| A large number of studies showed that oral administration of undenatured type ? collagen can inhibit the occurrence of arthritis or alleviate the symptoms of rheumatoid arthritis(RA)and osteoarthritis(OA)by inducing immune tolerance.At present,there are two main types of undenatured collagen,namely insoluble(IC ?)and soluble undenatured type ? collagen(SC ?).IC ? has poor solubility,but after oral administration,it may release some soluble type ? collagen to expose its immune activity epitopes under the action of enzyme and p H environment of gastrointestinal digestive system.By contrast,the solubility of SC ? is good,which makes it possible to fully expose its immune active epitopes.However,the stability of SC ? is poor,that is,the triple helix structure is easy to be destroyed,which may be accompanied by the loss of biological activity.Importantly,the stability and degradation of these two kinds of undenatured type ? collagen during gastrointestinal digestion have not been reported.Therefore,in this paper,we compared the structural properties and gastrointestinal digestive characteristics of IC ? and SC ?,and analyzed the key factors in the process of gastrointestinal digestion.Then,we explored the protective effect of polysaccharides on gastric digestion of SC ? and the mechanism of this protection,and further clarified the influence of p H,Na Cl and polysaccharide on the structure and thermal stability of SC ?.The main research contents and results are as follows:(1)The INFOGEST static in vitro digestion model was used to simulate the gastrointestinal digestion experiment on IC ? and SC ?.The structural properties and gastrointestinal digestive characteristics of IC ? and SC ? were analyzed by sodium dodecyl sulfonate polyacrylamide gel electrophoresis(SDS-PAGE),Fourier transform infrared spectroscopy(FTIR),X-ray diffraction(XRD),circular dichroism spectroscopy(CD),transmission electron microscopy(TEM)and atomic force microscopy(AFM).Results showed that,after gastric digestion,especially under low p H conditions,some thick and short collagen fibers were dissolved from IC ?,which was accompanied by the release of soluble protein with triple helix structure.As to SC ?,when gastric digestion was performed under higher p H conditions,collagen fibers were mildly thinned and curved,and the triple helix structure was slightly destroyed.However,those changes hardly occurred during the intestinal digestion.Moreover,the undenatured type ? collagen existing in SC ? digestive supernatant was 1.2-12.4 times higher than that of IC ? depending on the p H conditions.These results suggested that,it is more likely for SC ? to exert its activity in relieving arthritis,and it should be taken when the p H of gastric environment is high.(2)According to the previous chapter,the gastric digestive stability of SC ? under p H 2.0was poor.In order to reduce the degradation of SC ?,the protective effects of guar gum(GG),xanthan gum(XG)and carrageenan(CG),which have different molecular weight,particle size and charge properties,on the gastric digestion of SC ? at p H 2.0 were investigated.The results of SDS-PAGE and circular dichroism spectrum showed that guar gum had limited protective effect on the gastric digestion of SC ?.Xanthan gum had weak protective effect at the ratio of0.1:1-0.2:1,and the protective effect was significantly enhanced at 0.4:1-1.5:1,that is,the content of triple helix structure in XG-SC ? was 2.48-3.10 times of that in SC ? after digestion.Carrageenan had significant protective effect at 0.1:1-0.2:1(1.99-2.48 times),and the protective effect slightly enhanced at 0.4:1-1.5:1(2.50-2.73 times).Combined with the analysis of rheology,particle size,?-potential,microscope,confocal laser scanning microscopy(CLSM)and circular dichroism spectrum,it was found that there was no significant correlation between viscosity and ellipticity,which could not directly reflect and predict the protective effect of polysaccharides on gastric digestion of SC ?.At the ratio of 0.1:1-0.2:1,carrageenan can combine with SC ? to form soluble complex,which could improve the thermal stability of SC ? at p H 2.0,and might also reduce the interaction between pepsin and SC ?,and thus produced a significant protective effect on SC ?.However,xanthan gum had no effect on the thermal stability of SC ?,but could combine with SC ? to form insoluble complexes,which might prevent the binding of pepsin and slightly protect SC ?.At the ratio of 0.4:1-1.5:1,both carrageenan and xanthan gum could form insoluble complexes with SC ?,which might hinder the binding of pepsin with SC ?,and thus inhibited the gastric digestion of SC ?.Therefore,through electrostatic interaction,a small amount of carrageenan could have a great protective effect on the gastric digestion of SC ? under p H 2.0,while XG required a higher dosage.(3)Finally,the effect of p H,Na Cl and polysaccharide on the structure and thermal stability of SC ? were investigated by circular dichroism spectroscopy and rheometer,and the correlation between triple helix structure and viscosity of SC ? was analyzed.Results showed that,at p H 2.0-4.0,the triple helix structure of SC ? was complete,and the denaturation temperature was 36.5-40 °C,suggesting the low thermal stability of SC ?.At p H 5.0-9.0,the triple helix structure was partially untwisted or curled(28%-57%),and the denaturation temperature was 40-43 °C,suggesting its higher thermal stability.At p H 10.0,the triple helix structure of SC ? was restored,and the degree of untwisting or curling was about 7%.In addition,the viscosity of SC ? increased first and then decreased during p H 2.0-5.0,unchanged at p H 5.0-9.0 and increased again at p H 10.0.With the change of p H,the correlation between viscosity and triple helix structure of SC ? was highly significant(P<0.01),which reflected the stretching and curling state of SC ?.They also had significant correlation(except p H 5.0,P<0.05)with the change of temperature,which reflected the degree of irreversible denaturation of SC ?,that is,the higher the temperature,the smaller the viscosity,and the greater the denaturation of SC ?.At p H 2.0,Na Cl(0-0.5 mol/L)and carrageenan(0.1:1-0.2:1)had little effect on the triple helix structure of SC ?,but Na Cl slightly decreased the denaturation temperature of SC ?(from 36.5 °C to 35 °C),while carrageenan increased the complete denaturation temperature from 37.5 °C to 42.5 °C.At p H 7.0,Na Cl,xanthan gum and carrageenan could promote the extension of the triple helix structure of SC ?,and Na Cl also decreased the denaturation temperature of SC ?(from 45 °C to 41 °C),while xanthan gum and carrageenan had little effect on the thermal stability of SC ?.Therefore,in order to maintain the complete triple helix structure of type ? collagen,it is necessary to strictly control the p H value,Na Cl concentration and temperature during the extraction and processing process,and the thermal stability of soluble undenatured type ? collagen under acidic conditions can be improved by adding carrageenan. |
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