Preparation and tissue structure analysis of horse bone collagen peptide

Preparation and tissue structure analysis of horse bone collagen peptide | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Preparation and tissue structure analysis of horse bone collagen peptide Jindi Wu, Heya Na, Fan Bai, Siyu Li, Hao Gao, Rina Sha This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4512011/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Oct, 2024 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract Horse bone is rich in collagen, with a composition similar to that of human collagen. Collagen peptides supply nutrients needed for human growth that act as antioxidants, lower blood pressure. This study explored the extraction of collagen and the preparation of collagen short peptides from Mongolian horse bones. Bones were collected from horses of varying ages, and the collagen content along with calcium salt distribution were observed through staining and imaging analyses. Next, the bones were processed into a powder and then subjected to ultra-high-pressure processing for degreasing. The degreasing conditions were optimised by single-factor and orthogonal tests. Following this, collagen was extracted using an acid-enzymatic method, and its structural characteristics and thermal stability were assessed. The collagen short peptides were extracted from the collagen samples, and the effects of the enzymatic hydrolysis time, temperature, pH, and enzyme amount on the extraction rate were evaluated. Finally, the resulting collagen peptides were analysed for antioxidant activity. In summary, this experiment optimised the extraction conditions for horse bone collagen, demonstrating that the ultra-high-pressure method minimally affects collagen structure, and the extraction rate was high. Hence our method has significant development potential. Biological sciences/Molecular biology Health sciences/Health care Mongolian horse bones Hard tissue section Ultra-high pressure Collagen peptide Antioxidant activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Lay summary Horse bones are a natural source of collagen, and at present, there are few reports on the development and research of bone-related food products. Compared with other livestock, horse bones have higher nutrient content ratios, making them a very high-quality collagen and calcium resource. Moreover, the bone quality of horse bones is dense, invigorating, detoxifying, and healing, and it has been used as a traditional medicine to treat fractures and arthritis, etc.The present study investigated a novel source of collagen and developed a methodology for extracting collagen peptides. Mongolian horse bones were identified as an abundant source of calcium and protein with low fat content. This study explored the extraction of collagen using ultra-high pressure processing, along with the evaluation of short peptides derived from Mongolian horse bones. 1. Introduction Ultra-high-pressure processing (UHP), also known as hydrostatic pressure technology, is a food processing technology that has attracted attention in the Chinese food industry, with substantial efforts and investments dedicated towards its research and development. UHP involves shaping or sizing food through vacuum packaging. In this process, food is placed in ultra-high pressure treatment equipment according to the basic principles of Le Chatelier and Pascal. A liquid medium is then employed to sterilise the food at pressures exceeding 100 MPa, effectively eliminating enzymes and modifying the food’s characteristics. This physical food processing method extends the shelf life of food (Kazutaka 2017). In essence, ultra-high-pressure treatment disrupts non-covalent bonds in food substances including hydrogen bonds, molecular bonds, hydrophobic effects, and electrostatic effects as well as the intricate organisational tertiary structures of the biomolecules in the food. Notably, covalent bonds of small molecules, such as vitamins and amino acids, remain unaffected. Consequently, the flavour, nutrition, and colour of the food are preserved (Nan et al. 2018). Currently, UHP is the preferred sterilisation method in the food industry (Patrignani et al. 2019), as it retains the food’s original composition (Xi et al. 2021). Moreover, the freshness of the food is not affected and can even be extended. As a result, UHP demonstrates considerable developmental potential (Aganovic et al. 2021). Dong et al. (2014) studied the effects of flavour enzymes on chicken bone hydrolysis over time, finding that the proportion of small molecular peptides (400–1000 Da) in products hydrolysed for 8 hours was 74 times higher than that in those hydrolysed for 1 hour. Kim et al. (2014) investigated the physicochemical properties of type Ⅰ collagen from bovine bone and studied the antioxidant activity of low-molecular-weight peptides from the leg bone of Jeju horses. Leon-Lopez et al. (2019) explored how the hydrolysis time affected collagen extraction and enzymolysis from sheep skin, as well as its structure and antioxidant activity. Moreover, Miroslava et al. (2019) separated squid collagen peptide using ultrafiltration membranes, finding that the hydrolysate with a molecular weight of less than 5 kDa had a strong antioxidant activity. Finally, Hou et al. (2023) found that components in the collagenase hydrolysate (with a molecular weight of 1–5 kDa) of sturgeon skin had a strong superoxide anion-scavenging ability. Horse bone, often used in traditional medicine to treat bone diseases, fractures, and arthritis, has a higher nutrient content ratio than bone from other domestic animals and is rich in high-quality collagen and calcium (Lee et al. 2007). However, limited studies have explored bones as a collagen source. Recently, efforts have been made to industrialise the Chinese horse industry (Fig. 1 ). However, progress has been slow due to a lack of attention and failure to emphasise its social and economic benefits. Consequently, the industry remains in the initial stages of industrialisation. Given the benefits of collagen and the rich nutrients derived from bone, this study explored the extraction and preparation of collagen short peptides from Mongolian horse bones, aiming to expand the use of horse bones in the industry. 2. Material and methods 2.1. Materials The materials for this experiment were the ribs and tibia of 2-, 8- and 13-years-old horses, which were provided by Shandong Dezhou Demu Meat Processing Co., LTD. Sheep bones and cattle bones were provided by Zhongke Wanxin Selenium-Enriched Halal Food Co., LTD., Ordos City, Inner Mongolia. 2.2. Reagents Table 1 Table 1 Reagents (and their manufacturers) used in the laboratory Reagent Manufacturer Ethyl acetate Tianjin Zhiyuan Chemical Reagent Co., LTD Petroleum ether Sinopharm Chemical Reagent Co. LTD Glacial acetic acid Tianjin Damao Chemical Reagent Factory Pepsinum Alkaline protease Bovine Serum Albumin□ Coomath bright blue G-250 Solarbio Solarbio Tianjin Yongda Chemical Reagent Co., LTD Tianjin Yongda Chemical Reagent Co., LTD MD34 dialysis bag Solarbio Absolute ethyl alcohol Xylene Sirius scarlet dye Alizarin red dye solution Permount TM Mounting Medium Glycol diethyl ether acetate Sinopharm Chemical Reagent Co. LTD Sinopharm Chemical Reagent Co. LTD Servicebio Servicebio Sinopharm Chemical Reagent Co. LTD Macklin Hydroxyproline (HYP) content detection kit Solarbio EDTA Potassium ferricyanide Solarbio Tianjin Yongda Chemical Reagent Co., LTD 2.3. Hard tissue section preparation and staining Fresh equine tibia of various ages were chosen, and the bone tissue was fixed with 4% paraformaldehyde solution (PFA) for 48 h. Subsequently, the tissues were subjected to dehydration by adding different concentrations of alcohol successively, and each gradient was maintained for 24–72 h. The sliced tibia of the horse was put into an embedding bottle, with an appropriate amount of embedding solution added. The system was vacuum-sealed for 5 h, and a water bath was set at 37 ℃. The bone tissue was sliced into approximately 10 µm using a slicer, and these slices were oven-roasted at 60 ℃ overnight. The sections were dehydrated with ethylene glycol ether acetate and ethanol of different gradients. The treated tissues were stained with Alizarin red and Sirius scarlet, respectively. 2.4. Ultra-high-pressure treatment The crushed horse bone was vacuum-packed and placed inside the ultra-high-pressure equipment. Distilled water was used as the medium for ultra-high-pressure treatment. (Control group: defatted horse bones without ultra-high-pressure treatment) 2.5. Extraction of collagen from horse bone meal The horse bone meal was mixed with ethyl acetate solution at a ratio of 1:15 g/mL, and the solution was shaken and degreased at 4℃ for 24 h. The decalcified horse bone meal was added into 0.1 mol/L NaOH solution at 1:10 g/mL, and the protein was removed by continuous agitation at 4℃ for 24 h. After the horse bone collagen was extracted, the supernatant was removed by centrifugation at 11171.56 x g for 20 min at 4℃. The precipitation continues to undergo enzymolysis, and this process is repeated twice to incorporate the supernatant. The supernatant was adjusted to a pH value of 7.4 with 3 mol/L NaOH solution. Then the NaCl solid powder was slowly added to the collagen extraction solution until the concentration reached 0.9 mol/L. The powder was placed in a chromatography tank at 4℃ for 24 h for salting out and centrifuged at 11171.56 x g for 20 min to retain the precipitation. The resulting precipitates were dissolved with 0.5 mol/L glacial acetic acid and put into a pre-treated dialysis bag. At 4℃, they were dialysed with 5 mmol/L glacial acetic acid solution for 48 h and then with distilled water for 24 h. The collagen obtained after dialysis was injected into a glass plate, frozen overnight in a refrigerator at − 80℃, and then dried in a vacuum freeze-dryer for approximately 24 h to obtain white powdered crystalline collagen, which was refrigerated at − 20℃ for use. The absorption value was measured at wavelength 560 nm, and a standard curve was drawn. 2.6. Thermal denaturation temperature analysis of horse bone collagen Approximately 3 mg of horse bone collagen samples of different ages were weighed into an aluminium crucible, pressed, and sealed, and an empty crucible was used as a reference for determination using differential scanning calorimeter (DSC). The temperature was increased from 25℃ to 100℃ at 2℃/min. FTIR analysis of the infrared spectrum was done following Rozi et al. ’s (2018) method. The collagen samples from the ribs and tibia of horses of different ages were mixed with potassium bromide crystals in a ratio of 1:100, and the freeze-dried collagen was extruded into sheets by a manual mechanical extrusion device. The wavenumber employed was in the range of 400–4000 cm − 1 , the number of scans used was 64, the speed was 0.2 cm/s, and the resolution was 4 cm − 1 (Xu et al. 2017). 2.7. Preparation of horse bone collagen peptide The freeze-dried horse bone collagen was dissolved in 0.5 mol/L glacial acetic acid, and the substrate concentration was 10%. The optimal pH value of alkaline protease was adjusted, and collagen enzymolysis was conducted. Upon completion, the mixture was placed in a water bath at 100 ℃ for 15 min, followed by cooling to room temperature (approximately 24 ℃). The solution was then centrifuged at 11171.56 x g under at 4℃ for 20 min, allowing the removal of the precipitate and collection of the supernatant. After overnight freezing in 80℃, vacuum freeze-drying was conducted. The protein concentration was determined using the Coomassie brilliant blue method and calculated using BCA method. The trichloroacetic acid soluble nitrogen method (TCA method) was utilised, and nitrogen content was determined using the Kjeldahl method. 2.8. Study on antioxidant activity of horse bone collagen peptide The obtained enzymatic hydrolysate was fractionated using ultrafiltration centrifuge tubes with molecular weight cutoffs of 10, 5, 3, and 1 kDa, respectively. Subsequently, the collected extracellular filtrate underwent vacuum freeze-drying. In test tubes, 1 mL of samples was mixed with 2.5 mL each of phosphate buffer (0.2 mol/L, pH 6.6) and 1% potassium ferricyanide solution. The mixture was then blended and placed in a water bath at 50℃ for 20 min. Following this, 2.5 mL 10% trichloroacetic acid solution was added and centrifuged at 335.37 x g for 10 min. After centrifugation, 2.5 mL of supernatant was removed, and 0.5 mL of 0.1% ferric trichloride solution and 2.5 mL of distilled water was sequentially added. The absorbance was measured at 700 nm after allowing for a 10-min rest period at (approximately 24℃) (Xie et al. 2021). 