Chondroitin sulfate (CS) has been widely used as a dietary supplement in the food industry at present. However, there is incomplete absorption in the body, due to the large molecular weight, abundant hydrophilic groups, and low lipid solubility. As a result, CS exhibits low bioavailability (typically less than 13%), when taken orally. The bioavailability of oral CS formulations is closely linked to the molecular weight. Furthermore, the intact CS is still challenging to absorb in the digestive tract, where the low molecular weight CS (LMCS) can be absorbed through the intestine. LMCS can be expected to serve as the superior bioactivity and bioavailability, compared with undegraded CS. Therefore, the preparation of LMCS by degrading CS is of great significance for the further development and utilization of CS. There are several ways to prepare the LMCS so far, including free radical, enzyme, and acid degradation. Among them, the Fenton process is one type of free radical degradation with a commonly used and cost-effective reaction. It can be expected to degrade the CS by catalytic H2O2 while retaining the main polysaccharide structure. In this study, LMCS from different sources was prepared using the Fenton reaction, in order to investigate the potential degradation mechanism. The molecular weight, sulfate, and glyoxylate contents of chondroitin sulfate (BCS) of bovine origin were analyzed under different degradation conditions. The optimal CS degradation was determined using single-factor experiments. Subsequently, chondroitin sulfate derived from chicken (CCS), bovine (BCS), and shark (SCS) were reacted under optimal degradation conditions for 2, 4, 6, and 8 hours. The contents of sulfate and uronic acid, as well as the mono- and disaccharide composition and structure of the degradation products, were determined using Fourier transform infrared (FT-IR) spectroscopy and high-performance liquid chromatography (HPLC). For the first time, the potential mechanism of CS degradation by the Fenton reaction was analyzed from the perspective of molecular conformation and bond energy using molecular dynamics (MD) and standard density flooding theory (DFT). The results showed that the optimal conditions for CS degradation were a copper acetate concentration of 0.5 mmol, pH 7.5, and a reaction temperature of 45 ℃, respectively. The chemical composition of degradation products showed that the molecular weight of CS was positively correlated with the sulfate, glyoxylate, and GlcA content. The ratio of 4S/6S in the degraded CCS and BCS showed a decreasing trend with increasing degradation time, while there was no outstanding pattern for the degradation products of SCS. This infers that the GlcA residues of chondroitin-4sulfate (CSA) were preferentially attacked by free radicals in the Fenton degradation reaction. The FT-IR results of the degradation products indicated that the absorption peaks were related to the carboxyl derivatives formed by the free radical degradation of GlcA. MD experiments show that the conformational changes of CSA in H2O occurred more frequently than those of non-sulfated chondroitin (CSO) and chondroitin-6sulfate (CSC), while the opposite result occurred in ·OH, where the RG and SASA values of CSA reached the minimum in ·OH. DFT calculations show that the C1-H bond of GlcA with radicals required a lower energy for the hydrogen abstraction reaction to occur, compared with GalNAc. Therefore, the C1-H of GlcA in CSA was more likely to undergo a hydrogen abstraction reaction with ·OH. These findings can provide promising guidance for the production of LMCS.
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Open Access
Issue
To gain a deep understanding of the taste characteristics of bone peptides from different species of livestock and poultry and the differences between them, and to explore the feasibility of using electronic tongue in their rapid qualitative identification.
Totally 16 samples of yak bone peptides (YBPs), bovine bone peptides (BBPs), pig bone peptides (PBPs) and chicken bone peptides (CBPs), four samples from each species, were collected for analysis of nutritional components and amino acid composition to clarify the material basis of the taste difference between them, and then the data of their taste characteristics were quantitatively discriminated by electronic tongue combined with multivariate statistical analysis.
The nutritional composition and amino acid composition of bone peptides from different animal species were different. Principal component analysis (PCA) and discriminant factor analysis (DFA) distinguished bone peptides from different animal species, and DFA was significantly more effective than PCA. Supervised partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA) and Fisher discriminant analysis model all had good discriminant capability for the taste characteristics of bone peptides, and the discriminant accuracy of the Fisher model was 68.8%.
