Effects of Sheep Bone Peptide-Chelated Calcium on Calcium Absorption and Bone Deposition in Rats Fed a Low-Calcium Diet

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Introduction
Calcium comprises 1.5-2% of the human body weight, as the predominant mineral element within the human body [1].Calcium serves as a messenger to regulate cell division and apoptosis, was involved in maintaining capillary and cell membrane permeability, and is essential for the formation of bone and teeth.It also actively participates to diverse physiological activities including nerve conduction, cell proliferation, immune regulation, and muscle contraction.
Despite the presence of elemental calcium in foods (such as dairy products, vegetables, starchy foods, and dried fruits), dietary intake often falls short of meeting the recommended calcium requirements attributed to factors such as lactose intolerance and personal dietary preferences [2,3].Although the reference values in the diet range from 1.0 to 1.3 g, the average global calcium intake in humans is less than 0.6 g per day [4,5].Numerous studies have confrmed that inadequate calcium intake is linked to an elevated risk of osteoporosis, rickets, and other bone metabolic diseases and even induces kidney stones and colon cancer [6].To prevent and treat calcium defciency and mitigate the adverse efects of inadequate calcium intake, more and more calcium supplements are entering the market, including inorganic calcium, calcium gluconate, and amino acid calcium.However, a majority of them exhibit low calcium ion concentration or limited bioavailability [7].Over the past few years, some bioactive peptides extracted from diverse proteins sources, such as whey protein [8], tilapia skin and scale [9], pacifc cod skin [10], and soybean [11,12], can chelate with calcium ions that form peptide calcium chelates that are stable in the gastrointestinal tract and can prevent calcium precipitation in the body and efectively increase calcium absorption.In a previous study, porcine collagen peptide calcium chelate was formulated, and the calcium chelation rate was 78.38%.Te outcomes from the Caco-2 cell monolayer model indicated a substantial enhancement in calcium ion transport by the complexes, mitigating the adverse impacts of phosphate and phytate [1,13].Wang et al. [14] prepared bovine bone collagen peptide calcium chelate and found that it was stable during heat treatment and gastrointestinal digestion, suggesting that it holds the potential function of enhancing calcium bioavailability.A large number of studies confrmed that calcium-chelated peptides can improve calcium absorption bioavailability and bone biomechanics in mice [15][16][17][18].
China's animal husbandry is very developed and is the world's most promising and fastest-growing meat market.As the main byproduct of sheep slaughter and processing, sheep bones represent about 10-20% of sheep carcasses.Te collagen peptide produced after collagen hydrolysis is a kind of biologically active peptide that can protect the gastric mucosa [19], prevent and treat ulcers [20], promote sebum metabolism [21], reduce blood pressure [22], promote bone formation [23], improve bone density, prevent and treat osteoporosis [24], and other efects.Referring to previous research reports [25][26][27], we found that animal bones, as byproducts after slaughter, are typically discarded as waste, with only a small amount used for commercial purposes, primarily for the preparation of boiled soup, gelatin, and lower-value fertilizers.Terefore, the use of sheep bone to produce high-value-added products is an emerging challenge.Wang et al. [14] obtained peptide calcium chelate from sheep bones and it remained stable in diferent temperature ranges and during simulated gastrointestinal digestion.Furthermore, the good calcium-binding capabilities of the enzymatic breakdown products of sheep bone were demonstrated in a previous paper by our research group [28].However, its efectiveness in facilitating calcium uptake and deposition in bone in living organisms remains unexplored.
Te objective of this investigation was to generate peptide calcium chelate from sheep bone by stepwise enzymatic hydrolysis of alkaline protease and favor protease, refne the optimal preparation parameters for SBP-Ca, elucidate the chelation ions' chelation sites with SBP, determine the calcium chelating ability of SBP-Ca, and characterize its properties and bioactivity.Tis study suggests that SBP-Ca can be used as a functional calciumfortifed food to improve calcium absorption.

