Doctoral thesis (Dissertations and theses)
Study on green preparation of chondroitin sulfate and antiosteoporosis activity of chondroitin sulfate calcium complex
Shen, Qingshan
2021
 

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Keywords :
CS; CSCa; thermal liquefaction; steam explosion; hot-pressure; chondrocyte proliferation; gut microbiota; bone mineral density; bone turnover markers; anti-osteoporosis
Abstract :
[en] China is the largest producer and exporter of chondroitin sulfate (CS), accounting for 80% of the world's CS production. For traditional CS isolation procedures, it is generally believed that high concentration alkaline treatment is required to break down the core protein. This produces a lot of effluent during industrial production, which is not environmentally friendly. About 30,000 tons of chicken sternal cartilage are produced in China each year, and most of them are usually processed into snacks or other low-value products. However, the constituents of chicken sternal cartilage are still undervalued and deserve to be considered for more economically competitive exploitation. Furthermore, the bioactivities of chondroitin sulfate metal complex, especially chondroitin sulfate calcium complex (CSCa), are poorly reported in vitro and in vivo. Based on the above-mentioned problems, the environmentally friendly preparation of CS from chicken sternal cartilage was proposed, and the bioactivities of CSCa were studied in vitro and in vivo. First, steam explosion (steam cracking) technology was used to liquefy chicken sternal cartilage for CS green preparation. The effects of experimental parameters, including pressure and holding time, on cartilage liquefaction were investigated. Part of the cartilage can be liquefied. The microstructural changes of the solid residue of the steam explosion treated cartilage were verified by scanning electron microscopy, and CS was obtained from the liquid fraction by enzymolysis and membrane separation technologies. The chemical and structural properties of CS were confirmed by Fourier transform infrared spectroscopy, agarose gel electrophoresis, nuclear magnetic resonance and chromatography. The results suggest that steam explosion can be used as a pretreatment method to liquefy cartilage for environmentally friendly isolation of CS. Next, another technology was proposed to completely liquefy chicken sternal cartilage. Hot-pressure technology with low temperature and long processing time was used to pretreat the cartilage for green CS preparation. After complete liquefaction by hot pressure, co-production of CS and peptides was achieved in an environmentally friendly manner by dual enzymatic hydrolysis and combined membrane separation technologies. The effects of hot-pressure parameters, including temperature and time, on the liquefaction of cartilage were investigated. Microstructural changes and migration of constituents in the solid residue of hot-pressure treated cartilage were verified by scanning electron microscopy and alcian blue staining. The chemical and structural properties of the CS sample were confirmed by Fourier transform infrared spectroscopy, agarose gel electrophoresis, chromatography and nuclear magnetic resonance. The results suggest that hot pressing can be used as a pretreatment method to liquefy cartilage for environmentally friendly industrial co-production of CS and peptides. In addition, chondroitin sulfate-calcium complex (CSCa) was fabricated, and the structural characteristics of CSCa and its proliferative bioactivity for chondrocyte were studied in vitro. The fabrication of CSCa was performed to study the maximum calcium retention capacity. The structural properties of CSCa were studied by Fourier transform infrared spectroscopy and nuclear magnetic resonance. The characterization of CSCa was analyzed by scanning electron microscopy, energy dispersive spectrometer, ultraviolet and visible spectrophotometer, atomic force microscope and X-ray diffraction. Thermal properties were analyzed by thermogravimetric analysis and differential scanning calorimetry. Additionally, the effects of CSCa on chondrocyte growth, cell cycle and apoptosis were studied in vitro. The intracellular calcium ion level of the chondrocyte was assessed by fluorescent probe staining. And some proliferative-related genes influenced by CSCa were studied by real-time quantitative polymerase chain reaction. The results suggest that calcium ions can bind to sulfate or carboxyl groups of CS chains forming a polysaccharide-metal complex. CSCa could probably interact with the calcium-sensing receptor, increasing intracellular calcium ions and influencing the cell cycle. CSCa-induced secretion of TGF-β1 could activate the TGF-β/Smads pathway and alter the expression of proliferation-associated genes ultimately leading to chondrocyte proliferation. Finally, the anti-osteoporosis bioactivity of CSCa was studied in vivo. The ovariectomized rat model was constructed by bilateral ovariectomy to simulate estrogen deficiency-induced osteoporosis in postmenopausal women. Bone microarchitecture and bone mineral density were analyzed by microcomputed tomography. Bone turnover markers, including bone formation and resorption markers in serum, were determined by enzyme-linked immunosorbent assay. In addition, multi-omics methods including microbiome and metabolome were used to investigate the alteration of microbiota and changes of metabolic profiles in the faeces from ovariectomized rats subjected to a dietary intervention of CSCa for twelve weeks. The results suggest that, compared with the osteoporosis rats, CSCa, instead of CS, can improve bone mineral density and microstructure of the femur, and alter the level of bone turnover markers in serum. Additionally, CSCa altered the composition of gut microbiota as well as metabolite profiles in the faeces by 16S rRNA sequencing and metabolomics. Specifically, after CSCa intervention, the relative abundance of Acidobacteria, Chloroflexi, and Gemmatimonadetes increased while that of Bacteroidetes, Actinobacteria, and the Bacteroidetes/Firmicutes ratio decreased at the phylum level as well as some specific changes in the gut microbiota community at the genus level. Enriched metabolites of lipid metabolism, amino acid metabolism, and biodegradation and xenobiotic metabolism were distinguished from those of model rats. Correlation analysis showed that certain taxa of the gut microbiota were significantly correlated with osteoporosis phenotypes and enriched metabolites. Overall, dietary CSCa intervention has the potential to alleviate osteoporosis and associated symptoms by acting on gut microbiota composition or metabolite profiles, as demonstrated in rats. This study provides scientific evidence of the potential effects of dietary CSCa on osteoporosis.
Disciplines :
Food science
Author, co-author :
Shen, Qingshan ;  Université de Liège - ULiège > TERRA Research Centre
Language :
English
Title :
Study on green preparation of chondroitin sulfate and antiosteoporosis activity of chondroitin sulfate calcium complex
Alternative titles :
[en] Green preparation of chondroitin sulfate and bioactivities of chondroitin sulfate calcium complex
Defense date :
06 December 2021
Number of pages :
XXii, 183
Institution :
ULiège - Université de Liège
Degree :
DOCTEUR EN SCIENCES AGRONOMIQUES ET INGENIERIE BIOLOGIQUE
Promotor :
Richel, Aurore  ;  Université de Liège - ULiège > Département GxABT > Smart Technologies for Food and Biobased Products (SMARTECH)
Everaert, Nadia ;  Université de Liège - ULiège > Département GxABT
Zhang, Chunhui
President :
Francis, Frédéric  ;  Université de Liège - ULiège > GxABT : Services généraux du site > Site GxABT - Relations internationales
Jury member :
Haubruge, Eric  ;  Université de Liège - ULiège > GxABT : Services généraux du site > Site GxABT - Cabinet du Vice-recteur
Jacquet, Nicolas  ;  Université de Liège - ULiège > Département GxABT > Smart Technologies for Food and Biobased Products (SMARTECH)
Huang, Feng
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since 29 November 2021

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