The taste characteristics of the four bone peptides differ umami, sweetness and bitterness. Electronic tongue taste detection combined with multivariate statistical analysis can provide a rapid and reliable means for the discrimination of bone peptides from different animal species.
Open Access
Issue
To prepare a calcium supplement for efficient absorption and utilization in the human body and to explore its effect on the osteogenic activity of MC3T3-E1 cells.
Bovine bone peptide was chelated with calcium, and the optimal reaction conditions were determined by response surface methodology (RSM). The structural characteristics and stability of peptide-calcium chelate were studied by infrared spectroscopy, X-ray diffraction and atomic absorption spectrometry. The effects of bovine bone peptide-calcium chelate on the proliferation, differentiation and mineralization of MC3T3-E1 cells were investigated.
The optimum conditions for the preparation of bovine bone peptide calcium chelate were peptide/calcium mass ratio 2.97, temperature 62.54 ℃ and pH 9.06. Under these conditions, the calcium chelating capacity was 55.65 μg/mg. The results of spectral analysis showed that amino nitrogen, hydroxyl oxygen and carboxyl oxygen were the major calcium-chelating sites. Bovine bone peptide could form coordinate bonds with calcium, and the molecular mass of the peptide increased after chelation with calcium. In addition, the stability of bovine bone peptide-calcium chelate was little affected by temperature, but sensitive to pH. The peptide-calcium chelate could significantly promote the proliferation, differentiation and mineralization of MC3T3-E1 cells.
Bovine bone peptide-calcium chelate has the potential to promote the osteogenic activity of MC3T3-E1 cells.
Chondroitin sulfate (CS) is widely distributed in animal cartilage tissues, such as thoracic cartilage, laryngeal bone, and nasal bone. The market demand for CS products is ever increasing with the global aging and aging of chronic diseases in recent years, due to the various functional characteristics, such as antioxidant, anti-inflammatory, and lipid-lowering. It is very necessary to promote the extraction of specific active substances. Natural deep eutectic solvents (NADESs) are often simply synthesized to design their structure for high extraction efficiency. The solvents can also be recycled and reused after use, in order to make them green and environmentally friendly. This study aims to improve the utilization rate of cartilage raw materials and reduce the burden of waste treatment. A natural deep eutectic solvent method was also proposed to extract the CS from the bovine laryngeal cartilage, taking betaine and choline chloride as hydrogen bond acceptors and six common natural substances as hydrogen bond donors. Twelve NADESs were prepared to combine them in pairs. Among them, NADES-3 (betaine/urea, molar ratio 1:2) had the highest extraction rate of CS, which was 16.49%. The reason was that the weak hydrogen bonds were formed in the solvent, due to the small molecular weight of urea, and then the betaine counteracted the denaturation of protein by urea, indicating the synergistic effect. The extracted CS was characterized by monosaccharide analysis, infrared spectroscopy, and molecular weight analysis, in order to confirm the extracted CS with an excellent structure. Subsequently, the effects of four single factors on CS extraction were studied, including liquid-solid ratio (10:1, 20:1, 30:1, 40:1, and 50:1 mL/g), moisture content (5%, 20%, 40%, 60%, and 80%), temperature (90, 100, 110, 120, and 130 ℃), and time (60, 90, 120, 150, and 180 min). The optimal extraction conditions were: liquid-solid ratio of 30:1 mL/g, moisture content of 40%, extraction temperature of 110 ℃, and extraction time of 120 minutes. The CS extraction rate reached 17.33%, which was 2.88 times higher than the traditional water extraction. A correlation analysis was made between the physicochemical properties of NADESs and the CS recovery rate. The extraction rate was negatively correlated with the viscosity, whereas, that was positively correlated with pH and polarity, and there was no significant correlation with the density. The density and viscosity of the solvent decreased after dilution with water, and the hydrogen bonding interaction between NADES components gradually weakened to enhance the mass transfer movement of the cartilage matrix to the solvent, thereby improving the extraction rate of CS. Excessive water disrupted the tight hydrogen bonding structure of NADESs, leading to the formation of simple aqueous solutions and the extraction efficiency of solvents. Finally, NADES-3 with a moisture content of 40% was determined as the optimal solvent for the CS extraction. Subsequently, NADES-3 was recycled to verify the reusability. The extraction rate of CS still reached 15.89% after five repetitions. The excellent extraction was maintained after multiple extractions, where some collagen residues in the solvent increased the steric hindrance between molecules, and weakened the hydrogen bonding interaction between them. The NADES extraction can effectively green-recover CS from laryngeal cartilage. This finding can provide new ideas for recyclable extractants, in order to extract bioactive compounds.
