A Review of Chondroitin Sulfate's Preparation, Properties, Functions, and Applications.

Chondroitin sulfate (CS) is a natural macromolecule polysaccharide that is extensively distributed in a wide variety of organisms. CS is of great interest to researchers due to its many in vitro and in vivo functions. CS production derives from a diverse number of sources, including but not limited to extraction from various animals or fish, bio-synthesis, and fermentation, and its purity and homogeneity can vary greatly. The structural diversity of CS with respect to sulfation and saccharide content endows this molecule with distinct complexity, allowing for functional modification. These multiple functions contribute to the application of CS in medicines, biomaterials, and functional foods. In this article, we discuss the preparation of CS from different sources, the structure of various forms of CS, and its binding to other relevant molecules. Moreover, for the creation of this article, the functions and applications of CS were reviewed, with an emphasis on drug discovery, hydrogel formation, delivery systems, and food supplements. We conclude that analyzing some perspectives on structural modifications and preparation methods could potentially influence future applications of CS in medical and biomaterial research.

Comparative Analysis of the Bioactive Compounds in Chicken Cartilage: Protective Effects of Chondroitin Sulfate and Type II Collagen Peptides Against Osteoarthritis Involve Gut Microbiota.

This study was designed to explore osteoarthritis (OA) treatment from bioactive compounds of chicken cartilage food supplements. The OA rat model induced by sodium iodoacetate was used to evaluate the treatment effect in vivo. In this study, we used animal experiments to show that oral chondroitin sulfate (CS), cartilage powder, and type II collagen peptides could increase the athletic ability of rats and reduce inflammatory cytokine levels in serum or synovial fluid, including prostaglandin E2, tumor necrosis factor-α, interleukin (IL) 1ß, IL-6, and IL-17. CS displayed the best treatment effect against OA. The morphological structure of articular cartilage indicated that CS could significantly improve cartilage tissue morphology and reduce OA score. Oral CS slowed down the development of OA by modulating gut microbiota. These results provided a useful scientific basis for the high-value utilization of chicken cartilage.

A sustainable and efficient recycling strategy of feather waste into keratin peptides with antimicrobial activity.

The study aimed to propose an efficient and eco-friendly strategy to improve the utilization of feather waste and converting it into high-valued antimicrobial products. Under the synergistic effect of instant catapult steam explosion (ICSE) (1.5 MPa-120 s), over 90% of chicken feather powder (CFP) was degraded into soluble peptides via keratinolysis within 3 h, about 90% of which were smaller than 3 kDa, indicating an overwhelming advantage than general proteolysis. Importantly, the keratinolysis hydrolysate of CFP was able to inhibit E. coli growth, among which the fraction < 3 kDa exhibited highest antimicrobial activity with a minimal inhibitory concentration of 30 mg/mL. Compared to other fractions, the fraction < 3 kDa contained higher content of hydrophobic amino acids (364.11 mg/g), in which about 79% of peptides had more than 60% hydrophobic ratio, potentially contributing to its antimicrobial activity. ICSE-keratinolysis process holds potential in reducing both protein resource waste and environmental pollution by valorizing feathers into antimicrobial product.

An advanced strategy for efficient recycling of bovine bone: Preparing high-valued bone powder via instant catapult steam-explosion.

As the major byproduct of meat processing, bovine bone are produced in large amounts annually. However, the inefficient utilization with low-added value resulted in serious resource waste. The study aims to prepare high-value bovine bone power (BBP) via instant catapult steam-explosion (ICSE) treatment, taking ball milling (BM) method as control. Results showed that ICSE treatment deconstructed bovine bone with more holes emerging, and effectively promoted mineral dissolution and protein degradation while reduced energy consumption. Compared with BM-BBP, ICSE-BBP possessed more protein and essential minerals, presenting in regular elliptical shapes with narrow distribution of particle size (0.1 âˆ¼ 40 µm), and owned better solution stability and protein solubility. ICSE-BBP also exhibited higher mineral release and protein digestibility during GI digestion while revealed no obvious cytotoxicity, indicating the potential applicability in nutrition-fortified foods. Taken together, ICSE technology holds promise in reusing bovine bone, providing an efficient and eco-friendly process for BBP industrial production.