2.9. Statistical analysis The software SPSS 26.0 was used for data analysis (P < 0.05); The mapping software Origin 2021 and image processing software ImageJ were used to map the test data. 3. Results and discussion 3.1. Horse bone composition The protein content in the bones of horses varied with age and exhibited a noticeable trend. It initially increased and then decreased with increasing age, as shown in Fig. 2 . Additionally, the bones were rich in calcium; however, the crude fat content was low. 3.2. Bone tissue composition Figures 3 and 4 and Table 2 depict the bone tissue compositions. In each tissue section, the area with the most collagen fibres was selected as the field of vision and used in the analysis with Image J. Picric acid scarlet staining (Fig. 3 ) revealed collagen fibres with a yellow background, and Sirius scarlet staining (Fig. 4 ) revealed dark red calcium salt deposits and a light red or nearly colourless background. Table 2 Collagen fibre content in bone tissue Collagen fiber area(%) 2-year-old horse bone 8-year- old horse bone 13-year -old horse bone 6-year-old sheep bone 8-year-old cow bone 71.88 ± 0.87 ab 79.99 ± 3.43 a 48.63 ± 2.10 d 79.39 ± 3.26 a 65.21 ± 2.40 c The collagen fibrillar fibres of the 13-year-old horse bone tissue were in loose bundles, were disorganised, and had an uneven density distribution. Furthermore, the collagen fibre area was 48.63%, significantly differing from that of the 2- and 8-year-old horses ( P < 0.05). In contrast, the fibrillar fibres of the 2- and 8-year-old horse bone tissue were curly strips with a uniform fibre density. The bone biopsy results indicated that the fibrillar fibres of the horse bone first increased and then decreased with age, suggesting that the bone grows and matures rapidly during youth; however, the collagen fibres do not change much during adulthood. As the horses age, the bones loosen, and the collagen fibres become disorganised. Moreover, the collagen fibres of the horse bone were compact, regularly shaped, and formed by more plates, consistent with the results of Zedda et al. (2020). Further, the horse bone fibrils were normal, with uniform density and orderly arrangement. Notably, the collagen fibre area of adult horse bones significantly differed from that of ox bones of the same age ( P < 0.05); goat bones, contrastingly, had a looser bone structure. As the bone develops with age, its density increases, as does calcification, owing to the deposition and maturation of calcium phosphate minerals and increased calcium salt distribution (Isaksson et al. 2010). Calcium salt distributions were higher in the bones of 2- and 8-year-old horses than in those of the 13-year-old horses (Fig. 4 ). The calcium salt distribution sizes were compared among species of the same age, with the distribution size slightly lower in horse bone than in ox bone; however, the collagen fibre area in horse bone was approximately 15% larger than that in ox bone. Therefore, Mongolian horse bone is highly dense and nutrient-rich, containing calcium, protein, and minimal fat. Furthermore, Mongolian horse bone is richer in collagen than the bones of other domestic animals, and the composition is similar to that of human collagen. 3.3. Degreasing horse bone meal The optimal parameters for degreasing horse bone powder using ultra-high pressure were explored. First, various pressures were tested (time: 9 min; solid-to-liquid ratio: 1:15 g/mL), and the degreasing rate peaked at 300 MPa (Fig. 5 A). Then, various times were tested (pressure: 300 MPa; solid-to-liquid ratio: 1:15 g/mL), and the degreasing rate increased significantly from 5 to 9 min ( P < 0.05), peaking at 9 min under pressure (Fig. 5 B). Finally, various solid-to-liquid ratios were tested (time: 9 min; pressure: 300 MPa). The degreasing rate significantly increased from solid-to-liquid ratios of 1:5 to 1:15 g/mL ( P < 0.05), peaking at a ratio of 1:15 g/mL (Fig. 5 C). Likely, 1:15 g/mL was the optimal ratio because the oil in the horse bone meal dissolved after adding enough degreasing solvent. Overall, the highest degreasing rate was 73.56%, achieved with 300 MPa of pressure for 9 min, with a solid-to-liquid ratio of 1:15 g/mL. The highest ethyl acetate degreasing rate was 70.24%, suggesting that degreasing horse bone powder is more effective with ultra-high pressure than with ethyl acetate. 3.4. Enzymatic collagen extraction from horse bones The optimal parameters for enzymatic collagen extraction from horse bones were explored next. First, the extraction times were tested with a 6% enzyme (pepsin) dose and a 1:15 g/mL solid/liquid ratio. The collagen extraction rate peaked (23.03%) after 24 h (Fig. 6 A). Next, various pepsin doses were tested with a 24-h extraction time and 1:15 g/mL solid/liquid ratio, and the extraction rate peaked at 6% pepsin (Fig. 6 B). Finally, various solid/liquid ratios were tested with a 24-h extraction time and 6% pepsin dose; the extraction rate peaked (21.57%) at 1:15 g/mL (Fig. 6 C). Overall, the highest collagen extraction rate was 26.65%, achieved with an enzyme dosage of 6%, extraction time of 24 h, and solid/liquid ratio of 1:15 g/mL. Collagen dissolution is the transition from solid to solute, during which pepsin promotes the release of collagen into the extraction solution. Thus, the extraction rate gradually increases as the extraction time increases (Ali et al. 2018). High enzyme concentrations also increase the extraction rate; however, it eventually decreases, as in our study (at 6% pepsin), perhaps because of insufficient substrate to continue the reaction. At this point, a further increase in the protease amount causes excessive enzymatic hydrolysis, breaking small-molecule protein fragments into free amino acids without increasing the collagen extraction rate (Lee et al. 2022). Furthermore, we found that solid-to-liquid ratios greater than 1:15 g/mL decreased the extraction rate. Excess solvent may weaken the stability of collagen, breaking the peptide chain and resulting in many peptide chain fragments, thus reducing the extraction rate (Matinong et al. 2022). Vidal et al. (2020) investigated yak bone collagen extraction, finding that the extraction rate initially increased and then decreased as the solid-to-liquid ratio increased; their extraction rate also peaked at a 1:15 g/mL solid-to-liquid ratio. Thus, too much solvent volume is not conducive to collagen extraction. 3.5. Structural characterisation and resolution of horse bone collagen Figure 7 presents the results of the infrared spectrum analysis of collagen extracted from horse bone powder from 400 to 4000 cm − 1 . The amide A, Ⅰ, Ⅱ, and Ⅲ band intensities in ultra-high pressure-treated collagen samples from the ribs of an 8-year-old horse were slightly lower than those of untreated bones (Fig. 7 A). Moreover, the absorption peaks of the rib and tibia decreased slightly in the amide zone with age (Figs. 7 B, C). We also measured the denaturation temperature of the collagen samples using differential scanning calorimetry and found that the denaturation temperature increased slightly with age (Table 3 ). Moreover, the denaturation temperatures of collagen extracted from the rib and tibia of 8-year-old horses treated with ultra-high-pressure were 30.54 ℃ and 32.40 ℃, respectively, whereas the temperature for untreated collagen was 36.52 ℃. Table 3 Thermostability of collagen extracted from horse bones age Denaturation temperature (T/℃) No ultra-high-pressure treatment Horse rib Horse tibia 2 32.05 ± 0.12 c 28.25 ± 0.21 c 29.35 ± 0.37 c 8 36.52 ± 0.25 b 30.54 ± 0.20 b 32.40 ± 0.27 b 13 43.23 ± 0.17 a 35.37 ± 0.47 a 36.74 ± 0.08 a Note: Significance is indicated by different lowercase letters in the same column (P < 0.05) Infrared spectroscopy can detect nearly all infrared spectra of biological materials under various conditions, making it one of the most effective methods to study protein hydrogen bonding (Jie et al. 2018). We found that ultra-high-pressure treatment had little effect on the structure of collagen extracted from horse bone meal, consistent with the results of Nan et al. (2018). The absorption of the amide A band is related to the N-H stretch vibration, and the absorption peak usually appears in the 3400–3440 cm − 1 region. However, when the N-H stretch vibration combines with a hydrogen bond, it may shift to a lower frequency, moving towards 3300 cm − 1 (Singh et al. 2011). The amide A bands in the untreated and treated collagen samples were at 3439.07 cm − 1 and 3435.22 cm − 1 , respectively, indicating that the collagen extracted from horse bone meal contains a hydrogen bond structure. Moreover, the amide B band peaks in the untreated and treated samples were at 2928.57 cm − 1 and 2947.23 cm − 1 , respectively, caused by the asymmetric stretching vibration of CH 2 in the molecule (Hazeena et al. 2022). The characteristic frequency range of the amide Ⅰ band is 1600–1700 cm − 1 , which is mainly related to the stretching vibration of the C = O bond along the peptide’s main chain. The amide Ⅰ band can be used to analyse the secondary structure of proteins; the larger the frequency value, the higher the order of the peptide chain skeleton (Gao et al. 2018). The absorption peaks of untreated and treated collagen samples were at 1656.06 cm − 1 and 1649.13 cm − 1 , respectively. Moreover, the absorption peaks for the amide II bands in the untreated and treated samples were at 1543.05 cm − 1 and 1541.23 cm − 1 , respectively, caused by the N-H bending vibration coupled with the C-N stretching vibration of collagen. High amide Ⅱ frequencies indicate the presence of many intermolecular hydrogen bonds in collagen, especially in the helical part of the collagen structure (Yu et al. 2018). The absorbance peaks of the untreated and treated collagen samples at 1483.18 cm − 1 and 1477.17 cm − 1 are closer to absorbance 1.0, indicating that the tertiary structure of collagen is intact. Finally, the amide Ⅲ bands for the untreated and treated samples were observed at 1220.94 cm − 1 and 1217.08 cm − 1 , respectively. This finding suggests that glycine, proline (Pro), and hydroxyproline content in collagen was relatively high, which makes horse bones treated in different ways have unique absorption peaks at 1200–1400 cm − 1 due to the C-N stretching and N-H bending vibrations, consistent with the CH 2 rocking vibration peak attributed to the glycine skeleton and Pro side chain (Ye et al. 2021). In conclusion, we found that the secondary structure of the extracted equine bone collagen was intact (Kobrina et al. 2010; Sarin et al. 2019; Ferraro et al. 2017). We also found that the rib and tibia absorption peaks decreased slightly with age in the amide zone, suggesting that horse bones have a complete triple helix structure, regardless of age. Kobrina et al. (2010) analysed the infrared spectral absorption peak of equine cartilage collagen from newborn (0 days) to immature age (5 to 11 months) and from immature age to adult age (6 to 10 years), observing that the infrared spectral absorption peaks increased with age, consistent with our results. We also found that the collagen’s denaturation temperature increased slightly with age, possibly due to a higher degree of crosslinking of the bone fibrillar fibres and the size of mineral crystals, which change with age, resulting in a slight increase in the thermal denaturation temperature of horse bone collagen (Ferraro et al. 2017). Notably, the denaturation temperature was lower in collagen samples from older horses treated with ultra-high pressure than in untreated samples. This might be due to fractured collagen crosslinking bonds in the bone meal after treatment, weakening the stability and, thus, decreasing thermal stability. 3.6. Extracting short peptides from horse bone collagen After characterising the horse bone collagen, we attempted to extract short peptides from the collagen samples and optimise the alkaline protease conditions. We measured the yield after varying the pH (constants: hydrolysis time: 4 h, temperature: 50 ℃, and enzyme [i.e., alkaline protease] dose: 3% by mass); enzymatic hydrolysis time (constants: temperature: 50 ℃, pH: 9.0, and enzyme dose: 3%); temperature (constants: hydrolysis time: 4 h, pH: 9.0, and enzyme dose: 3%); and enzyme dose (constants: hydrolysis time: 4 h, temperature: 50 ℃, and pH: 9.0) (Fig. 8 ). The optimal conditions for a maximal short peptide yield were pH 9 (Fig. 8 A), 4 h of enzymolysis (Fig. 8 B), 50 ℃ temperature (Fig. 8 C), and 3% enzyme dose (Fig. 8 D). The pH for the optimal short peptide yield was 9.0, indicating that pH primarily affects protease activity. Each protease has an optimal pH range, outside of which the activity decreases, and the enzymatic hydrolysis efficiency will be correspondingly low, decreasing the short peptide yield (Jiang et al. 2014). Furthermore, we found that reaction times exceeding 4 h decreased the yield. Likely, a longer reaction resulted in more enzymolysis products and complexes, which inhibited the enzymolysis reaction, decreased the reaction speed, and thus decreased the yield. Furthermore, the peptide’s reaction site becomes saturated over time; some peptides are decomposed into amino acids, and the decomposition rate exceeds the protein peptide formation rate, decreasing the peptide yield (Vidal et al. 2022). We also found that the short peptide yield increased and then decreased as the temperature increased, peaking at 50 ℃ (yield: 63.78%). When the temperature rises, the protein conversion rate increases; however, the enzyme protein will slowly denature or deactivate, decreasing the enzyme reaction rate and, thus, the peptide yield (Beaubier et al. 2021). Finally, we found the highest yields with a 3% enzyme dose (yield: 62.63%). Adding enzymes to the reaction increases the probability that the enzyme and collagen interact, which increases the yield. However, the yield will no longer increase once the substrate is saturated. 