Crosslinking is crucial to enhance the physicochemical properties of collagen-based edible scaffolds for cell-cultured meat (CCM). This study aims to systematically investigate the precise and controllable enhancement of cross-linking agents proanthocyanidins (PA), sodium tripolyphosphate (TPP) and different cross-linking means (PA/TPP/PA+TPP) on the performance of edible CCM scaffolds. Bovine bone collagen (BBC)/chitosan (CS) self-crosslinked fibrillar gels (B/C) were first prepared at the optimal stoichiometric ratio of 4∶1 (w/w). Afterward, BBC/CS scaffolds, including PA-crosslinked BBC/CS scaffolds (B/C/P), TPP-crosslinked BBC/CS scaffolds (B/C/T), and PA+TPP-crosslinked BBC/CS scaffolds (B/C/PT) were prepared by immersing B/C in PA for 2 h, TPP for 2 h, and a combination of PA for 1 h followed by TPP for 1 h, respectively. Subsequently, a comparative assessment was conducted on the molecular structure (hydrogen bonding, degree of molecular order, triple-helical structure integrity, and degree of crystallinity), physicochemical properties (porosity, thermal stability, crosslinking degree, swelling/water retention ratio, dissolution degree, enzymatic resistance capacity, and mechanical strength), and biocompatibility (seeding efficiency and cell viability) of the samples before (B/C) and after crosslinking (B/C/P, B/C/T, and B/C/PT). Results from Fourier Transform Infrared Spectroscopy (FTIR) and X-ray Diffraction (XRD) showed that the crosslinking improved the molecular structure of the BBC/CS scaffolds without affecting the triple-helical structure integrity of BBC. The BBC/CS scaffolds exhibited a higher degree of molecular order and crystallinity, as well as more and tighter hydrogen bonding interactions, compared with B/C. Scanning electron microscopy (SEM) revealed that the B/C/P (98.3%/136 μm), B/C/PT (96.5%/175 μm), and B/C/T (94.8%/203 μm) shared higher porosity but smaller pore sizes, compared with the BBC/CS fibrillar gels (93.2%/265 μm). Differential scanning calorimetry (DSC) results showed that the crosslinking improved the thermal stability of the BBC/CS scaffolds within a smaller temperature range (10~100 ℃) rather than a larger temperature range (30~500 ℃), compared with B/C. The thermal stability of crosslinked scaffolds was ranked as follows: B/C/P (Tm 66.8 ℃, ΔH 13.52 J/g), B/C/PT (Tm 62.2 ℃, ΔH 10.26 J/g), and B/C/T (Tm 60.6℃, ΔH 8.71 J/g), compared with the B/C (Tm 55.6 ℃, ΔH 5.26 J/g) and BBC (Tm 51.3 ℃, ΔH 3.11 J/g). The cross-linked BBC/CS scaffolds exhibited a lower dissolution ratio (B/C/P: 17.3%, B/C/PT: 31.6%, and B/C/T: 35.9%), while a higher degree of crosslinking (B/C/P: 46.53%, B/C/PT: 38.96%, B/C/T: 30.61%), swelling/water retention ratio (B/C/P: 68/7.0 times, B/C/PT: 45/5.3 times, B/C/T: 37/4.4 times, weight after moisture absorption equilibrium to initial dry weight), enzymatic resistance capacity (B/C/P: 11.1%, B/C/PT: 20.6%, B/C/T: 23.3%, degradability), mechanical strength (B/C/P: 12.5 kPa, B/C/PT: 11.9 kPa, B/C/T: 10.2 kPa), and biocompatibility (B/C/P: 86.37%/ 118.33%, B/C/PT: 79.33%/ 115.68%, B/C/T: 63.52%/ 112.82%, seeding efficiency and cell viability, respectively), compared with the B/C (dissolution ratio 55.6%; swelling/water retention ratio 25/2.9 times; enzyme resistance capacity 35.2%; mechanical strength 5.6 kPa; seeding efficiency 56.59%/cell viability 100%). Generally, BBC/CS scaffolds shared superior physicochemical properties, compared with B/C. The B/C/P showed better performance than B/C/T and B/C/PT. The potential crosslinking mechanisms of PA and TPP predominantly relied on the extensive hydrogen bonding between the abundant hydroxyl groups of PA and BBC, as well as the ionic interactions between the phosphate ions of TPP and the amino groups of CS. The three different crosslinking means (PA, TPP, PA+TPP) can accurately improve the performance of BBC/CS scaffolds with different costs and degrees based on their different mechanisms. Therefore, in the production of different edible CCM scaffolds, appropriate crosslinking methods can be comprehensively considered based on the specific performance requirements, production costs, and crosslinking efficiency to achieve precise control. The study provide reference for the construction of edible CCM scaffolds with excellent properties and controllable cost based on PA and TPP cross-linking.