Physicochemical properties, digestibility and anti-osteoporosis effect of yak bone powder with different particle sizes.

As a kind of promising resource, animal bone has been widely processed into functional foods. However, there is little research about the effect of particle size on the physicochemical properties and digestibility of yak bone powder (YBP), as well as its anti-osteoporosis activity. In this study, the YBP with median particle sizes (MPS) ranging from 19.68 to 128.37 µm were prepared, and their digestibility and anti-osteoporosis activity were investigated. The results showed that smaller MPS significantly increased water holding capacity and protein solubility without changing composition. The MPS reduction greatly promoted protein digestion, producing more peptides<3 kDa and free amino acids while decreased Ca2+ and P5+ release during gastrointestinal digestion. The in vivo results revealed the positive effect of YBP on ovariectomy-induced osteoporosis in rats. The bone mineral density of ovariectomized (OVX) rats was obviously improved by regulating bone turnover markers (B-ALP, OCN, S-CTX, ES and TRAP), thus potentially shedding light on osteoporosis remission. However, different MPS exhibited a weak effect on osteoporosis in OVX rats. Therefore, YBP could be produced in relatively large particle size without sacrificing food sensory quality, the processing time of which could also be shortened for higher productivity and lower cost.

Modulation of gut microbiota by chondroitin sulfate calcium complex during alleviation of osteoporosis in ovariectomized rats.

Although chondroitin sulfate calcium complex (CSCa) was claimed to have the bioactivity for bone care in vitro, its anti-osteoporosis bioactivity was little reported in vivo. Here, the effects of CSCa on osteoporosis rats were investigated. Results showed that, compared with the osteoporosis rats, CSCa could improve the bone mineral density and microstructure of femur, and change the bone turnover markers level in serum. 16S rRNA sequencing and metabolomics analysis indicated CSCa intervention altered the composition of gut microbiota along with metabolite profiles in ovariectomized rat faeces. The correlation analysis showed some gut microbiota taxa were significantly correlated with osteoporosis phenotypes and the enriched metabolites. Taken together, dietary CSCa intervention has the potential to alleviate the osteoporosis and related symptoms probably involving gut microbiota or the metabolite profiles as demonstrated in rats. This study provides some scientific evidence for the potential effects of CSCa as the food supplement on the osteoporosis.

Discrimination of geographical origin and species of China's cattle bones based on multi-element analyses by inductively coupled plasma mass spectrometry.

Consumers have an increasing concern in the provenance of the foods they consume. Methods for discriminating geographical origins and species of cattle bone product are essential to provide veracious information for consumers and avoid the adulteration and inferior problems. In this study, 50 element contents of a total of 143 cattle bone samples from eight producing regions in China, were determined by inductively coupled plasma mass spectrometry (ICP-MS). Element contents were used as chemical indicators to discriminate species and geographical origins of cattle bone samples by multivariate data analysis, including hierarchical cluster analysis (HCA) and linear discriminant analysis (LDA). The K-fold cross validation accuracy for species and geographical origin discrimination was 99.3% and 94.5%, respectively. This study reveals that multi-element analysis accompanied by LDA is an effective technique to ensure the information reliability of cattle bone samples, and this strategy may be a potential tool for standardizing market.

Fabrication of chondroitin sulfate calcium complex and its chondrocyte proliferation in vitro.