3.7. Short peptide antioxidant activity We isolated four groups of horse collagen peptides with different molecular weights: 5–10, 3–5, 1–3, and < 1 KDa. The reducing abilities of horse bone collagen short peptide with different molecular weights extracted from horses of different ages gradually increased as the concentration increased from 5 to 10 mg/mL ( P < 0.05; Tables 4 and 5 ). The reducing abilities decreased as the molecular weights increased; 1–3 kDa components had the strongest abilities, whereas 5–10 kDa components had the weakest abilities. These results were compared with the reducing power of the antioxidant VC. However, compared with that of the antioxidant VC at the same concentration, the reducing power was weaker with reducing molecular weights, and the difference was significant ( P < 0.05). Moreover, the hydroxyl radical-scavenging ability of peptides with the same molecular weight differed based on the age of the bones, and the activity was the strongest in the extracts from 8-year-old bones (Tables 6 and 7 ). Finally, with the increase in the concentration of collagen peptide solution, the O 2 free radical clearance rate increased for each ultrafiltration component (Tables 8 and 9 ). Table 4 Reduction capacity of peptides isolated from collagen extracted from horse rib bones age reducing force concentration (mg/mL) 5–10 KDa 3–5 KDa 1–3 KDa < 1 KDa 2 5 0.0800 ± 0.004 d 0.0890 ± 0.001 bc 0.1037 ± 0.001 a 0.0940 ± 0.002 b 10 0.0870 ± 0.001 c 0.0903 ± 0.005 c 0.1130 ± 0.002 a 0.0947 ± 0.002 b 8 5 0.0883 ± 0.011 c 0.0920 ± 0.002 c 0.1350 ± 0.007 a 0.1013 ± 0.015 ab 10 0.1090 ± 0.012 d 0.1217 ± 0.005 c 0.1813 ± 0.005 a 0.1597 ± 0.003 b 13 5 0.0813 ± 0.005 c 0.0803 ± 0.001 c 0.1043 ± 0.025 a 0.0997 ± 0.010 b 10 0.0903 ± 0.004 c 0.0983 ± 0.011 bc 0.1800 ± 0.010 a 0.1130 ± 0.002 b Vc 5 3.7210 ± 0.010 10 3.8077 ± 0.051 Note: Peer reduction ability represented by different lowercase letters is significantly correlated at the 0.05 level. Table 5 Reduction capacity of peptides isolated from collagen extracted from horse tibia bones age reducing force concentration(mg/mL) 10 KDa-5KDa 5 KDa-3KDa 3 KDa-1KDa < 1KDa 2 5 0.0787 ± 0.002 c 0.0853 ± 0.001 bc 0.1077 ± 0.008 a 0.0873 ± 0.001 b 10 0.0830 ± 0.002 cd 0.0877 ± 0.001 bc 0.1213 ± 0.002 a 0.0923 ± 0.002 b 8 5 0.0833 ± 0.001 cd 0.0893 ± 0.0005 c 0.1030 ± 0.008 a 0.0917 ± 0.001 b 10 0.0877 ± 0.001 c 0.0943 ± 0.001 b 0.1250 ± 0.002 a 0.0963 ± 0.004 ab 13 5 0.0733 ± 0.001 d 0.0873 ± 0.002 c 0.1007 ± 0.003 a 0.0913 ± 0.002 b 10 0.0840 ± 0.025 c 0.0903 ± 0.0005 b 0.1117 ± 0.006 a 0.0940 ± 0.001 b Note: Different lowercase letters indicate a significant correlation with reduction capacity (P < 0.05). Table 6 Hydroxyl radical-scavenging ability of peptides isolated from collagen extracted from horse rib bones age Hydroxyl radical scavenging power concentration(mg/mL) 10 KDa-5KDa 5 KDa-3KDa 3 KDa-1KDa < 1KDa 2 5 68.90 ± 0.387 d 73.90 ± 0.590 c 88.81 ± 1.686 a 84.58 ± 0.572 b 10 71.64 ± 1.394 cd 77.58 ± 0.997 c 98.21 ± 0.614 a 88.08 ± 1.397 b 8 5 69.80 ± 0.554 c 81.65 ± 2.159 b 92.39 ± 0.775 a 85.41 ± 0.964 ab 10 73.15 ± 2.015 c 86.57 ± 0.757 b 98.65 ± 0.675 a 92.84 ± 1.027 ab 13 5 70.47 ± 1.340 c 80.10 ± 1.023 b 90.82 ± 2.358 a 84.56 ± 1.778 ab 10 81.20 ± 1.368 c 88.59 ± 2.685 bc 97.94 ± 0.741 a 89.07 ± 1.045 g Vc 5 10 90.94 ± 1.806 92.85 ± 2.057 Note: Different lowercase letters indicate a significant correlation with hydroxyl radical clearance (P < 0.05). Table 7 Hydroxyl radical-scavenging ability of peptides isolated from collagen extracted from horse tibia bones age Hydroxyl radical scavenging power concentration(mg/mL) 10 KDa-5KDa 5 KDa-3KDa 3 KDa-1KDa < 1KDa 2 5 66.66 ± 2.046 c 76.95 ± 1.547 b 92.41 ± 1.066 a 82.96 ± 1.211 b 10 75.39 ± 1.394 c 78.10 ± 2.685 bc 95.27 ± 2.060 a 86.57 ± 1.340 b 8 5 75.16 ± 2.926 c 83.34 ± 2.203 b 94.50 ± 1.049 a 90.53 ± 1.345 ab 10 75.16 ± 2.926 c 83.44 ± 2.358 bc 96.15 ± 2.425 a 93.28 ± 2.006 ab 13 5 72.93 ± 0.779 cd 78.29 ± 2.536 b 93.96 ± 1.160 a 87.60 ± 1.346 ab 10 75.61 ± 0.779 c 82.43 ± 0.851 b 95.68 ± 2.679 a 89.48 ± 0.774 ab Note: Different lowercase letters indicate a significant correlation with hydroxyl radical clearance (P < 0.05). Table 8 Superoxide anion-removal capacity of peptides isolated from collagen extracted from horse rib bones age Superoxide anion scavenging capacity concentration(mg/mL) 10 KDa-5KDa 5 KDa-3KDa 3 KDa-1KDa < 1KDa 2 5 19.84 ± 2.745d 26.19 ± 2.380 c 41.27 ± 1.377 a 34.12 ± 0.374 b 10 20.63 ± 0.634d 27.77 ± 2.380 c 46.82 ± 1.374 a 39.68 ± 1.371 b 8 5 29.36 ± 1.231d 34.91 ± 0.663 c 46.03 ± 1.072 a 41.27 ± 0.635 b 10 35.71 ± 2.385d 40.50 ± 2.004 c 51.99 ± 0.656 a 49.77 ± 2.412 b 13 5 22.96 ± 0.764d 27.77 ± 1.371 c 41.27 ± 1.376 a 37.33 ± 1.406 b 10 23.75 ± 1.166 d 34.91 ± 0.374 c 45.12 ± 0.412 a 39.68 ± 0.517 ab Vc 5 60.52 ± 0.745 10 79.74 ± 1.011 Note: Different lowercase letters indicate a significant correlation with superoxide anion clearance (P < 0.05). Table 9 Superoxide anion-removal capacity of peptides isolated from collagen extracted from horse tibia bones age Superoxide anion scavenging capacity concentration(mg/mL) 10 KDa-5KDa 5 KDa-3KDa 3 KDa-1KDa < 1KDa 2 5 25.25.±0.685 d 29.46 ± 1.610 c 40.95 ± 0.685 a 37.08 ± 0.707 b 10 28.57 ± 0.387 d 33.33 ± 2.380 c 46.03 ± 1.374 a 39.43 ± 0.906 b 8 5 31.39 ± 1.231 d 33.65 ± 0.736 c 41.75 ± 1.537 a 38.68 ± 0.987 b 10 37.08 ± 0.707 c 40.76 ± 2.004 c 54.73 ± 2.415 a 45.12 ± 0.190 b 13 5 26.39 ± 1.199 d 30.37 ± 0.345 c 45.12 ± 0.190 a 38.11 ± 1.716 b 10 34.13 ± 0.589 d 39.47 ± 0.393 c 49.10 ± 0.412 a 42.20 ± 0.517 ab Note: Different lowercase letters indicate a significant correlation with superoxide anion clearance (P < 0.05). Reducing capacity and antioxidant activity are somewhat related; thus, the antioxidant efficacy of collagen peptides can be assessed by measuring their reducing capacity (Wang et al. 2023). In Fe 3+ and Fe 2+ reaction systems, the greater the light absorption value at 700 nm, the stronger the reducing power (Bo et al. 2017). Our results were consistent with those of Chen et al. (2023), who found that low-molecular-weight polypeptides of collagen peptide had a strong reducing ability. A dose–effect relationship exists for collagen peptides of different molecular weights and their ability to clear hydroxyl radicals (Lee et al. 2022). We found that peptides with the same molecular weight had variable hydroxyl radical-scavenging abilities (antioxidant activities), based on the age of the bone; extract from 8-year-old horses had the strongest activity. Moreover, when the concentration of the collagen peptide solution was the same, the hydroxyl radical-scavenging ability of short peptides (molecular weight: 1–3 KDa) was significantly higher than that of other molecular weight components. In addition, the hydroxyl free radical-scavenging ability was slightly higher with an equivalent concentration of VC than that of VC alone. The hydroxyl free radical-scavenging abilities of the collagen peptides extracted from the horse tibia and ribs demonstrated that these peptides could scavage hydroxyl free radicals, consistent with the finding of Hernández-Ruiz et al. (2023), who reported that the small molecular weight collagen peptide components had superior activity than the large molecular weight components. However, this result is controversial as others have reported conflicting results. For example, Shen et al. (2021) reported that the 5–10 KDa molecular weight pig skin collagen antioxidant peptides have good antioxidant activity, suggesting that the antioxidant activity of collagen peptides is related to their relative molecular weight and is influenced by their amino acid composition and sequence (Sun et al. 2021). Superoxide anion radicals (O2-·) are produced during processes such as oxygen transport in haemoglobin, autoxidation, mitochondrial electron transport chain, and cytochrome reactions. When light induces melanin production, too many lone electron pairs are formed, and O 2 ·can form. In addition, superoxide cation radicals can induce the formation of other autonomous groups, such as hydroxyl radicals (Zu et al. 2023). Finally, we found that as the concentration of collagen peptide solution increased, the O 2 free radical clearance rate increased for each ultrafiltration component, suggesting a dose–effect relationship (Zu et al. 2022). Furthermore, for peptides with the same molecular weight, the antioxidant activity of the peptides differed based on the age of the bones, and the superoxide anion-scavenging activity was the strongest with the peptides extracted from an 8-year-old horse bone. When the concentration of collagen peptide solution was equivalent, the O 2 scavenging activity decreased as the molecular weights increased; 1–3 kDa components had the strongest abilities, whereas 5–10 kDa components had the weakest abilities. Thus, horse bone meal collagen peptides with different molecular weights can clear O 2 at rates similar to those of VC, although 5–10 KDa components have a lower superoxide anion clearance rate than VC. Similar results were reported in a study by Irshad et al. (2015), who found that 1–3 KDa bovine spinal cord polypeptides had better antioxidant activity than other components. 4. Conclusion In this study, we extracted and characterised collagen from horse bone, followed by short peptide extraction from these collagen samples. An imaging analysis first confirmed significant differences in the fibrillar fibres of horse bones of different ages. Furthermore, the collagen fibres of adult horse bones were regularly shaped, uniformly dense, arranged orderly, and had a higher bone density than other animal bones (cattle and goat). The thermal stability of the horse bones also increased with age. Moreover, ultra-high-pressure treatment improved the horse bone degreasing rate compared to the use of ethyl acetate but decreased the denaturation temperature of horse bone collagen. However, the treatment left the tertiary structure of collagen intact. Finally, we isolated four groups of horse collagen peptides with different molecular weights, which had different antioxidant capacities; the components with the lowest molecular weights (1–3 KDa) had the highest activities. Moreover, age affected the peptide’s antioxidant activity independent of molecular weight, and the antioxidant activity of the peptides from 8-year-old horse bones was the strongest. Domestic research has been conducted based on the national conditions of China, according to the regional characteristics of the horse industry. However, there are few studies on the high-quality resource, Mongolian horse bone. Therefore, how to use bioengineering technology reasonably to develop and study the precious resource of horse bone and explore its economic value is a research topic with broad prospects and practical significance. At present, our team is focusing on the study of bovine and sheep bone collagen and peptides. Based on this, collagen was extracted from Mongolian horse bone and prepared into collagen short peptides, and its antioxidant activity was determined. This study lays the foundation for further development, utilisation, and industrial upgrading of horse bone collagen peptides. Abbreviations UHP: Ultra-high-pressure processing; DSC, differential scanning calorimeter; Declarations The authors declare no conflict of interest. All data generated or analysed during this study are included in this published article. Statement of Ethics : All animal experiments were performed according to the regulations of the Administration of Affairs Concerning Experimental Animals in China. The experimental protocol was approved by the Animal Welfare and Research Ethics Committee of the Inner Mongolia Agricultural University (approval ID: 20154617–8). Author Contribution Jindi Wu, propose research topics, design research plans, thesis writingHeya Na, Statistical analysis dataFan Bai, collect sample Siyu Li , Statistical analysis dataHao Gao, Statistical analysis data Rina Sha, propose research topics, design research plans,obtain research funding Acknowledgements: This work has been funded by the Postgraduate Training fund (RC1900004555) and the Natural Science Foundation of Inner Mongolia (Grant No. 2022QN03026). We would like to thank Elsevier Language Editing Services for English language editing. 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05:23:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4512011/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4512011/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-75960-7","type":"published","date":"2024-10-28T15:57:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59872178,"identity":"3bad996b-16ce-4cba-a77d-800fcf4f8956","added_by":"auto","created_at":"2024-07-08 17:11:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":59142,"visible":true,"origin":"","legend":"\u003cp\u003eCurrent application direction for horses.