Preparation of Defatted Sheep Bone Meal.
After manually removing nonbone residues, such as meat, sheep bones were rinsed and pressure cooked at 121 °C for 40 min, followed by multiple hot water washes and then subjected to drying at 80 °C for 12 h.Subsequently, the sheep bones were crushed and sieved through an 80-mesh sieve to produce powder, which was soaked in anhydrous ethanol and agitated at room temperature for 24 h (change the solution once); the ratio of the sample to the solution was 1 : 10 (w/v).After washing with distilled water, the mixture was subjected to centrifugation at 8000 r/min for 15 min at 4 °C.Te precipitate was placed in a drying oven and subjected to drying at 70 °C for 6 h to acquire defatted sheep bone powder, which was stored at −20 °C.

Preparation of Sheep Bone Peptide (SBP).
According to previous studies [29], defatted sheep bone meal was prepared as a 6% solution (w/v) in deionized water and then hydrolyzed with alkaline protease (50 °C, 2 h, and pH 9) followed by favor protease (50 °C, 2 h, and pH 7.5).Following this, the substrate underwent heating at 100 °C for 15 min to deactivate the enzyme, cooled to ambient temperature, and then centrifuged at 8000 r/min for 15 min at 4 °C.Te resulting supernatant was collected and further processed through vacuum freeze-dried to obtain SBP powder.

Preparation of Sheep Bone Peptide-Chelated Calcium (SBP-Ca).
Te freeze-dried SBP (10 mg/ml) was reconstituted in deionized water to a total volume of 20 mL.Add CaCl 2 to achieve SBP: CaCl 2 ratios of (0.5 : 1, 1 : 1, 2 : 1, 3 : 1, 4 : 1, 5 : 1), adjust the solution pH to 8, and react at 50 °C for 50 min to determine the optimal SBP: CaCl 2 ratio.Subsequently, at a SBP: CaCl 2 ratio of 1 : 1, vary the solution pH (5, 6, 7, 8, 9, and 10), react at 50 °C for 50 min to determine the optimal pH; with a SBP: CaCl 2 ratio of 1 : 1, solution pH 7, perform water bath treatments at various temperature (40, 45, 50, 55, and 60 °C) for 50 min to determine the optimal chelation temperature; and with a SBP: CaCl 2 ratio of 1 : 1, solution pH 7, react at 45 °C for varying durations (30,40,50,60, and 70 min) to determine the optimal reaction time.After the chelation reaction, absolute ethanol (8 times the volume of the reaction mixture) was then introduced into the reactant solutions for the removal of unreacted free calcium ions and SBP.Te mixtures obtained were subjected to centrifugation at 8000 r/min for 15 min at 4 °C.Te precipitate was freeze-dried and designated as SBP-Ca.Te supernatant and reactant solution were digested using a mixture of acids (HNO 3 : perchloric acid, 4 : 1, v/v) and adjusted to a fxed volume using lanthanum solution (20 g/L).Te calcium content in both the solution and the supernatant was analyzed using a fame atomic absorption spectrophotometer (ZEEnit 700P, Germany).Te calculation method for the calcium chelation rate is outlined in the following: Calcium chelation rate(%) � Total calcium content-Calcium content in the supernatant Total calcium content × 100. (1)

Fourier Transform Infrared (FTIR) Spectroscopy
Analysis.FTIR analysis was used to examine the structural changes between SBP and SBP-Ca.SBP and SBP-Ca were thoroughly ground with dry KBr, mixed, and loaded onto the FTIR instrument (Nicolet iS20, Nicolet, USA).Te spectra were quantifed in the spectral range of 4000-400 cm −1 with 32 scans in which the spectra were obtained at a resolution of 4 cm −1 and this background spectrum was recorded by the transparent fraction of KBr [9].

Ultraviolet-Visible Absorption Spectroscopy (UV).
Te method outlined by Wu et al. [13] with some modifcations.In particular, SBP and SBP-Ca were confgured as a 1 mg/mL solution.Te spectra were obtained using a UV spectrophotometer (TU-1810, Beijing Pu-Analysis General Instrument Co., Ltd.) within the wavelength region of 190-400 nm.Te UV-vis spectrophotometer was blank calibrated using deionized water before measuring samples.

Diferential Scanning Calorimeter (DSC) Analysis.
Te thermal transition characteristics of SBP and SBP-Ca were determined using a diferential scanning calorimeter (JSM-7800F, Nippon Electronics Co., Ltd.) with certain adjustments to the method described by Wang et al. [14].Among them, SBP and SBP-Ca were weighed about 3.0 mg and sealed in an aluminum crucible.Utilizing an empty aluminum crucible as a reference, scanning was performed at a temperature increase rate of 10 °C/min within the temperature range of 25-200 °C.