Chondroitin sulfate (CS)-calcium complex (CSCa) was fabricated, and the structural characteristics of CSCa and its proliferative bioactivity to the chondrocyte were investigated in vitro. Results suggested calcium ions could bind CS chains forming polysaccharide-metal complex, and the maximum calcium holding capacity of CSCa reached 4.23 %. Characterization of CSCa was performed by EDS, AFM, FTIR, UV, XRD and 1H-NMR. It was found that calcium ions were integrated with CS by binding the sulfate or carboxyl groups. The thermal properties analysis indicated CSCa had a good thermal stability by TGA and DSC. CSCa could interact the calcium-sensing receptor increasing the intracellular calcium ions and influence the cell cycle. The TGF-ß1 secretion induced by CSCa could activate the TGF-ß/Smads pathway and change the genes associated proliferation expression ultimately leading to the chondrocyte proliferation. This research probably has an important implication for understanding the effect of CSCa on bone care as food supplements.

Metabolomics strategy reveals the osteogenic mechanism of yak (Bos grunniens) bone collagen peptides on ovariectomy-induced osteoporosis in rats.

Our previous work demonstrated that yak bone collagen peptides (YBP) possessed excellent osteogenic activity in vitro. However, associations between YBP and osteoporosis were less established, and the positive effect and underlying mechanism of YBP in the treatment of osteoporotic rats in vivo remained unclear. Herein, ovariectomized rats were intragastrically administered with YBP or 17ß-estradiol for 12 weeks. Bone turnover markers, bone biomechanical parameters and bone microarchitecture were investigated to identify the specific changes of potential antagonistic effects of YBP on ovariectomized rats. Then, serum samples were analyzed by UPLC/Q-TOF-MS to identify metabolites. The results showed that YBP treatment remarkably altered the content of serum bone turnover markers and prevented the ovariectomy-induced deterioration of bone mechanical and microarchitecture characteristics. A total of forty-one biomarkers for which levels changed markedly upon treatment have been identified based on non-targeted metabolomics. Among them, twenty-one metabolites displayed a downward expression level, while twenty metabolites showed an upward expression level in the YBP group and finally were selected as potential biomarkers. The levels of these biomarkers displayed significant alterations and a tendency to be restored to normal values in YBP treated osteoporotic rats. A systematic network analysis of their corresponding pathways delineated that the protective or recovery effect of YBP on osteoporosis occurred primarily by regulating the amino acid metabolism and lipid metabolism (especially unsaturated fatty acid). Collectively, these findings highlight that such peptides hold promise in further advancement as a natural alternative for functional and health-promoting foods, which could be potentially used in mediated treatment of osteoporosis.

Yak (Bos grunniens) bones collagen-derived peptides stimulate osteoblastic proliferation and differentiation via the activation of Wnt/ß-catenin signaling pathway.

BACKGROUND: As the world's population is transitioning gradually to an ageing stage, the incidence of osteoporosis is increasing annually. Yak bone is one of the major components of Tibetan medicine and it has mainly been associated with an improvement in bone health, for example against osteoporosis. However, the functional bioactive ingredients and the underlying mechanisms are still unclear. RESULTS: Sequential purification of yak-bone hydrolysates was achieved by ultrafiltration, size exclusion chromatography, and semi-preparative reverse-phase high-performance liquid chromatography. After this, 35 novel peptides were identified by mass spectrometry analysis, of which peptide GPAGPPGPIGNV (GP-12) displayed the highest osteoblast proliferation-promoting activity, with an increase of 42.7% in cell growth. An in vitro stability study demonstrated that GP-12 was digested into smaller peptides (GP-9, GV-9, AV-10 and GP-11) after simulated gastrointestinal digestion and absorption (Caco-2 cell monolayers) experiments. However, some of them still can be absorbed intact through the (Caco-2 cell monolayers by a paracellular route (Papp: 5.36 ± 0.34 cm s-1 ). Flow cytometry results indicated that GP-12 enhanced osteoblastic proliferation by inducing the alteration of the cell-cycle progression both from the G0/G1 to the S phase and from the S to the G2/M phase. Quantitative real-time polymerase chain reaction (PCR) and western blot results revealed that GP-12 induced osteoblastic proliferation and differentiation in a dose-response manner through the activation of a Wnt/ß-catenin signaling pathway. CONCLUSION: These findings highlighted that such peptides hold the promise of discovering candidates for functional and health-promoting foods, which could be potentially used for the treatment of osteoporosis. © 2020 Society of Chemical Industry.