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4512011/v1/f65779bd6f5ac396844087a0.jpg"},{"id":59871152,"identity":"b07ec279-2c71-408a-ab3d-d205d84f04d1","added_by":"auto","created_at":"2024-07-08 17:03:31","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":84413,"visible":true,"origin":"","legend":"\u003cp\u003eBasic bone composition of Mongolian horses of different ages.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4512011/v1/ff4753170d26820ce5e18d2e.jpg"},{"id":59871155,"identity":"e37c14ca-e6e1-4e59-92e3-f9d90f3a166c","added_by":"auto","created_at":"2024-07-08 17:03:32","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":134583,"visible":true,"origin":"","legend":"\u003cp\u003ePicric acid scarlet staining of bone tissue collected from Mongolian horses, oxen, and goats (20´ magnification).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4512011/v1/1c3748b5ac65d2bb3ead489c.jpg"},{"id":59873576,"identity":"eea68695-4dbc-4879-b4a5-ae8d1e15a4e1","added_by":"auto","created_at":"2024-07-08 17:27:32","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":103488,"visible":true,"origin":"","legend":"\u003cp\u003eSirius scarlet staining of bone tissue collected from Mongolian horses, oxen, and goats (20´ magnification).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4512011/v1/75ff915b53df1550c93df647.jpg"},{"id":59871153,"identity":"e921768c-dc63-4b58-bce2-8133103ae300","added_by":"auto","created_at":"2024-07-08 17:03:31","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":42544,"visible":true,"origin":"","legend":"\u003cp\u003eOptimising the horse bone powder degreasing rate. Effects of (A) ultra-high pressure (time: 9 min; solid-to-liquid ratio: 1:15 g/mL); (B) time under pressure (pressure: 300 MPa; solid-to-liquid ratio: 1:15 g/mL); and (C) the solid/liquid ratio (time: 9 min; pressure: 300 MPa).\u003c/p\u003e\n\u003cp\u003ea: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; b: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; c: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4512011/v1/ca93a9ccc41aee891a8b8067.jpg"},{"id":59872179,"identity":"b92fde02-f2bf-4183-a9bf-ddafd2c421d8","added_by":"auto","created_at":"2024-07-08 17:11:32","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":52208,"visible":true,"origin":"","legend":"\u003cp\u003eOptimising the collagen extraction rate from horse bones. Effects of (A) the extraction time (enzyme [pepsin] dose: 6%; solid-to-liquid ratio: 1:15 g/mL); (B) the enzyme dose (time: 24 hours; solid-to-liquid ratio: 1:15 g/mL); and (C) the solid-to-liquid ratio (time: 24 hours; enzyme dose: 6%).\u003c/p\u003e\n\u003cp\u003ea: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; b: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; c: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4512011/v1/9c8e7d7b70fa2e307b12dc68.jpg"},{"id":59873180,"identity":"e4eb52a9-a85d-45ee-880d-ddfe6e714b9f","added_by":"auto","created_at":"2024-07-08 17:19:32","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":79590,"visible":true,"origin":"","legend":"\u003cp\u003eInfrared spectrum analysis (400–4000 cm\u003csup\u003e-\u003c/sup\u003e1) of collagen extracted from horse bone powder. (A) Collagen extracted from 8-year-old horse rib bone (a) without and (b) with ultra-high-pressure treatment. (B) Collagen extracted from the ribs of horses of different ages: (a, black) 2 years, (b, red) 8 years, and (c, blue) 13 years. (C) Collagen extracted from the tibia of horses of different ages: (a, black) 2 years, (b, red) 8 years, and (c, blue) 13 years.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4512011/v1/7ac929191ef57ff541c1b063.jpg"},{"id":59871159,"identity":"cebe7af0-a362-4627-8a57-5f17db1ac3b4","added_by":"auto","created_at":"2024-07-08 17:03:32","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":52508,"visible":true,"origin":"","legend":"\u003cp\u003eOptimising the short peptide yield from collagen extracted from horse bones. Effects of (A) pH (constants: hydrolysis time: 4 h, temperature: 50 ℃, and enzyme [i.e., alkaline protease] amount: 3% by mass); (B) enzymatic hydrolysis time (constants: temperature: 50 ℃, pH: 9.0, and enzyme dose: 3%); (C) temperature (constants: hydrolysis time: 4 h, pH: 9.0, and enzyme dose: 3%); and (D) the enzyme dose (constants: hydrolysis time: 4 h, temperature: 50 ℃, and pH: 9.0).\u003c/p\u003e\n\u003cp\u003ea: P \u0026lt; 0.001; b: P \u0026lt; 0.01; c: P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4512011/v1/907ba05ddebe23d6eada244c.jpg"},{"id":68206642,"identity":"151f5ee0-ab22-427b-9990-7df26a615b54","added_by":"auto","created_at":"2024-11-04 16:33:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1583588,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4512011/v1/2ca7dd57-d65b-4003-9bfa-0c4f13d2c276.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preparation and tissue structure analysis of horse bone collagen peptide","fulltext":[{"header":"Lay summary","content":"\u003cp\u003eHorse bones are a natural source of collagen, and at present, there are few reports on the development and research of bone-related food products. Compared with other livestock, horse bones have higher nutrient content ratios, making them a very high-quality collagen and calcium resource. Moreover, the bone quality of horse bones is dense, invigorating, detoxifying, and healing, and it has been used as a traditional medicine to treat fractures and arthritis, etc.The present study investigated a novel source of collagen and developed a methodology for extracting collagen peptides. Mongolian horse bones were identified as an abundant source of calcium and protein with low fat content. This study explored the extraction of collagen using ultra-high pressure processing, along with the evaluation of short peptides derived from Mongolian horse bones.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eUltra-high-pressure processing (UHP), also known as hydrostatic pressure technology, is a food processing technology that has attracted attention in the Chinese food industry, with substantial efforts and investments dedicated towards its research and development. UHP involves shaping or sizing food through vacuum packaging. In this process, food is placed in ultra-high pressure treatment equipment according to the basic principles of Le Chatelier and Pascal. A liquid medium is then employed to sterilise the food at pressures exceeding 100 MPa, effectively eliminating enzymes and modifying the food\u0026rsquo;s characteristics. This physical food processing method extends the shelf life of food (Kazutaka 2017). In essence, ultra-high-pressure treatment disrupts non-covalent bonds in food substances including hydrogen bonds, molecular bonds, hydrophobic effects, and electrostatic effects as well as the intricate organisational tertiary structures of the biomolecules in the food. Notably, covalent bonds of small molecules, such as vitamins and amino acids, remain unaffected. Consequently, the flavour, nutrition, and colour of the food are preserved (Nan \u003cem\u003eet al.\u003c/em\u003e 2018). Currently, UHP is the preferred sterilisation method in the food industry (Patrignani \u003cem\u003eet al.\u003c/em\u003e 2019), as it retains the food\u0026rsquo;s original composition (Xi \u003cem\u003eet al.\u003c/em\u003e 2021). Moreover, the freshness of the food is not affected and can even be extended. As a result, UHP demonstrates considerable developmental potential (Aganovic \u003cem\u003eet al.\u003c/em\u003e 2021).\u003c/p\u003e \u003cp\u003eDong \u003cem\u003eet al.\u003c/em\u003e (2014) studied the effects of flavour enzymes on chicken bone hydrolysis over time, finding that the proportion of small molecular peptides (400\u0026ndash;1000 Da) in products hydrolysed for 8 hours was 74 times higher than that in those hydrolysed for 1 hour. Kim \u003cem\u003eet al.\u003c/em\u003e (2014) investigated the physicochemical properties of type Ⅰ collagen from bovine bone and studied the antioxidant activity of low-molecular-weight peptides from the leg bone of Jeju horses. Leon-Lopez \u003cem\u003eet al.\u003c/em\u003e (2019) explored how the hydrolysis time affected collagen extraction and enzymolysis from sheep skin, as well as its structure and antioxidant activity. Moreover, Miroslava \u003cem\u003eet al.\u003c/em\u003e (2019) separated squid collagen peptide using ultrafiltration membranes, finding that the hydrolysate with a molecular weight of less than 5 kDa had a strong antioxidant activity. Finally, Hou \u003cem\u003eet al.\u003c/em\u003e (2023) found that components in the collagenase hydrolysate (with a molecular weight of 1\u0026ndash;5 kDa) of sturgeon skin had a strong superoxide anion-scavenging ability.\u003c/p\u003e \u003cp\u003eHorse bone, often used in traditional medicine to treat bone diseases, fractures, and arthritis, has a higher nutrient content ratio than bone from other domestic animals and is rich in high-quality collagen and calcium (Lee \u003cem\u003eet al.\u003c/em\u003e 2007). However, limited studies have explored bones as a collagen source. Recently, efforts have been made to industrialise the Chinese horse industry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, progress has been slow due to a lack of attention and failure to emphasise its social and economic benefits. Consequently, the industry remains in the initial stages of industrialisation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven the benefits of collagen and the rich nutrients derived from bone, this study explored the extraction and preparation of collagen short peptides from Mongolian horse bones, aiming to expand the use of horse bones in the industry.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eThe materials for this experiment were the ribs and tibia of 2-, 8- and 13-years-old horses, which were provided by Shandong Dezhou Demu Meat Processing Co., LTD. Sheep bones and cattle bones were provided by Zhongke Wanxin Selenium-Enriched Halal Food Co., LTD., Ordos City, Inner Mongolia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Reagents\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eReagents (and their manufacturers) used in the laboratory\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReagent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eManufacturer\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthyl acetate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTianjin Zhiyuan Chemical Reagent Co., LTD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePetroleum ether\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSinopharm Chemical Reagent Co. LTD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlacial acetic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTianjin Damao Chemical Reagent Factory\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePepsinum\u003c/p\u003e \u003cp\u003eAlkaline protease\u003c/p\u003e \u003cp\u003eBovine Serum Albumin□\u003c/p\u003e \u003cp\u003eCoomath bright blue G-250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSolarbio\u003c/p\u003e \u003cp\u003eSolarbio\u003c/p\u003e \u003cp\u003eTianjin Yongda Chemical Reagent Co., LTD\u003c/p\u003e \u003cp\u003eTianjin Yongda Chemical Reagent Co., LTD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMD34 dialysis bag\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSolarbio\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsolute ethyl alcohol\u003c/p\u003e \u003cp\u003eXylene\u003c/p\u003e \u003cp\u003eSirius scarlet dye\u003c/p\u003e \u003cp\u003eAlizarin red dye solution\u003c/p\u003e \u003cp\u003ePermount TM Mounting Medium\u003c/p\u003e \u003cp\u003eGlycol diethyl ether acetate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSinopharm Chemical Reagent Co. LTD\u003c/p\u003e \u003cp\u003eSinopharm Chemical Reagent Co. LTD\u003c/p\u003e \u003cp\u003eServicebio\u003c/p\u003e \u003cp\u003eServicebio\u003c/p\u003e \u003cp\u003eSinopharm Chemical Reagent Co. LTD\u003c/p\u003e \u003cp\u003eMacklin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHydroxyproline (HYP) content detection kit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSolarbio\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEDTA\u003c/p\u003e \u003cp\u003ePotassium ferricyanide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSolarbio\u003c/p\u003e \u003cp\u003eTianjin Yongda Chemical Reagent Co., LTD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Hard tissue section preparation and staining\u003c/h2\u003e \u003cp\u003eFresh equine tibia of various ages were chosen, and the bone tissue was fixed with 4% paraformaldehyde solution (PFA) for 48 h. Subsequently, the tissues were subjected to dehydration by adding different concentrations of alcohol successively, and each gradient was maintained for 24\u0026ndash;72 h. The sliced tibia of the horse was put into an embedding bottle, with an appropriate amount of embedding solution added. The system was vacuum-sealed for 5 h, and a water bath was set at 37 ℃. The bone tissue was sliced into approximately 10 \u0026micro;m using a slicer, and these slices were oven-roasted at 60 ℃ overnight. The sections were dehydrated with ethylene glycol ether acetate and ethanol of different gradients. The treated tissues were stained