Scanning Electron Microscopy (SEM).
Te microstructure features of both SBP and SBP-Ca were analyzed using scanning electron microscopy (S-3400N, Hitachi, Japan).An appropriate amount of SBP and SBP-Ca powder samples were evenly attached to the specimen holder and coated with gold-plated flm.Te microscopic images were captured at a working distance ranging from 12.7 to 12.8 mm and an accelerating voltage of 25 kV.rats in each group).Group Z was fed standard AIN-93 animal feed (calcium content: 0.5%) for the entire 4-week duration of the experiment, while the other groups received the same amount of low-calcium AIN-93 diet (calcium content: 0.1%).Te low-calcium diet formula was slightly modifed according to the standard of the AIN-93 feed formulation and was produced by Sino Biotechnology Co., Ltd.(Hunan, China).Te formula was (in mass fraction): casein 20.00%, cystine 0.30%, corn starch 39.70%, maltodextrin 13.20%, sucrose 10.00%, cellulose 5.00%, soybean oil 7.00%, multimineral M1003 3.50%, multidimensional V1001 1.00%, and tartaric acid choline 0.25%.Starting from the second week until the end of the experiment, the target calcium intake from calcium sources (CaCl 2 and SBP-Ca) was established at fve times the recommended daily intake for humans calculated based on the recommended daily calcium intake of 800 mg Ca/60 kg body weight.Tis corresponded to a calcium intake of 13.33 × 5 mg Ca/(day•kg bw).Calcium sources were solubilized in distilled water and administered individually from the feed using the gavage feeding method.
2.6.2.Sample Collection.After the experimental period, all rats were subjected to a 12-hour fasting period, and their body length and weight were measured.Subsequently, the weight growth rate and body length growth rate were calculated.Te rats were anesthetized using isofurane, blood samples were then obtained from the eyeball, and the blood collected was centrifuged for serum separation.Rats were sacrifced via cervical dislocation, followed by removal of the heart, liver, spleen, kidneys, and lungs, rinsed with cold 0.9% saline, wiped with flter paper, and weighed to calculate the visceral index [31].Te right and left femur and tibia were dissected, and the surrounding soft tissues were cleaned and enveloped in saline-soaked gauze before being stored at −20 °C for subsequent studies. Weight After drying, the femurs underwent carbonization in an electric furnace and were ashed at 800 °C for 2 h in a mufe.Te quantifcation of the bone calcium content was carried out using fame atomic absorption spectrometry (ZEEnit 700P, Germany).

Microcomputed Tomography (Mico-CT).
Te left femur of the rat was immersed and fxed in a 4% paraformaldehyde solution for 24-48 h, removed and rinsed three times with PBS (bufer), and stored in a 75% alcohol solution at 4 °C.Ten, it was scanned and analyzed using a Micro-CT (SkyScan 1176, Bruker, Germany) scanning imaging system, and 171-200 layers (18 μm pixel size) of the femur were scanned from the fusion of the bone tissue growth plate [3].3D images were obtained by 3D reconstruction using CTVOX software and the scanning parameters were analyzed using CTAn software.Te main indices included bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.T), trabecular number (Tb.N), and trabecular spacing (Tb.Sp).

Hematoxylin and Eosin (H&E) Stained.
Following the Micro-CT measurement, the left femur underwent fxation in methanol for decalcifcation.Subsequently, it was embedded and stained to facilitate morphological analysis.Both transverse and longitudinal sections of the femur were stained with hematoxylin and eosin [32] and then the bone morphology was examined using an optical electron microscope (DM3000, Leica Instruments, Germany).

Statistical Analysis.
Data analysis was performed utilizing SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and Origin software (Origin Lab, Northampton, MA, USA).Te normality of the data distribution was examined through the Shapiro-Wilk test.Diferences between experimental groups and between process conditions were performed by one-way ANOVA.Data were considered signifcantly diferent at the level of P < 0.05.

