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.

The interaction between muscle proteins and aroma compounds during processing can lead to a loss of aroma in the meat products, thereby altering the aroma profile for the consumer preference. Heat treatment has been commonly used in meat processing. The addition of spices during stewing meat can be used to eliminate the off-flavor for the enhanced color and the fragrance. Among them, terpenoid compounds are the key volatile flavor compounds in spices. But it is unclear on the mechanism of their interaction with muscle proteins. Myofibrillar protein (MP) and sarcoplasmic protein (SP) also play important roles as components of muscle proteins. This study aims to clarify the patterns and mechanisms of the interaction between muscle proteins and terpenoid compounds during heat treatment. Various physicochemical properties of MP and SP were determined at different heating times, such as surface hydrophobicity, total sulfhydryl content, particle size, and secondary structure content. Three terpenoid compounds (3-carene, limonene, and linalool) which significantly contributed to the aroma of stewed pork, were selected to investigate the interaction between aroma compounds and muscle proteins. Solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) was used to study the changes in the aroma absorption ability of proteins with heating time. The correlation was examined between aroma absorption and conformational changes of MP and SP. Moreover, the molecular docking analysis was also implemented to reveal the mechanism of muscle proteins binding to the terpenoid compounds. The results indicated that the heat treatment significantly altered the conformation of MP and SP. The surface hydrophobicity and total sulfhydryl content of MP and SP initially increased and then decreased, as the heating time increased, due to their unfolding and aggregation behaviors. Both MP and SP exhibited an increase in particle sizes during heating, particularly with a higher degree of SP aggregation, due to the lower thermal stability. Furthermore, the secondary structure content analysis showed that the intramolecular hydrogen bond of proteins was rearranged into intermolecular hydrogen bond, and then the ordered structure was transformed into a random coil conformation following heating, resulting in a decrease of α-helix structure content and an increase of β-sheet and random coil structure content. MP and SP demonstrated enhanced aroma adsorption during the initial 5 min of heating, due to their unfolded secondary structures and exposed binding sites. However, the continued heating led to the aggregation of MP and SP, burying the binding sites, and reducing their adsorption ability. Although the MP and SP exhibited similar adsorption within 0-5 min of heating, the SP displayed weaker adsorption than the MP after 10-60 min of heating. This difference was attributed to the higher degree of aggregation in the SP, which resulted in greater steric hindrance and the burial of more aroma-binding sites. Molecular docking results demonstrated that the hydrophobic interactions dominated the interactions between 3-carene/limonene and muscle proteins, whereas the linalool bound to muscle proteins through hydrophobic interactions and hydrogen bonds. Overall, the heat treatment can be expected to modify the conformation of muscle proteins, and then regulate the exposure or burial of aroma binding sites. Consequently, there was some impact on the interaction between muscle proteins and terpenoid compounds. The findings can provide theoretical guidance to control the aroma retention/release behavior during the thermal processing of meat products for the higher quality of meat products.

Bone collagen peptides prepared by enzymatic hydrolysis are mixed peptides with a variety of potential biological activities. However, there is a lack of research on targeted screening of bone collagen peptides with the ability to promote osteoblast proliferation. In this study, the molecular mass and sequence of yak bone collagen peptides prepared by enzymatic hydrolysis were determined by high performance liquid chromatography (HPLC) and nano-liquid chromatography-tandem mass spectrometry (nano LC-MS/MS), respectively. The semi-flexible docking method CDOCKER based on epidermal growth factor receptor (EGFR) was used for the targeted screening of bone collagen peptides with the ability to promote osteoblast proliferation. Finally, in vitro cell tests were used to verify the molecular docking results. Results indicated that a total of 78 yak bone collagen peptides were identified, the low-molecular-mass ones being predominant (those with molecular mass less than 3000 Da accounted for 91.8%). GPAGPQGPRGDKGETGEQ (GP-18, 1736.815 Da), TPEVDDEALEKFDK (TP-14, 1634.775 Da), GPAGPQGPRGDKGETGE (GP-17, 1608.757 Da), GKSGDRGETGPAGPAGPIGPV (GK-21, 1875.951 Da), and GKSGDRGETGPAGPAGPIGPVG (GK-22, 1932.973 Da) had the potential to promote osteoblast proliferation. The relative proliferation rate of mouse MC3T3-E1 osteoblasts treated with 3.0 mg/mL GK-22 was 106%, which was significantly higher than that of the control group (P < 0.05). The results of this study provide a theoretical reference for targeted screening and industrial preparation of bioactive peptides with the ability to promote osteoblast proliferation.

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 (T_m 66.8 ℃, ΔH 13.52 J/g), B/C/PT (T_m 62.2 ℃, ΔH 10.26 J/g), and B/C/T (T_m 60.6℃, ΔH 8.71 J/g), compared with the B/C (T_m 55.6 ℃, ΔH 5.26 J/g) and BBC (T_m 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.

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.