Liquefaction of porcine hoof shell to prepare peptone substitute by instant catapult steam explosion.

Instant catapult steam explosion (ICSE) was proposed as a method to liquefy porcine hoof shell (PHS) and prepare a peptone substitute for fermentation culture, achieving environmentally friendly animal by-product recycling. The liquefaction of PHS was conducted at various pressures (0.5-2.3 MPa) for 5-30 min. As evidenced by the scanning electron microscopy analysis, ICSE caused randomly cracks changing the morphological structure of the solid fraction, and ultimately led to protein migration from the solid to liquid phase. Moreover, the chromatographic analysis revealed that the main constituents of the liquid fraction were short peptides (<2 kDa, 84.72%) and amino acids (1.68 mg/mL) at the pressure of 2.3 MPa for 30 min. Subsequently, liquid fractions were prepared as a PHS peptone substitute for fermentation culture. Results suggested the PHS peptone substitute as the main nitrogen source in media was more suitable for the growth of fungus. Therefore, ICSE provides a possibility of large-scale environmentally sustainable management of animal by-products through liquefaction.

Co-production of chondroitin sulfate and peptide from liquefied chicken sternal cartilage by hot-pressure.

Co-production of chondroitin sulfate (CS) and peptides was realized from the liquid fraction of chicken sternal cartilage subjected to hot-pressure (HP) by membrane combination separation technology. Cartilage was liquefied via the HP treatment at 110 °C (0.07 MPa) and 120 °C (0.1 MPa) for 0.5 - 2.5 h, respectively. The optimized co-production procedure was as follows: enzymolysis temperature, 61.2 °C; the enzyme ratio of trypsin and papain, 1.3:1 (W/W); enzymolysis time ratio, 2:2 (h/h), under which the highest yields of CS and peptides were 18.85% and 67.99%, and the recoveries were 93.63% and 92.69%. The average molecular weight of CS sample was 67.79 kDa. CS sample was confirmed using agarose-gel electrophoresis, and the structure was analyzed by Fourier transform infrared spectroscopy, chromatography and nuclear magnetic resonance. Taken together, HP can be as a pretreatment method to liquefy cartilage for the industrial co-production of CS and peptides with eco-friendly.

Liquefaction of chicken sternal cartilage by steam explosion to isolate chondroitin sulfate.

Chondroitin sulfate (CS), together with peptide, was isolated from the liquid fraction of chicken sternal cartilage subjected to steam explosion (SE) by membrane separation. Cartilage was liquefied via the SE conditions, including various pressures (1.0-1.6 MPa) and times (60-140 s). The extraction procedure was optimized as follows: the amount of papain added, 0.11%; enzymolysis time, 10.5 h; and enzymolysis temperature, 56.5 °C, under which the highest recovery and total yield of CS were 92.15% and 18.55% at 1.4 MPa for120 s, and the counterparts of peptides were 87.35% (1.0 MPa, 140 s) and 63.07% (1.6 MPa, 140 s). The average molecular weight of CS samples ranged from 30 to 35 kDa. CS sample was confirmed using agarose-gel electrophoresis, and the structure was analysed Fourier transform infrared spectroscopy, chromatography and nuclear magnetic resonance. Taken together, SE can be an eco-friendly pretreatment method to liquefy cartilage for CS isolation.

Chicken leg bone as a source of chondroitin sulfate.