Efect of Diferent Process Conditions on the Calcium-Chelating Capacity of SBP-Ca.
As the chelation temperature increased from 40 °C to 45 °C, the calcium chelation rate increased signifcantly from 80.15% to 84.86% (P < 0.05), reaching the maximum value (Figure 1(a)).Te moderate increase in temperature favors molecular movement increases the chance of molecular collision and enhances the probability of binding between calcium ions and amino acid residues, increasing the calcium chelation rate.However, the chelation reactions themselves are exothermic [33].Terefore, since the temperature exceeded 45 °C, the structures of the peptides and their complexes with calcium can be easily disrupted [34], leading to partial or complete changes in the molecular structure of SBP.Aggregation between molecules occurs, resulting in a reduction in available chelating groups for calcium ions, thus causing a decrease in the chelation efciency.An analogous trend was noticed by Wu et al. [13], when investigating pig bone collagen peptide with calcium chelation, where the calcium-binding capacity peaked at 50 °C, followed by a decrease with a further increase in temperature.
Te impact of chelation time on the rate of calcium chelation is depicted in Figure 1(b).As the chelation time extended from 30 to 60 min, the chelation rate of calcium rapidly increased from 74.80% to 85.13% (P < 0.05), reaching its maximum value.However, a further extension of the reaction time resulted in a decrease in the chelation rate to 82.77% (P < 0.05).Also, Zhai et al. [35], in the preparation of mung bean peptide-calcium chelates, also found that the rate of calcium chelation showed a tendency to increase and then stabilize as the chelation time increased and reached a maximum at 60 min.Probably, as suggested by Fang et al. [36], with an increase in the reaction time, the contact between calcium ions and peptides increases, leading to better efciency of the chelation reaction.However, once a certain time threshold is reached, the chelate becomes stable and the calcium chelation rate ceases to increase.Xun et al. [37] concluded that a long reaction time may lead to chelate instability and decomposition, thus reducing the chelation rate.As the mass ratio of SBP/CaCl 2 was escalated from 0.5 : 1 to 1 : 1, the chelation rate increased signifcantly from 85.69% to 87.50%, reaching the maximum value (P < 0.05; Figure 1(c)).Tis phenomenon could be attributed to the increasing content of peptides in the reaction system as the SBP/CaCl 2 mass ratio increases, facilitating greater contact between calcium ions and the peptide ligand, thereby promoting the reaction in the forward direction and increasing the chelation rate.However, when the SBP/CaCl 2 mass ratio exceeded 1 : 1, saturation of calcium binding to peptides occurred, hindering a further improvement of the chelation rate.Similar variations in the chelation rate with the peptide-to-calcium mass ratio were observed by Cui et al. [38] in the preparation of calcium complexes with sea cucumber egg hydrolysates.
Te calcium chelation rate increased from pH 5 to 7 (P < 0.05), reaching a maximum value of 87.96% and then decreased (Figure 1(d)).In acidic environments, a rise in pH leads to a decline in H + concentration, increasing the electron-donating capacity of potential metal ion chelation sites (-COOH and -NH 2 ).In alkaline environments, high concentrations of OH-tend to form Ca(OH) 2 precipitates with calcium ions, resulting in a reduction in the calciumbinding capacity of SBP.Terefore, a neutral environment is the best condition for SBP to bind calcium ions, aligning with the observations of Luo et al. [39].In conclusion, the optimal chelation process (pH � 7, temperature � 45 °C, SBP/ CaCl 2 mass ratio � 1 : 1, and time � 60 min) was selected for the preparation of SBP-Ca, with a chelation rate as high as 89.24%, and used for subsequent analysis and animal experiments.