with Alizarin red and Sirius scarlet, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Ultra-high-pressure treatment\u003c/h2\u003e \u003cp\u003eThe crushed horse bone was vacuum-packed and placed inside the ultra-high-pressure equipment. Distilled water was used as the medium for ultra-high-pressure treatment. (Control group: defatted horse bones without ultra-high-pressure treatment)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Extraction of collagen from horse bone meal\u003c/h2\u003e \u003cp\u003eThe horse bone meal was mixed with ethyl acetate solution at a ratio of 1:15 g/mL, and the solution was shaken and degreased at 4℃ for 24 h. The decalcified horse bone meal was added into 0.1 mol/L NaOH solution at 1:10 g/mL, and the protein was removed by continuous agitation at 4℃ for 24 h. After the horse bone collagen was extracted, the supernatant was removed by centrifugation at 11171.56 x \u003cem\u003eg\u003c/em\u003e for 20 min at 4℃. The precipitation continues to undergo enzymolysis, and this process is repeated twice to incorporate the supernatant. The supernatant was adjusted to a pH value of 7.4 with 3 mol/L NaOH solution. Then the NaCl solid powder was slowly added to the collagen extraction solution until the concentration reached 0.9 mol/L. The powder was placed in a chromatography tank at 4℃ for 24 h for salting out and centrifuged at 11171.56 x \u003cem\u003eg\u003c/em\u003e for 20 min to retain the precipitation. The resulting precipitates were dissolved with 0.5 mol/L glacial acetic acid and put into a pre-treated dialysis bag. At 4℃, they were dialysed with 5 mmol/L glacial acetic acid solution for 48 h and then with distilled water for 24 h. The collagen obtained after dialysis was injected into a glass plate, frozen overnight in a refrigerator at \u0026minus;\u0026thinsp;80℃, and then dried in a vacuum freeze-dryer for approximately 24 h to obtain white powdered crystalline collagen, which was refrigerated at \u0026minus;\u0026thinsp;20℃ for use. The absorption value was measured at wavelength 560 nm, and a standard curve was drawn.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Thermal denaturation temperature analysis of horse bone collagen\u003c/h2\u003e \u003cp\u003eApproximately 3 mg of horse bone collagen samples of different ages were weighed into an aluminium crucible, pressed, and sealed, and an empty crucible was used as a reference for determination using differential scanning calorimeter (DSC). The temperature was increased from 25℃ to 100℃ at 2℃/min. FTIR analysis of the infrared spectrum was done following Rozi \u003cem\u003eet al.\u003c/em\u003e\u0026rsquo;s (2018) method. The collagen samples from the ribs and tibia of horses of different ages were mixed with potassium bromide crystals in a ratio of 1:100, and the freeze-dried collagen was extruded into sheets by a manual mechanical extrusion device. The wavenumber employed was in the range of 400\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the number of scans used was 64, the speed was 0.2 cm/s, and the resolution was 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Xu \u003cem\u003eet al.\u003c/em\u003e 2017).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Preparation of horse bone collagen peptide\u003c/h2\u003e \u003cp\u003eThe freeze-dried horse bone collagen was dissolved in 0.5 mol/L glacial acetic acid, and the substrate concentration was 10%. The optimal pH value of alkaline protease was adjusted, and collagen enzymolysis was conducted. Upon completion, the mixture was placed in a water bath at 100 ℃ for 15 min, followed by cooling to room temperature (approximately 24 ℃). The solution was then centrifuged at 11171.56 x \u003cem\u003eg\u003c/em\u003e under at 4℃ for 20 min, allowing the removal of the precipitate and collection of the supernatant. After overnight freezing in 80℃, vacuum freeze-drying was conducted. The protein concentration was determined using the Coomassie brilliant blue method and calculated using BCA method. The trichloroacetic acid soluble nitrogen method (TCA method) was utilised, and nitrogen content was determined using the Kjeldahl method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Study on antioxidant activity of horse bone collagen peptide\u003c/h2\u003e \u003cp\u003eThe obtained enzymatic hydrolysate was fractionated using ultrafiltration centrifuge tubes with molecular weight cutoffs of 10, 5, 3, and 1 kDa, respectively. Subsequently, the collected extracellular filtrate underwent vacuum freeze-drying. In test tubes, 1 mL of samples was mixed with 2.5 mL each of phosphate buffer (0.2 mol/L, pH 6.6) and 1% potassium ferricyanide solution. The mixture was then blended and placed in a water bath at 50℃ for 20 min. Following this, 2.5 mL 10% trichloroacetic acid solution was added and centrifuged at 335.37 x \u003cem\u003eg\u003c/em\u003e for 10 min. After centrifugation, 2.5 mL of supernatant was removed, and 0.5 mL of 0.1% ferric trichloride solution and 2.5 mL of distilled water was sequentially added. The absorbance was measured at 700 nm after allowing for a 10-min rest period at (approximately 24℃) (Xie \u003cem\u003eet al.\u003c/em\u003e 2021).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe software SPSS 26.0 was used for data analysis (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); The mapping software Origin 2021 and image processing software ImageJ were used to map the test data.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Horse bone composition\u003c/h2\u003e \u003cp\u003eThe protein content in the bones of horses varied with age and exhibited a noticeable trend. It initially increased and then decreased with increasing age, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Additionally, the bones were rich in calcium; however, the crude fat content was low.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Bone tissue composition\u003c/h2\u003e \u003cp\u003eFigures \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e depict the bone tissue compositions. In each tissue section, the area with the most collagen fibres was selected as the field of vision and used in the analysis with Image J. Picric acid scarlet staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) revealed collagen fibres with a yellow background, and Sirius scarlet staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) revealed dark red calcium salt deposits and a light red or nearly colourless background.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCollagen fibre content in bone tissue\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCollagen fiber area(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-year-old horse bone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8-year- old horse bone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13-year -old horse bone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6-year-old sheep bone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8-year-old cow bone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e71.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e79.99\u0026thinsp;\u0026plusmn;\u0026thinsp;3.43\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.63\u0026thinsp;\u0026plusmn;\u0026thinsp;2.10\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e79.39\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e65.21\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe collagen fibrillar fibres of the 13-year-old horse bone tissue were in loose bundles, were disorganised, and had an uneven density distribution. Furthermore, the collagen fibre area was 48.63%, significantly differing from that of the 2- and 8-year-old horses (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, the fibrillar fibres of the 2- and 8-year-old horse bone tissue were curly strips with a uniform fibre density. The bone biopsy results indicated that the fibrillar fibres of the horse bone first increased and then decreased with age, suggesting that the bone grows and matures rapidly during youth; however, the collagen fibres do not change much during adulthood. As the horses age, the bones loosen, and the collagen fibres become disorganised. Moreover, the collagen fibres of the horse bone were compact, regularly shaped, and formed by more plates, consistent with the results of Zedda \u003cem\u003eet al.\u003c/em\u003e (2020). Further, the horse bone fibrils were normal, with uniform density and orderly arrangement. Notably, the collagen fibre area of adult horse bones significantly differed from that of ox bones of the same age (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); goat bones, contrastingly, had a looser bone structure.\u003c/p\u003e \u003cp\u003eAs the bone develops with age, its density increases, as does calcification, owing to the deposition and maturation of calcium phosphate minerals and increased calcium salt distribution (Isaksson \u003cem\u003eet al.\u003c/em\u003e 2010). Calcium salt distributions were higher in the bones of 2- and 8-year-old horses than in those of the 13-year-old horses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The calcium salt distribution sizes were compared among species of the same age, with the distribution size slightly lower in horse bone than in ox bone; however, the collagen fibre area in horse bone was approximately 15% larger than that in ox bone. Therefore, Mongolian horse bone is highly dense and nutrient-rich, containing calcium, protein, and minimal fat. Furthermore, Mongolian horse bone is richer in collagen than the bones of other domestic animals, and the composition is similar to that of human collagen.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Degreasing horse bone meal\u003c/h2\u003e \u003cp\u003eThe optimal parameters for degreasing horse bone powder using ultra-high pressure were explored. First, various pressures were tested (time: 9 min; solid-to-liquid ratio: 1:15 g/mL), and the degreasing rate peaked at 300 MPa (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Then, various times were tested (pressure: 300 MPa; solid-to-liquid ratio: 1:15 g/mL), and the degreasing rate increased significantly from 5 to 9 min (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), peaking at 9 min under pressure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Finally, various solid-to-liquid ratios were tested (time: 9 min; pressure: 300 MPa). The degreasing rate significantly increased from solid-to-liquid ratios of 1:5 to 1:15 g/mL (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), peaking at a ratio of 1:15 g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Likely, 1:15 g/mL was the optimal ratio because the oil in the horse bone meal dissolved after adding enough degreasing solvent. Overall, the highest degreasing rate was 73.56%, achieved with 300 MPa of pressure for 9 min, with a solid-to-liquid ratio of 1:15 g/mL. The highest ethyl acetate degreasing rate was 70.24%, suggesting that degreasing horse bone powder is more effective with ultra-high pressure than with ethyl acetate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Enzymatic collagen extraction from horse bones\u003c/h2\u003e \u003cp\u003eThe optimal parameters for enzymatic collagen extraction from horse bones were explored next. First, the extraction times were tested with a 6% enzyme (pepsin) dose and a 1:15 g/mL solid/liquid ratio. The collagen extraction rate peaked (23.03%) after 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Next, various pepsin doses were tested with a 24-h extraction time and 1:15 g/mL solid/liquid ratio, and the extraction rate peaked at 6% pepsin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Finally, various solid/liquid ratios were tested with a 24-h extraction time and 6% pepsin dose; the extraction rate peaked (21.57%) at 1:15 g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Overall, the highest collagen extraction rate was 26.65%, achieved with an enzyme dosage of 6%, extraction time of 24 h, and solid/liquid ratio of 1:15 g/mL.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCollagen dissolution is the transition from solid to solute, during which pepsin promotes the release of collagen into the extraction solution. Thus, the extraction rate gradually increases as the extraction time increases (Ali \u003cem\u003eet al.\u003c/em\u003e 2018). High enzyme concentrations also increase the extraction rate; however, it eventually decreases, as in our study (at 6% pepsin), perhaps because of insufficient substrate to continue the reaction. At this point, a further increase in the protease amount causes excessive enzymatic hydrolysis, breaking small-molecule protein fragments into free amino acids without increasing the collagen extraction rate (Lee \u003cem\u003eet al.\u003c/em\u003e 2022). Furthermore, we found that solid-to-liquid ratios greater than 1:15 g/mL decreased the extraction rate. Excess solvent may weaken the stability of collagen, breaking the peptide chain and resulting in many peptide chain fragments, thus reducing the extraction rate (Matinong \u003cem\u003eet al.\u003c/em\u003e 2022). Vidal \u003cem\u003eet al.