Open Access
Basic Research
Issue
Constructing an edible extracellular matrix (ECM) scaffold with good thermal stability, self-assembly properties and biocompatibility is crucial for manufacturing structured cell cultured meat products. In this study, carboxymethyl chitosan (CMCS) was introduced into the bovine bone collagen (BBC) system. Using ultraviolet (UV) absorption, infrared(IR) and fluorescence spectroscopy, it was found that the interaction between BBC and CMCS was enhanced with the increase in CMCS concentration, without affecting the triple-helical structure. The results of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) showed that the introduction of CMCS reinforced the thermal stability of BBC. Turbidity test, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) confirmed an increase in the degree of fibrillogenesis and aggregation behavior and changes in self-assembly rate; a looser and twisted three-dimensional structure with a larger fibril diameter and a wider diameter distribution was observed after the introduction of CMCS. However, the introduction of CMCS did not significantly affect the formation and length of D-periodicity (a characteristic alternating light/dark transverse stripe structure formed during the self-assembly process of collagen fibers) in BBC, and there was no significant difference in the cell compatibility of the system before and after the introduction of CMCS. The BBC-CMCS interaction might be dominated more by electrostatic forces than covalent interactions and hydrogen bonds with the increase in CMCS concentration. These results indicated that the introduction of CMCS improved the thermal stability and self-assembly properties of BBC without affecting its biocompatibility or triple-helical integrity. This study provides a reference for the development of excellent edible collagen-based ECM as a biomimetic scaffold for cultured meat and for the deep processing and high-value utilization of livestock and poultry bone by-products.
Open Access
Basic Research
Issue
In order to investigate the application potential of prolyl endopeptidase (PEP) in the enzymatic preparation of bone collagen peptides, the amino acid sequence characteristics of bovine bone collagen (BBC), porcine bone collagen (PBC) and chicken bone collagen (CBC) were comparatively analyzed, and their potential enzymatic cleavage sites and theoretical hydrolysis degrees were predicted. Collagen hydrolyzed by PEP at 55 ℃ and pH 8.0 was characterized by hydrolysis degree, molecular mass distributionand scanning electron microscopy (SEM). Infrared spectroscopy, X-ray diffraction (XRD) and circular dichroism (CD) spectroscopy were used to explore the structural changes of collagen during enzymatic digestion. The results showed that PEP could hydrolyze the three collagens. The hydrolysis degree of PBC was the highest (51.35%), followed by those of BBC (22.81%) and CBC (29.81%). The molecular masses of the three collagen hydrolysates were mostly distributed below 500 Da. Spectroscopic analysis showed that PEP destroyed the triple helix structure of collagen, and then degraded it. Therefore, PEP can efficiently enzymatically hydrolyze collagen into small molecule peptides, which provides a basis for the enzymatic preparation of functional collagen-derived peptides.