FTIR Analysis.
As a powerful tool for studying peptide structure [40], FTIR technology has been used to further study the potential mechanism of the peptide-calcium reaction.As illustrated in Figure 2, after the addition of calcium ions, the FTIR spectrum of the SBP changed signifcantly; this may be caused by the change in the vibrational frequency of the amino acid moiety [41].SBP exhibits a characteristic absorption peak at 1398.72 cm −1 , and a stretching change occurs in the SBP-Ca, moving to 1415.52 cm −1 , corresponding to the characteristic absorption region of carboxylate, indicating that the -COOH in SBP chelated with calcium ions.Simultaneously, the absorption peak of C�O in SBP increased from 2943.14 cm −1 to 2957.13 cm −1 , which indicated that the carbonyl of the amide (peptide bond) contributes to the establishment of the peptide calcium chelates.Tis result is similar to that observed with calcium-chelated sheep bone collagen peptide obtained by the enzymatic-fermentation method [42].Furthermore, the absorption peak corresponding to the -NH 2 telescopic vibration moves from 1130.78 cm −1 to 1146.30cm −1 and the wave bandwidth becomes wider.In summary, it could be speculated that the main afnity sites of SBP encompass carboxyl oxygen, amino nitrogen, and oxygen within the peptide bond.Tese functional groups chelate with calcium ions to form a novel compound SBP-Ca.

Ultraviolet-Visible Absorption Spectroscopy (UV)
Analysis.Ultraviolet spectroscopy is an analytical method employed to examine alterations in substances and ascertain the emergence of novel compounds [43].Interactions between metal ions and organic ligands may result in the appearance of new absorption peaks or the shift or disappearance of the initial absorption peaks [38].Te absorption peaks of SBP and SBP-Ca were observed at 296 nm and 308 nm, respectively, as shown in Figure 3. Tis suggests that the conjugation efect occurs involving the π electron of carbonyl double bonds and the electrons in the lone pair on groups such as -NH 2 and -OH when binding with calcium ions.Tis alteration afected the internal electron transitions of the ligand and the valence electron transitions of the corresponding atomic, resulting in a decrease in the transition energy of n ⟶ σ * [44,45].Consequently, chelation resulted in an enhancement of absorption intensity and a redshift in the UV spectrum.Hence, it could be concluded that a new substance is formed through the chelation of peptides with calcium.

Diferential Scanning Calorimetry Analysis.
Te SBP showed a peak of heat absorption at 172 °C, which is mainly due to the breakage of chemical bonds in the SBP (Figure 4).When the SBP was chelated with calcium ions, the peak of heat absorption occurred at 155 °C.Te diference between the two heat absorption peaks may be ascribed to the different positions of the C-N bond and the diferent energy and temperature required to break the C-N bond at elevated temperatures [42].6 Journal of Food Biochemistry 3.5.Analysis of SEM.Te microstructures of SBP and SBP-Ca are displayed in Figure 5; the surface of SBP is smooth, the tissue state is delicate, and the sheet structure is present.On the contrary, the surface of SBP-Ca is rough and loose and exhibits numerous spherical formations.Te variations in microstructure between SBP and SBP-Ca are probably due to the change in internal structure induced by the coordination bond formed between peptide and calcium [46].

Growth Status and Calcium Absorption in Rats.
Troughout the experiment, the rats in group D exhibited reduced food intake and hair loss after 2 weeks.Rats in the other groups were fexible, had regular food intake, and exhibited normal coat color.Tere was no death or diarrhea in rats during the experiment.Te weight growth rate of group D exhibited a marked reduction compared to the other groups (P < 0.05; Table 1).No signifcant diferences were observed in body length and body length growth rate    Z: rats fed with AIN-93 diet; D: rats fed with a low calcium AIN-93 diet; C: rats fed with a low calcium AIN-93 diet and CaCl 2 ; T: rats fed with a low calcium AIN-93 diet and SBP-Ca.Te values in columns with diferent superscript letters are signifcantly diferent (P < 0.05).Journal of Food Biochemistry among the groups (P > 0.05).Calcium is vital for maintaining body growth and metabolism, so a defcient intake of calcium could have reduced the weight growth rate of the animals [14].Te visceral index is a common evaluation index in toxicological tests, which increases or decreases to varying degrees when the viscera of the animal changes, such as congestion, edema, hypertrophy, or atrophy of the viscera [47].Tere were no statistically signifcant diferences observed in the cardiac, lung, and kidney indices between the groups (P > 0.05; Table 2).Te liver index in group D exhibited a higher value compared to the normal group and the other groups, but the disparity was not statistically signifcant (P > 0.05).A high liver index may be related to liver disorders [48].Tese results suggest that SBP-Ca supplementation had no impact on the normal growth and health of rats.