\u003c/em\u003e (2020) investigated yak bone collagen extraction, finding that the extraction rate initially increased and then decreased as the solid-to-liquid ratio increased; their extraction rate also peaked at a 1:15 g/mL solid-to-liquid ratio. Thus, too much solvent volume is not conducive to collagen extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Structural characterisation and resolution of horse bone collagen\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e presents the results of the infrared spectrum analysis of collagen extracted from horse bone powder from 400 to 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The amide A, Ⅰ, Ⅱ, and Ⅲ band intensities in ultra-high pressure-treated collagen samples from the ribs of an 8-year-old horse were slightly lower than those of untreated bones (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Moreover, the absorption peaks of the rib and tibia decreased slightly in the amide zone with age (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also measured the denaturation temperature of the collagen samples using differential scanning calorimetry and found that the denaturation temperature increased slightly with age (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Moreover, the denaturation temperatures of collagen extracted from the rib and tibia of 8-year-old horses treated with ultra-high-pressure were 30.54 ℃ and 32.40 ℃, respectively, whereas the temperature for untreated collagen was 36.52 ℃.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThermostability of collagen extracted from horse bones\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eDenaturation temperature (T/℃)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo ultra-high-pressure treatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHorse rib\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHorse tibia\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: Significance is indicated by different lowercase letters in the same column (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eInfrared spectroscopy can detect nearly all infrared spectra of biological materials under various conditions, making it one of the most effective methods to study protein hydrogen bonding (Jie \u003cem\u003eet al.\u003c/em\u003e 2018). We found that ultra-high-pressure treatment had little effect on the structure of collagen extracted from horse bone meal, consistent with the results of Nan \u003cem\u003eet al.\u003c/em\u003e (2018). The absorption of the amide A band is related to the N-H stretch vibration, and the absorption peak usually appears in the 3400\u0026ndash;3440 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e region. However, when the N-H stretch vibration combines with a hydrogen bond, it may shift to a lower frequency, moving towards 3300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Singh \u003cem\u003eet al.\u003c/em\u003e 2011). The amide A bands in the untreated and treated collagen samples were at 3439.07 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3435.22 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, indicating that the collagen extracted from horse bone meal contains a hydrogen bond structure. Moreover, the amide B band peaks in the untreated and treated samples were at 2928.57 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2947.23 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, caused by the asymmetric stretching vibration of CH\u003csub\u003e2\u003c/sub\u003e in the molecule (Hazeena \u003cem\u003eet al.\u003c/em\u003e 2022). The characteristic frequency range of the amide Ⅰ band is 1600\u0026ndash;1700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which is mainly related to the stretching vibration of the C\u0026thinsp;=\u0026thinsp;O bond along the peptide\u0026rsquo;s main chain. The amide Ⅰ band can be used to analyse the secondary structure of proteins; the larger the frequency value, the higher the order of the peptide chain skeleton (Gao \u003cem\u003eet al.\u003c/em\u003e 2018). The absorption peaks of untreated and treated collagen samples were at 1656.06 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1649.13 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Moreover, the absorption peaks for the amide II bands in the untreated and treated samples were at 1543.05 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1541.23 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, caused by the N-H bending vibration coupled with the C-N stretching vibration of collagen. High amide Ⅱ frequencies indicate the presence of many intermolecular hydrogen bonds in collagen, especially in the helical part of the collagen structure (Yu \u003cem\u003eet al.\u003c/em\u003e 2018). The absorbance peaks of the untreated and treated collagen samples at 1483.18 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1477.17 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are closer to absorbance 1.0, indicating that the tertiary structure of collagen is intact. Finally, the amide Ⅲ bands for the untreated and treated samples were observed at 1220.94 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1217.08 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. This finding suggests that glycine, proline (Pro), and hydroxyproline content in collagen was relatively high, which makes horse bones treated in different ways have unique absorption peaks at 1200\u0026ndash;1400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e due to the C-N stretching and N-H bending vibrations, consistent with the CH\u003csub\u003e2\u003c/sub\u003e rocking vibration peak attributed to the glycine skeleton and Pro side chain (Ye \u003cem\u003eet al.\u003c/em\u003e 2021). In conclusion, we found that the secondary structure of the extracted equine bone collagen was intact (Kobrina \u003cem\u003eet al.\u003c/em\u003e 2010; Sarin \u003cem\u003eet al.\u003c/em\u003e 2019; Ferraro \u003cem\u003eet al.\u003c/em\u003e 2017).\u003c/p\u003e \u003cp\u003eWe also found that the rib and tibia absorption peaks decreased slightly with age in the amide zone, suggesting that horse bones have a complete triple helix structure, regardless of age. Kobrina \u003cem\u003eet al.\u003c/em\u003e (2010) analysed the infrared spectral absorption peak of equine cartilage collagen from newborn (0 days) to immature age (5 to 11 months) and from immature age to adult age (6 to 10 years), observing that the infrared spectral absorption peaks increased with age, consistent with our results.\u003c/p\u003e \u003cp\u003eWe also found that the collagen\u0026rsquo;s denaturation temperature increased slightly with age, possibly due to a higher degree of crosslinking of the bone fibrillar fibres and the size of mineral crystals, which change with age, resulting in a slight increase in the thermal denaturation temperature of horse bone collagen (Ferraro \u003cem\u003eet al.\u003c/em\u003e 2017). Notably, the denaturation temperature was lower in collagen samples from older horses treated with ultra-high pressure than in untreated samples. This might be due to fractured collagen crosslinking bonds in the bone meal after treatment, weakening the stability and, thus, decreasing thermal stability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Extracting short peptides from horse bone collagen\u003c/h2\u003e \u003cp\u003eAfter characterising the horse bone collagen, we attempted to extract short peptides from the collagen samples and optimise the alkaline protease conditions. We measured the yield after varying the pH (constants: hydrolysis time: 4 h, temperature: 50 ℃, and enzyme [i.e., alkaline protease] dose: 3% by mass); enzymatic hydrolysis time (constants: temperature: 50 ℃, pH: 9.0, and enzyme dose: 3%); temperature (constants: hydrolysis time: 4 h, pH: 9.0, and enzyme dose: 3%); and enzyme dose (constants: hydrolysis time: 4 h, temperature: 50 ℃, and pH: 9.0) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The optimal conditions for a maximal short peptide yield were pH 9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA), 4 h of enzymolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), 50 ℃ temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC), and 3% enzyme dose (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe pH for the optimal short peptide yield was 9.0, indicating that pH primarily affects protease activity. Each protease has an optimal pH range, outside of which the activity decreases, and the enzymatic hydrolysis efficiency will be correspondingly low, decreasing the short peptide yield (Jiang \u003cem\u003eet al.\u003c/em\u003e 2014). Furthermore, we found that reaction times exceeding 4 h decreased the yield. Likely, a longer reaction resulted in more enzymolysis products and complexes, which inhibited the enzymolysis reaction, decreased the reaction speed, and thus decreased the yield. Furthermore, the peptide\u0026rsquo;s reaction site becomes saturated over time; some peptides are decomposed into amino acids, and the decomposition rate exceeds the protein peptide formation rate, decreasing the peptide yield (Vidal \u003cem\u003eet al.\u003c/em\u003e 2022). We also found that the short peptide yield increased and then decreased as the temperature increased, peaking at 50 ℃ (yield: 63.78%). When the temperature rises, the protein conversion rate increases; however, the enzyme protein will slowly denature or deactivate, decreasing the enzyme reaction rate and, thus, the peptide yield (Beaubier \u003cem\u003eet al.\u003c/em\u003e 2021). Finally, we found the highest yields with a 3% enzyme dose (yield: 62.63%). Adding enzymes to the reaction increases the probability that the enzyme and collagen interact, which increases the yield. However, the yield will no longer increase once the substrate is saturated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Short peptide antioxidant activity\u003c/h2\u003e \u003cp\u003eWe isolated four groups of horse collagen peptides with different molecular weights: 5\u0026ndash;10, 3\u0026ndash;5, 1\u0026ndash;3, and \u0026lt;\u0026thinsp;1 KDa. The reducing abilities of horse bone collagen short peptide with different molecular weights extracted from horses of different ages gradually increased as the concentration increased from 5 to 10 mg/mL (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Tables\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The reducing abilities decreased as the molecular weights increased; 1\u0026ndash;3 kDa components had the strongest abilities, whereas 5\u0026ndash;10 kDa components had the weakest abilities. These results were compared with the reducing power of the antioxidant VC. However, compared with that of the antioxidant VC at the same concentration, the reducing power was weaker with reducing molecular weights, and the difference was significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, the hydroxyl radical-scavenging ability of peptides with the same molecular weight differed based on the age of the bones, and the activity was the strongest in the extracts from 8-year-old bones (Tables\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Finally, with the increase in the concentration of collagen peptide solution, the O\u003csub\u003e2\u003c/sub\u003e free radical clearance rate increased for each ultrafiltration component (Tables\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and \u003cspan refid=\"Tab9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eReduction capacity of peptides isolated from collagen extracted from horse rib bones\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003ereducing force\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003econcentration (mg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026ndash;10 KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u0026ndash;5 KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u0026ndash;3 KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1 KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0800\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0890\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1037\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0940\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0870\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0903\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1130\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0947\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0883\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0920\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1350\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1013\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1090\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1217\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1813\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1597\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0813\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0803\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1043\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0997\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0903\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0983\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1800\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1130\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003e3.7210\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003e3.8077\u0026thinsp;\u0026plusmn;\u0026thinsp;0.051\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: Peer reduction ability represented by different lowercase letters is significantly correlated at the 0.05 level.