| There are many factors which could affect the tenderness,such as myofibril protein and connective tissue.Until now,there are many researches focused on the change of the muscle fibers,and the researches aimed to explore the contribution of connective tissue to beef tenderness are rare.The relationship between maturity and intramuscular fat deposition,and cross-links and proteoglycans in connective tissue are rarely reported.There also are some controversies on the degradation of muscle fiber caused by cathepsin L,and few reports on the weakening mechanism of connective tissue.Acid marinating can weak connective tissue in beef.However,existing reports did not involve the changes of proteoglycans during processing.In this paper,the main concern is the changes of cross-links and proteoglycans in connective tissue in different physiological maturities and marbling levels.SDS-PAGE,western blot,scanning electron microscopy(SEM),transmission electron microscope(TEM),light microscope,infrared spectra and DSC technique were employed to explore the mechanism of weakened myofibril protein,connective tissue after treated by cathepsin L and organic acid.The specific contents and results are as follows.1.The relationship between mature cross-links,decorin,glycosaminoglycans(GAGs)the properties of connective tissue and shear force in bovine longissimus thoracic muscleIn the beef with the same teeth maturity,the content of cross-links decorin and glycosaminoglycans(GAGs)decreased during intramuscular fat deposited from marbling level C,B,A to S,which could improve the tenderness of beef by increasing the heat solubility of collagen.Scanning electron microscopy(SEM)images showed that collagen fiber dispersed into filaments with a loose structure in the highly marbled beef samples.Meanwhile,in the beef with the same marbling level,the content of cross-links,decorin and GAGs increased with the increase of physiological maturity(0,1,2,3&4 pairs of permanent incisors),which reduced the heat solubility of connective tissue and toughened the beef partly.Collagen fibers arranged closely and distributed in flake or clusters,showing tightly pattern.Multivariate statistical results showed that the content of mature cross-links,decorin and GAGs was negatively correlated with collagen heat solubility and was a significantly positive correlated with shear force.The heat solubility of collagen was prior to the content of collagen in connective tissue,both of which decided beef shear force.2.Reaserch on Cathepsin L degrading myofibril and connective tissueCathepsin L was firstly purified by acidification,ammonium sulfate precipitation,dialysis,enrichment,DEAE-Sephacel,Sephacryl S-100,SP Sepharose Fast Flow,Blue Sepharose Fast Flow,ConA Sepharose 4B chromatography.The molecular weight of purified cathepsin L was about 30 kDa,and the optimum pH was 5.5.SDS-PAGE showed that myosin heavy chain(MHC),troponin T,tropomyosin and myosin light chain(MLC1)could be hydrolyzed by cathepsin L.The degradations of Actin,a-actin,desmin,troponin I were not obvious.TEM figures showed that cathepsin L mainly acted on A-bands(mainly MHC)of muscle fiber;Nevertheless,the degradation of Z-line area(actin,cytoskeletal protein)was weak.The model of hydrolysis caused by cathepsin was different from calpains.CD spectrum showed that,the relative content of a-helix dropped by 13.2%,and the(3-sheet content increased by 108.6%in the process of myofibril hydrolysis.The thermal transition temperature of the connective tissue incubated by cathepsin L in vitro could be reduced significantly.In the control group of pH 5.0 and 5.5(citrate buffer),the To value decreased from 60.63?to 57.87?and 59.73?respectively.In the enzyme treatment group of pH 5.0,after 36 h incubation,the To value decreased to 41.41?(P<0.05)significantly.In the treatment of pH 5.5,the To value decreased by 3.16?(P<0.05)in control group.After 36 h incubation(pH 5.0),the Tp value decreased significantly from 65.38?to 43.79?(P<0.05).Tp decreased slightly to 60.79?in pH 5.5(P>0.05).It indicated that the lower the pH was,the more obvious cathepsin L degradated connective tissue.The SDS-PAGE analysis of CNBr-digested peptides did not show significantly different,which indicated that the degradation of collagen in IMCT treated with cathepsin L could take place on the terminal non-helical peptides rather than the triple helix region of a-chains.Meanwhile,decorin could be degraded by cathepsin L,and the efficiency of cathepsin L hydrolysis was higher in lower pH.Combined with all the results above,the prosperities changes in IMCT were attributed to the degradation of collagen and decorin,thus affecting beef shear force.3.Reaserch on the effect of marination with 2-hydroxy succinic acid on the properties of IMCT and the shear force of beefMarinating with 2-hydroxy succinic acid could reduce shear force and collagen content in bovine longissimus thoracis muscle.The collagen content reduced from 4.7 mg/g to 3.5 mg/g and the heat solubility increased from 17%to 24%in the acid treated group after 24 h marinating.The observation of histology showed that primary perimysium and secondary perimysium thickness were all reduced,and even collapse occurred in the primary perimysium after 24 h treatment.Meanwhile,the initial denaturation temperature and peak denaturation temperature(TO and TP)of the connective tissue decreased significantly(P<0.05),and reduced from 60.63?and 65.38?to 37.55?and 40.15?,respectively.In addition,decorin in connective tissue could be dissociated gradually after treated with acid.The ATR-FTIR showed that the changes of secondary structure in collagen were not obvious after marinating connective tissue with only a small transformation among 310-helix,anti aggreganted strands and Turn structure,which might be related to the dissociation of decorin.Part shifts in conformations of the "thermal stability area" of collagen made the collagen triple helix chain unwinding more easily and collagen protein thermal-denatured more easily. |
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