Efect of SBP-Ca on Serum Biochemical Markers.
Compared with group D, the serum calcium and phosphorus levels in groups C and T exhibited elevated serum calcium and phosphorus levels, yet the variance was not statistically signifcant (P > 0.05; Figures 6(a) and 6(b)).Tis may be due to the dynamic equilibrium between bone calcium and serum calcium.In the case of low daily calcium intake levels, bone calcium will be released into the blood to sustain serum calcium and serum phosphorus concentration at a normal level [49].ALP and OCN serve as biomarkers for bone formation, providing reliable indicators of osteoblast activity and the status of bone formation.Robison and Soames [50] revealed that ALP was crucial in the upregulation of bone calcifcation, and its expression increases abnormal calcifcation.Te highest ALP was found in group D (P < 0.05; Figure 6(c)), indicating that the rats in this group were severely calcium defcient and in state of high bone turnover.Tis also suggests that a low-calcium diet rat model was successfully established.When supplemented with CaCl 2 and SBP-Ca, ALP activity was signifcantly reduced (P < 0.05).Tis indicates a negative correlation was identifed between ALP activities and Ca intake.Tese fndings align with the observations made by Zhao et al. [51] and others, investigating the impact of desalted duck egg white peptides on calcium absorption in rats and found that serum ALP activity levels could be increased during a long-term low calcium intake.In addition, ALP activity decreased signifcantly (P < 0.05) in the SBP-Ca group (25.17 ± 1.19 U/L) compared to the CaCl 2 group (29.38 ± 1.15 U/L).Tese results were consistent with the fndings reported by Chen et al. [9].Te levels of OCN in group D were notably signifcantly reduced compared to the other groups (P < 0.05; Figure 6(d)).In group T, the OCN content was signifcantly (P < 0.05) higher (12.43 ± 0.69 ng/mL) than in group C (8.01 ± 1.82 ng/mL).Tese results further confrm that SBP-Ca supplementation promotes new bone formation and is absorbed more easily in rats [49], which is superior to CaCl 2 supplementation.
3.8.Biochemical Parameters of the Femur.Te assimilation and metabolism of calcium may induce alterations in femoral characteristics, so femoral characteristics are ideal monitoring indicators to evaluate the efect of calcium supplementation [52].Tere were no notable variations in femur length among the groups (P > 0.05; Figure 7(a)).Hernández-Becerra et al. [53] also highlighted that the femur length remained unafected by the dietary calcium Moreover, the bone calcium content within the SBP-Ca-fed group exhibited a notable superiority over that of the CaCl 2fed group (P < 0.05).From this point of view, the efect of dietary supplementation with SBP-Ca is higher than that of CaCl 2 .Tese results indicate that SBP-Ca supplementation has a positive efect on calcium absorption, favors the growth and development of rat bones, and can efectively alleviate bone calcium loss.In a similar low-calcium model experiment, Zhao et al. [3] observed a similar phenomenon in the phosvitin phosphopeptide-Ca complex-enhanced calcium absorption and deposition in the rat femur.
3.9.Morphological Analysis of Bone Tissue.Micro-CT has been extensively employed to investigate the alterations in the microstructure of femoral trabeculae in experimental subjects [55].As shown in Figure 8, group Z had a tight trabecular structure with good continuity, while group D had a severely disrupted trabecular structure with obvious large areas of trabecular-free bone.Te bone microstructure of rats supplemented with SBP-Ca showed more bone trabeculae, fewer areas of boneless trabecular bone, and more intact trabecular junction structures compared to group D. Tese fndings align with the substantial enhancement in bone microstructure and increased trabecular integrity reported by Hua et al. [54] using phosvitin phosphopeptide-Ca in low-calcium diets.Te relevant bone microstructural parameters verifed this result, as depicted in Figure 8, compared with group Z; group D exhibited a signifcantly reduced BMD of the femoral bone, BV/TV, Tb.T, and Tb.N (P < 0.05).Furthermore, the Tb.Sp in the bones of rats was highest in group D (P < 0.05), suggesting that the induction of the lowcalcium diet rat model was successful.Te BMD of rats fed SBP-Ca and CaCl 2 were 0.29 and 0.17 g/cm 3 , respectively.Tis indicated that SBP-Ca is more efective than CaCl 2 in bone remineralization.Compared with group D, SBP-Ca supplementation signifcantly improved femoral Tb.T and Tb.N and reduced Tb.Sp (P < 0.05).Tese results suggest that SBP-Ca supplementation improves BMD, increases bone mass, preserves bone microarchitecture, and has the potential to ameliorate osteoporosis in rats.A prior investigation suggested that the Dunaliella salina-derived peptide could enhance the BMD and trabecular number and decrease trabecular bone space in ovariectomy (OVX) rats [56]; this is similar to our fndings.
Furthermore, the histomorphological changes of the transverse section of the femur were examined using H&E staining and observed through an electron microscope [32].Te results of the H&E staining depicting the trabecular structure of the femur are illustrated in Figure 9. Te trabecular of the structure bone of the femur in group Z was closely and evenly arranged, and continuity was well-maintained.Te trabecular structure of group D showed dissolution and fractures.In group T, the restoration of bone quantity in the femur, resulting in increased trabecular bone thickness and reduced intertrabecular space compared to group D. Tese results indicated that SBP-Ca supplementation signifcantly increased BMD, improved bone microarchitecture, and enhanced the integrity of bone trabeculae in rats with a low-calcium diet, with a more pronounced efect compared to the use of inorganic calcium (group C).