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eReduction capacity of peptides isolated from collagen extracted from horse tibia bones\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003ereducing force\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003econcentration(mg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 KDa-5KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 KDa-3KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 KDa-1KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0787\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0853\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1077\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0873\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0830\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0877\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1213\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0923\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0833\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0893\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0005\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1030\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0917\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0877\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0943\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1250\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0963\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0733\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0873\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0913\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0840\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0903\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0005\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1117\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0940\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: Different lowercase letters indicate a significant correlation with reduction capacity (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHydroxyl radical-scavenging ability of peptides isolated from collagen extracted from horse rib bones\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e \u003cp\u003eHydroxyl radical scavenging power\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003econcentration(mg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 KDa-5KDa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 KDa-3KDa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 KDa-1KDa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1KDa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e68.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.387\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e73.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.590\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e88.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.686\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e84.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.572\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.394\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e77.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.997\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e98.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.614\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e88.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.397\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.554\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81.65\u0026thinsp;\u0026plusmn;\u0026thinsp;2.159\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e92.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.775\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e85.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.964\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e73.15\u0026thinsp;\u0026plusmn;\u0026thinsp;2.015\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.757\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e98.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.675\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e92.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.027\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.340\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.023\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e90.82\u0026thinsp;\u0026plusmn;\u0026thinsp;2.358\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e84.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.778\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.368\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88.59\u0026thinsp;\u0026plusmn;\u0026thinsp;2.685\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.741\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e89.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.045\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003e90.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.806\u003c/p\u003e \u003cp\u003e92.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eNote: Different lowercase letters indicate a significant correlation with hydroxyl radical clearance (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHydroxyl radical-scavenging ability of peptides isolated from collagen extracted from horse tibia bones\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eHydroxyl radical scavenging power\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003econcentration(mg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 KDa-5KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 KDa-3KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 KDa-1KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.046\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.547\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e92.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.066\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e82.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.211\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.394\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78.10\u0026thinsp;\u0026plusmn;\u0026thinsp;2.685\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.060\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e86.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.340\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.926\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83.34\u0026thinsp;\u0026plusmn;\u0026thinsp;2.203\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e94.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.049\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e90.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.345\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.926\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.358\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e96.15\u0026thinsp;\u0026plusmn;\u0026thinsp;2.425\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e93.28\u0026thinsp;\u0026plusmn;\u0026thinsp;2.006\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.779\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78.29\u0026thinsp;\u0026plusmn;\u0026thinsp;2.536\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e93.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.160\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e87.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.346\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.779\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.851\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.679\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e89.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.774\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eNote: Different lowercase letters indicate a significant correlation with hydroxyl radical clearance (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSuperoxide anion-removal capacity of peptides isolated from collagen extracted from horse rib bones\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c8\" namest=\"c4\"\u003e \u003cp\u003eSuperoxide anion scavenging capacity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003econcentration(mg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10 KDa-5KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 KDa-3KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3 KDa-1KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.745d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.19\u0026thinsp;\u0026plusmn;\u0026thinsp;2.380\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e41.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.377\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e34.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.374\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.634d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.380\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.374\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e39.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.371\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.231d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.663\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.072\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e41.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.635\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.71\u0026thinsp;\u0026plusmn;\u0026thinsp;2.385d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.004\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e51.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.656\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e49.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.412\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.764d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.371\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e41.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.376\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e37.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.406\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.166\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.374\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e45.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.412\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e39.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.517\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eVc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003e60.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.745\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003e79.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eNote: Different lowercase letters indicate a significant correlation with superoxide anion clearance (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab9\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSuperoxide anion-removal capacity of peptides isolated from collagen extracted from horse tibia bones\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eSuperoxide anion scavenging capacity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003econcentration(mg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 KDa-5KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 KDa-3KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 KDa-1KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.25.\u0026plusmn;0.685\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.610\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.685\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.707\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.387\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.380\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e46.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.374\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e39.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.906\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.231\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.736\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.537\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e38.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.987\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.707\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.76\u0026thinsp;\u0026plusmn;\u0026thinsp;2.004\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e54.73\u0026thinsp;\u0026plusmn;\u0026thinsp;2.415\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e45.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.190\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.199\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.345\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e45.