatase, bone γ-carboxyglutamate-containing protein, and type I procollagen C-peptide. The bone resorption markers included tartrate-resistant acid phosphatase, serum type I collagen C-terminal peptide, and urine deoxypyridinoline. Micro-CT was used to measure the bone morphomechanical parameters of the right femoral specimens, including bone mineral density, bone volume fraction, trabecular number, and trabecular spacing.

5. The method for determining the effect of multiple livestock and poultry-derived collagen peptides on osteoporosis according to claim 1, wherein: The concentration of collagen peptide that can significantly improve the proliferation activity of MC3T3-E1 osteoblasts damaged by oxidative stress is determined by the following method: The CCK-8 method was used to detect the effects of various livestock and poultry collagen peptides at concentration gradients of 0.05, 0.1, 0.5, 1.0, and 5.0 mg / mL on cell proliferation activity; Screening for the lowest effective concentration that increases cell proliferation activity by ≥30% compared to the negative control group and has a statistically significant difference; When the minimum effective concentrations of various collagen peptides were different, 1.0 mg / mL was used as the unified test concentration for subsequent in vitro experiments.

6. The method for determining the effect of multiple livestock and poultry-derived collagen peptides on osteoporosis according to claim 1, wherein: A bilaterally ovariectomized osteoporosis model was established in female SD rats, and various collagen peptides from different sources were administered orally for a period of time. The following methods were used: Three-month-old female SD rats underwent bilateral ovariectomy. After 4 weeks of postoperative recovery, they were given 1000 mg / kg of collagen peptides from various livestock and poultry species by oral gavage daily for 12 weeks.

7. The method for determining the effect of multiple livestock and poultry-derived collagen peptides on osteoporosis according to claim 1, wherein: In vivo animal experiments include the following steps: Establishment of a bilaterally ovariectomized osteoporosis model in female SD rats: 3-month-old female SD rats underwent bilateral ovariectomy. Serum estradiol levels and body weight recovery were monitored weekly for 4 weeks after surgery. The model was considered successfully established when serum estradiol levels were <15 pg / mL and body weight recovered to 95% ± 3% of the preoperative level. Determining the intervention dose of collagen peptides: The CCK-8 method was used to detect the improvement effect of each collagen peptide on the proliferation activity of MC3T3-E1 osteoblasts damaged by oxidative stress, and the minimum effective concentration (MEC) that increased cell activity by ≥30% compared with the negative control group and p < 0.05 was screened; According to the body surface area conversion formula, rat dose = human dose × 6.2, the MEC of each collagen peptide was converted into an equivalent oral dose; The patients were given an equivalent dose of collagen peptide by oral gavage daily, and serum bone-specific alkaline phosphatase B-ALP was measured every 2 weeks; If the B-ALP test value increases by ≥20% compared with the baseline for two consecutive times, the intervention will be terminated, with a minimum period of ≥8 weeks; otherwise, the intervention will be extended to 16 weeks.

8. The method for determining the effect of multiple livestock and poultry-derived collagen peptides on osteoporosis according to any one of claims 1 or 2, wherein: Bone collagen peptides from various livestock and poultry species include yak bone collagen peptide YBPs, cattle bone collagen peptide BBPs, pig bone collagen peptide PBPs and chicken bone collagen peptide CBPs, and the concentration used is 1.0 mg / mL.

9. Application of the determination method according to claim 1 in the development of functional bone-derived foods and high-value utilization of livestock and poultry bone by-products, characterized in that: By comparing the differences in anti-osteoporosis activity of collagen peptides from various livestock and poultry species, reasonably high-value collagen peptide raw materials can be screened out for the preparation of functional foods or drugs to improve osteoporosis.

10. The use according to claim 9, characterized in that The reasonably high-valued collagen peptide raw material is yak bone collagen peptide YBPs or cattle bone collagen peptide BBPs.

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