Conclusions
In this study, defatted sheep bone powder was used as the raw material to prepare SBP by double enzymatic hydrolysis.Te optimal chelation process of sheep bone peptides with calcium ions (pH � 7, temperature � 45 °C, SBP/CaCl 2 mass ratio � 1 : 1, and time � 60 min) was used to prepare sheep bone peptide-chelated calcium (SBP-Ca).Under these process conditions, the chelation rate of calcium in the SBP-Ca chelate can reach 89.24%.Structural characterization analysis revealed that SBP-Ca is a novel compound distinct from SBP. Te carboxyl oxygen atoms, amino nitrogen atoms, and oxygen atoms on the peptide bond in SBP were identifed to participate in the chelation of SBP with calcium ions.In addition, the surface of SBP-Ca exhibited a densifed structure.Using a rat model, the addition of SBP-Ca in lowcalcium diets could promote calcium absorption and increase bone growth and density.Histomorphological studies of bone showed that SBP-Ca could restore the structure of trabecular bone.Tese results indicate that SBP-Ca supplementation is efective in preventing calcium defciency and enhancing the bioavailability of calcium.Furthermore, SBP-Ca is more efective than CaCl 2 supplementation in promoting bone formation in rats with low-calcium levels with no side efects detected during the trial.Terefore, SBP-Ca can be used as a novel calcium absorption promoter and may fnd valuable applications in the prevention of calcium defciency.However, this study has some limitations, including a relatively short experimental period, a small sample size, and insufcient research depth.Terefore, in the next step, we will expand the sample size and extend the experimental period to 8-12 weeks.We will further explore the mechanism of the ameliorative efect of SBP-Ca on osteoporosis from the following three aspects: intestinal genes, skeletal genes and intestinal fora.

Figure 1 :
Figure 1: Efects of diferent process conditions on the calcium chelating rate of sheep bone peptide chelated calcium (SBP-Ca): (a) temperature, (b) time, (c) mass ratio of SBP/CaCl 2 , and (d) pH.Data are expressed as the mean ± sd.Diferent letters indicate a signifcant diference (P < 0.05).

Figure 9 :
Figure9: H&E staining of femoral transverse section of rats (×20 magnifcation); Z: rats fed with AIN-93 diet, control group; D: rats fed with a low calcium AIN-93 diet; C: rats fed with a low calcium AIN-93 diet and CaCl 2 ; T: rats fed with a low calcium AIN-93 diet and SBP-Ca.

Table 1 :
Rat body weight, body length, rate of body weight gain, and rate of body length growth.

Table 2 :
Organ indices of rat heart, liver, spleen, kidneys, and lungs.: rats fed with AIN-93 diet; D: rats fed with a low calcium AIN-93 diet; C: rats fed with a low calcium AIN-93 diet and CaCl 2 ; T: rats fed with a low calcium AIN-93 diet and SBP-Ca.Te values in columns with diferent superscript letters are signifcantly diferent (P < 0.05). Z