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.190\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e38.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.716\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.589\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.393\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e49.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.412\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e42.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.517\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: Different lowercase letters indicate a significant correlation with superoxide anion clearance (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eReducing capacity and antioxidant activity are somewhat related; thus, the antioxidant efficacy of collagen peptides can be assessed by measuring their reducing capacity (Wang \u003cem\u003eet al.\u003c/em\u003e 2023). In Fe\u003csup\u003e3+\u003c/sup\u003e and Fe\u003csup\u003e2+\u003c/sup\u003e reaction systems, the greater the light absorption value at 700 nm, the stronger the reducing power (Bo \u003cem\u003eet al.\u003c/em\u003e 2017). Our results were consistent with those of Chen \u003cem\u003eet al.\u003c/em\u003e (2023), who found that low-molecular-weight polypeptides of collagen peptide had a strong reducing ability.\u003c/p\u003e \u003cp\u003eA dose\u0026ndash;effect relationship exists for collagen peptides of different molecular weights and their ability to clear hydroxyl radicals (Lee \u003cem\u003eet al.\u003c/em\u003e 2022). We found that peptides with the same molecular weight had variable hydroxyl radical-scavenging abilities (antioxidant activities), based on the age of the bone; extract from 8-year-old horses had the strongest activity. Moreover, when the concentration of the collagen peptide solution was the same, the hydroxyl radical-scavenging ability of short peptides (molecular weight: 1\u0026ndash;3 KDa) was significantly higher than that of other molecular weight components. In addition, the hydroxyl free radical-scavenging ability was slightly higher with an equivalent concentration of VC than that of VC alone. The hydroxyl free radical-scavenging abilities of the collagen peptides extracted from the horse tibia and ribs demonstrated that these peptides could scavage hydroxyl free radicals, consistent with the finding of Hern\u0026aacute;ndez-Ruiz \u003cem\u003eet al.\u003c/em\u003e (2023), who reported that the small molecular weight collagen peptide components had superior activity than the large molecular weight components. However, this result is controversial as others have reported conflicting results. For example, Shen \u003cem\u003eet al.\u003c/em\u003e (2021) reported that the 5\u0026ndash;10 KDa molecular weight pig skin collagen antioxidant peptides have good antioxidant activity, suggesting that the antioxidant activity of collagen peptides is related to their relative molecular weight and is influenced by their amino acid composition and sequence (Sun \u003cem\u003eet al.\u003c/em\u003e 2021).\u003c/p\u003e \u003cp\u003eSuperoxide anion radicals (O2-\u0026middot;) are produced during processes such as oxygen transport in haemoglobin, autoxidation, mitochondrial electron transport chain, and cytochrome reactions. When light induces melanin production, too many lone electron pairs are formed, and O\u003csub\u003e2\u003c/sub\u003e\u0026middot;can form. In addition, superoxide cation radicals can induce the formation of other autonomous groups, such as hydroxyl radicals (Zu \u003cem\u003eet al.\u003c/em\u003e 2023).\u003c/p\u003e \u003cp\u003eFinally, we found that as the concentration of collagen peptide solution increased, the O\u003csub\u003e2\u003c/sub\u003e free radical clearance rate increased for each ultrafiltration component, suggesting a dose\u0026ndash;effect relationship (Zu \u003cem\u003eet al.\u003c/em\u003e 2022). Furthermore, for peptides with the same molecular weight, the antioxidant activity of the peptides differed based on the age of the bones, and the superoxide anion-scavenging activity was the strongest with the peptides extracted from an 8-year-old horse bone. When the concentration of collagen peptide solution was equivalent, the O\u003csub\u003e2\u003c/sub\u003e scavenging activity decreased as the molecular weights increased; 1\u0026ndash;3 kDa components had the strongest abilities, whereas 5\u0026ndash;10 kDa components had the weakest abilities. Thus, horse bone meal collagen peptides with different molecular weights can clear O\u003csub\u003e2\u003c/sub\u003e at rates similar to those of VC, although 5\u0026ndash;10 KDa components have a lower superoxide anion clearance rate than VC. Similar results were reported in a study by Irshad \u003cem\u003eet al.\u003c/em\u003e (2015), who found that 1\u0026ndash;3 KDa bovine spinal cord polypeptides had better antioxidant activity than other components.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, we extracted and characterised collagen from horse bone, followed by short peptide extraction from these collagen samples. An imaging analysis first confirmed significant differences in the fibrillar fibres of horse bones of different ages. Furthermore, the collagen fibres of adult horse bones were regularly shaped, uniformly dense, arranged orderly, and had a higher bone density than other animal bones (cattle and goat). The thermal stability of the horse bones also increased with age. Moreover, ultra-high-pressure treatment improved the horse bone degreasing rate compared to the use of ethyl acetate but decreased the denaturation temperature of horse bone collagen. However, the treatment left the tertiary structure of collagen intact. Finally, we isolated four groups of horse collagen peptides with different molecular weights, which had different antioxidant capacities; the components with the lowest molecular weights (1\u0026ndash;3 KDa) had the highest activities. Moreover, age affected the peptide\u0026rsquo;s antioxidant activity independent of molecular weight, and the antioxidant activity of the peptides from 8-year-old horse bones was the strongest. Domestic research has been conducted based on the national conditions of China, according to the regional characteristics of the horse industry. However, there are few studies on the high-quality resource, Mongolian horse bone. Therefore, how to use bioengineering technology reasonably to develop and study the precious resource of horse bone and explore its economic value is a research topic with broad prospects and practical significance. At present, our team is focusing on the study of bovine and sheep bone collagen and peptides. Based on this, collagen was extracted from Mongolian horse bone and prepared into collagen short peptides, and its antioxidant activity was determined. This study lays the foundation for further development, utilisation, and industrial upgrading of horse bone collagen peptides.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eUHP: Ultra-high-pressure processing; DSC, differential scanning calorimeter;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatement of Ethics\u003c/b\u003e:\u003c/p\u003e \u003cp\u003e All animal experiments were performed according to the regulations of the Administration of Affairs Concerning Experimental Animals in China. The experimental protocol was approved by the Animal Welfare and Research Ethics Committee of the Inner Mongolia Agricultural University (approval ID: 20154617\u0026ndash;8).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJindi Wu, propose research topics, design research plans, thesis writingHeya Na, Statistical analysis dataFan Bai, collect sample Siyu Li , Statistical analysis dataHao Gao, Statistical analysis data Rina Sha, propose research topics, design research plans,obtain research funding\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eThis work has been funded by the Postgraduate Training fund (RC1900004555) and the Natural Science Foundation of Inner Mongolia (Grant No. 2022QN03026). 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A first comparison of bone histomorphometry in extant domestic horses (Equus caballus) and a Pleistocene Indian wild horse (Equus namadicus). \u003cem\u003eIntegrative Zoology\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e\u003cstrong\u003e(\u003c/strong\u003e6\u003cstrong\u003e)\u003c/strong\u003e, 448\u0026ndash;460. https://doi.org/10.1111/1749-4877.12444\u003c/li\u003e\n\u003cli\u003eZu, X. Y., Zhao, Y. J., Fu, S. M., Liao, T., Li, H. L., \u0026amp; Xiong, G. Q. (2021). Physicochemical properties and biological activities of silver carp scale peptide and its nanofiltration fractions.\u003cem\u003e \u003c/em\u003e\u003cem\u003eFrontiers in Nutrition\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e, 812443. (in Chinese). https://doi.org/10.3389/fnut.2021.812443\u003c/li\u003e\n\u003cli\u003eZu, X., Huang, Y., Zhao, Y., Xiong, G., Liao, T., \u0026amp; Li, H. (2023). Peptide extraction from silver carp (Hypophthalmichthys molitrix) scales via enzymatic hydrolysis and membrane filtration. \u003cem\u003eItalian Journal of Food Science\u003c/em\u003e, \u003cem\u003e35\u003c/em\u003e\u003cstrong\u003e(\u003c/strong\u003e2\u003cstrong\u003e)\u003c/strong\u003e, 44\u0026ndash;53. (in Chinese). https://doi.org/10.15586/ijfs.v35i2.2248\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mongolian horse bones, Hard tissue section, Ultra-high pressure, Collagen peptide, Antioxidant activity","lastPublishedDoi":"10.21203/rs.3.rs-4512011/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4512011/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHorse bone is rich in collagen, with a composition similar to that of human collagen. Collagen peptides supply nutrients needed for human growth that act as antioxidants, lower blood pressure. This study explored the extraction of collagen and the preparation of collagen short peptides from Mongolian horse bones. Bones were collected from horses of varying ages, and the collagen content along with calcium salt distribution were observed through staining and imaging analyses. Next, the bones were processed into a powder and then subjected to ultra-high-pressure processing for degreasing. The degreasing conditions were optimised by single-factor and orthogonal tests. Following this, collagen was extracted using an acid-enzymatic method, and its structural characteristics and thermal stability were assessed. The collagen short peptides were extracted from the collagen samples, and the effects of the enzymatic hydrolysis time, temperature, pH, and enzyme amount on the extraction rate were evaluated. Finally, the resulting collagen peptides were analysed for antioxidant activity. In summary, this experiment optimised the extraction conditions for horse bone collagen, demonstrating that the ultra-high-pressure method minimally affects collagen structure, and the extraction rate was high. Hence our method has significant development potential.\u003c/p\u003e","manuscriptTitle":"Preparation and tissue structure analysis of horse bone collagen peptide","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-08 17:03:26","doi":"10.21203/rs.3.rs-4512011/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-09T13:20:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-08T17:14:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-03T11:17:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-03T09:33:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69694229976133404319561506023266286413","date":"2024-07-03T05:38:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234342641193726142927348955128877295318","date":"2024-07-02T09:00:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"229819542679856032725801827684540011838","date":"2024-07-02T07:53:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"289776611057920006262628113170903318298","date":"2024-07-02T03:30:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-01T17:12:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-01T17:08:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-06-13T08:53:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-12T05:32:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-06-01T05:22:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6a13293a-4f59-4ca0-b666-8795caec4e81","owner":[],"postedDate":"July 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":33731453,"name":"Biological sciences/Molecular biology"},{"id":33731454,"name":"Health sciences/Health care"}],"tags":[],"updatedAt":"2024-11-04T16:22:22+00:00","versionOfRecord":{"articleIdentity":"rs-4512011","link":"https://doi.org/10.1038/s41598-024-75960-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-10-28 15:57:03","publishedOnDateReadable":"October 28th, 2024"},"versionCreatedAt":"2024-07-08 17:03:26","video":"","vorDoi":"10.1038/s41598-024-75960-7","vorDoiUrl":"https://doi.org/10.1038/s41598-024-75960-7","workflowStages":[]},"version":"v1","identity":"rs-4512011","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4512011","identity":"rs-4512011","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}