The preparation of bone peptides (livestock and poultry bone peptides, LBPs) for the development of bone-derived functional foods is one of the important ways for the high-value utilization of livestock and poultry bone by-products. In this experiment, the leg bone from four main livestock and poultry species were used as raw materials to prepare LBPs based on the same process, and their physicochemical properties and bioactivities were compared and analyzed, so as to provide the reference for the high-value utilization of livestock and poultry bone resources and the development of bone-derived functional foods.
Yak bone peptides (YBPs), bovine bone peptides (BBPs), porcine bone peptides (PBPs) and chicken bone peptides (CBPs) were prepared from yak, bovine, porcine and chicken leg bones, respectively. Physicochemical properties were characterized, including basic nutritional components, amino acid compositions, molecular weight and particle size distribution. Moreover, the bioactivities of the four LBPs, including osteoblast promoting proliferation, immune regulation, angiotensin converting enzyme inhibitors (ACEI) and antioxidation, were compared and analyzed.
The relative contents of crude protein in YBPs, BBPs and PBPs were (89.70±0.77)%, (90.43±0.88)% and (89.36±1.32)%, respectively, which were significant higher than that of CBPs (79.18±1.49)%). The essential amino acids and sulfur-amino acids of CBPs were significant higher than those of YBPs, BBPs and PBPs. The four LBPs were mainly composed of small molecular peptides with MW<2 kD, accounting for about 90%. There was no significant difference in particle size distribution of LBPs powder, which was mainly concentrated in the range of 10-20 µm and 40-60 µm. The bioactivities of different LBPs were analyzed. It was found that YBPs had the most significant osteoblasts promoting proliferation effect, and the proliferation rate was 37.27% at 0.5 mg∙mL-1; BBPs had the strongest effect on macrophage proliferation, and the proliferation rate was 39.26% at 5 mg∙mL-1; PBPs had the strongest ACEI activity, and the inhibition rate of ACE activity was 82.37% at 15 mg∙mL-1; YBPs had the strongest comprehensive antioxidant capacity, compared with BBPs, PBPs and CBPs.
The physicochemical properties of LBPs from four distinct species were different, but they all met the demand of raw material for the development of bone-derived functional foods. The bioactivities of LBPs from different species were different, which were suitable for the development of different bone-derived functional foods: YBPs had the strongest osteoblasts promoting proliferation effect and comprehensive antioxidant capacity, which was more suitable for the development of bone health improving or antioxidant bone-derived functional foods; BBPs had the strongest effect on promoting macrophage proliferation, which was more suitable for the development of immunomodulatory bone-derived functional foods; PBPs had the strongest ACEI activity and was more suitable for the development of bone-derived functional foods with blood pressure control effect; CBPs had the better powder properties and higher mineral content, which could be used as dietary nutritional supplements.
Open Access
Research Article
Issue
Bone collagen hydrolysates (peptides) derived from byproduct of animal product processing have been used to produce commercially valuable products due to their potential antioxidant activity. Maillard glycosylated reaction is considered as a promising method to enhance the antioxidant activity of peptides. Hence, this research aims at investigating the Maillard glycosylation activity and antioxidant activity of bone collagen hydrolysates from different sources. In this study, 3 glycosylated bone collagen hydrolysates were prepared and characterized, and cytotoxicity and antioxidant activity were analyzed and evaluated. The free amino groups loss, browning intensity, and fluorescence intensity of G-Cbcp (glycosylated chicken bone collagen hydrolysates (peptides)) were the heaviest, followed by G-Pbcp (glycosylated porcine bone collagen hydrolysates (peptides)) and G-Bbcp (glycosylated bovine bone collagen hydrolysates (peptides)). The results of amino acid analysis showed that amino acid composition of different bone collagen hydrolysates was significantly different and the amino acid decreased to different degrees after Maillard glycosylated reaction, which may lead to differences in Maillard glycosylated reaction activity. Furthermore, the 3 glycosylated hydrolysates showed no significant cytotoxicity. The results showed that glycosylation process significantly increased the antioxidant activity of bone collagen hydrolysates, and G-Cbcp showed the strongest antioxidant activity, followed by G-Pbcp and G-Bbcp. Therefore, compared with the bone collagen hydrolysates, 3 glycosylated hydrolysates showed significant characteristic and structural changes, and higher antioxidant activity.
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