In this work, chondroitin sulfate (CS) was extracted from chicken leg bone soup using the heat-resin static adsorption extraction (HSAE) method. The HSAE method was optimized as follows: resin dosage, 10%; adsorption time, 4.3 h; eluent concentration, 2 M; eluent time, 1.3 h, under which the yield of CS1 from the bone soup reached 0.14% and the recovery rate was 67.35%. CS2, as reference, was obtained from the ends of chicken leg bone using enzymatic method. CS1 and CS2, together with other glycosaminoglycans, were confirmed using agarose-gel electrophoresis. The average molecular weight of CS1 and CS2 was 35.81 kDa and 37.18 kDa, respectively. The structures of CS1 and CS2 were compared using Fourier-transform infrared spectroscopy and high-performance liquid chromatography, and no significant difference was observed. Overall, the HSAE method was proposed to be a promising approach for the coproduction of CS and bone soup.

Preparation, identification and molecular docking study of novel osteoblast proliferation-promoting peptides from yak (Bos grunniens) bones.

The aim of this study was to isolate and identify osteogenic bioactive peptides from yak bones collagen, while simultaneously investigating their underlying mechanisms for promoting osteoblast proliferation. Response surface methodology (RSM) was employed to investigate the effect of hydrolysis variables on the production of peptides with osteoblast proliferation-promoting activity (OPPA). The concentration of soluble peptides reached 0.5169 mg mL-1, which was well matched with the value (0.5189 mg mL-1) predicted by the model, with the following optimized conditions: hydrolysis time, 3.6 h; pH, 6.12; hydrolysis temperature, 54 °C; E/S (enzyme to substrate) of 5637 U g-1. Hydrolysates were then separated using an ultrafiltration membrane system, and the peptides (<3 kDa) possessed excellent OPPA with a dose-response relationship. A total of 59 novel peptides were identified by HPLC-MS/MS with Mascot analysis. GPSGPAGKDGRIGQPG (GP-16) and GDRGETGPAGPAGPIGPV (GD-18) were selected for docking to investigate the underlying mechanisms of interaction. The molecular docking study revealed that osteoblast proliferation stimulation activity of GP-16 and GD-18 was mainly attributed to the formation of very strong hydrogen bonds with the epidermal growth factor receptor (EGFR). These results indicate that such peptides are promising in the discovery of potential candidates for the future industrial production of functional peptides, which could be used in the mediated treatment of osteoporosis.

Thymol Induces Conidial Apoptosis in Aspergillus flavus via Stimulating K+ Eruption.

Aspergillus flavus is a notorious foodborne fungus, posing a significant risk to humans in the form of hepatocellular carcinoma or aspergillosis. Thymol, as a food preservative, could efficiently kill conidia of A. flavus. However, the underlying mechanisms by which thymol kills A. flavus are not completely understood. With specific fluorescent dyes, we detected several apoptotic hallmarks, including chromatin condensation, phosphatidylserine externalization, DNA damage, mitochondrial depolarization, and caspase 9 activation in conidia exposed to 200 µg/mL of thymol, indicating that thymol induced a caspase-dependent conidial apoptosis in A. flavus. Chemical-protein interactome (CPI) and autodock analyses showed that KCNAB, homologue to the ß-subunit of the voltage-gated potassium channel (Kv) and aldo-keto reductase, was the potential target of thymol. Following studies demonstrated that thymol could activate the aldo-keto reductase activity of KCNAB in vitro and stimulate a transient K+ efflux in conidia, as determined using a Port-a-Patch. Blocking K+ eruption by 4-aminopyridine (a universal inhibitor of Kv) could significantly alleviate thymol-mediated conidial apoptosis, indicating that activation of Kv was responsible for the apoptosis. Taken together, our results revealed a K+ efflux-mediated apoptotic pathway in A. flavus, which greatly contributed to the development of an alternative strategy to control this pathogen.

Bactericidal activity of alpha-bromocinnamaldehyde against persisters in Escherichia coli.

Persisters are tolerant to multiple antibiotics, and widely distributed in bacteria, fungi, parasites, and even cancerous human cell populations, leading to recurrent infections and relapse after therapy. In this study, we investigated the potential of cinnamaldehyde and its derivatives to eradicate persisters in Escherichia coli. The results showed that 200 µg/ml of alpha-bromocinnamaldehyde (Br-CA) was capable of killing all E. coli cells during the exponential phase. Considering the heterogeneous nature of persisters, multiple types of persisters were induced and exposed to Br-CA. Our results indicated that no cells in the ppGpp-overproducing strain or TisB-overexpressing strain survived the treatment of Br-CA although considerable amounts of persisters to ampicillin (Amp) and ciprofloxacin (Cip) were induced. Chemical induction by carbonyl cyanide m-chlorophenylhydrazone (CCCP) led to the formation of more than 10% persister to Amp and Cip in the entire population, and Br-CA still completely eradicated them. In addition, the cells in the stationary phase, which are usually highly recalcitrant to antibiotics treatment, were also completely eradicated by 400 µg/ml of Br-CA. Further studies showed that neither thiourea (hydroxyl-radical scavenger) nor DPTA (Fe3+ chelator to block the hydroxyl-radical) affected the bactericidal efficiency of the Br-CA to kill E. coli, indicating a ROS-independent bactericidal mechanism. Taken together, we concluded that Br-CA compound has a novel bactericidal mechanism and the potential to mitigate antibiotics resistance crisis.

ROS Involves the Fungicidal Actions of Thymol against Spores of Aspergillus flavus via the Induction of Nitric Oxide.

Aspergillus flavus is a well-known pathogenic fungus for both crops and human beings. The acquisition of resistance to azoles by A. flavus is leading to more failures occurring in the prevention of infection by A. flavus. In this study, we found that thymol, one of the major chemical constituents of the essential oil of Monarda punctate, had efficient fungicidal activity against A. flavus and led to sporular lysis. Further studies indicated that thymol treatment induced the generation of both ROS and NO in spores, whereas NO accumulation was far later than ROS accumulation in response to thymol. By blocking ROS production with the inhibitors of NADPH oxidase, NO generation was also significantly inhibited in the presence of thymol, which indicated that ROS induced NO generation in A. flavus in response to thymol treatment. Moreover, the removal of either ROS or NO attenuated lysis and death of spores exposed to thymol. The addition of SNP (exogenous NO donor) eliminated the protective effects of the inhibitors of NADPH oxidase on thymol-induced lysis and death of spores. Taken together, it could be concluded that ROS is involved in spore death induced by thymol via the induction of NO.

Nanocapsular dispersion of cinnamaldehyde for enhanced inhibitory activity against aflatoxin production by Aspergillus flavus.

Cinnamaldehyde (CA) is marginally soluble in water, making it challenging to evenly disperse it in foods, and resulting in lowered anti-A. flavus efficacy. In the present study, nano-dispersed CA (nano-CA) was prepared to increase its aqueous solubility. Free and nano-dispersed CA were compared in terms of their inhibitory activity against fungal growth and aflatoxin production of A. flavus both in Sabouraud Dextrose (SD) culture and in peanut butter. Our results indicated that free CA inhibited the mycelia growth and aflatoxin production of A. flavus with a minimal inhibitory concentration (MIC) value of 1.0 mM, but promoted the aflatoxin production at some concentrations lower than the MIC. Nano-CA had a lower MIC value of 0.8 mM against A. flavus, and also showed improved activity against aflatoxin production without the promotion at lower dose. The solidity of peanut butter had an adverse impact on the antifungal activity of free CA, whereas nano-dispersed CA showed more than 2-fold improved activity against the growth of A. flavus. Free CA still promoted AFB1 production at the concentration of 0.25 mM, whereas nano-CA showed more efficient inhibition of AFB1 production in the butter.