| The curing pigments were prepared from hemoglobin to substitute the color fixation ofnitrite. Although the remnant of nitrite was reduced in the meat product, the stability anddispersion of the curing pigments prepared from Nitroso-Hemoglobin (NO-Hb) which hadbeen reported was very poor. So the application of them was limited. In this paper, the studywas focused on the improvement of the stability and dispersion of NO-Hb, the preparation ofNO-free curing pigment——the hemoglobin-histidine complex and ferrous heme-histidinecomplex prepared from hemoglobin, then the application of these pigments in sausage. Themajor research content and data was as follows:1.The stability and dispersion of NO-Hb was improved through the glycosylation. Thehemoglobin extracted from porcine blood was used as material to prepare NO-Hb. AfterNO-Hb reacted with different saccharide, the heat stability of production was detected. Theinfluences of concentration of NO-Hb and saccharide,pH of the solution,the heattemperature and time etc on glycosylation reaction were studied. The single factor and theorthogonal experiment indicated that pH influenced mostly on the reaction, the second wasthe concentration of NO-Hb. The best conditions were that concentration of NO-Hb was 5.0percent, concentration of chitosan was 1.0 percent, pH of reaction solution was 5.0, the heattemperature was 60℃, heat time was 20 min and stired slowly. There were 77.2 percenthemoglobin, 3.87 percent moisture, 3.41 percent ash and 865mg/kg nitrite in the powderpigment after the solution was sprayed drying. It could dissolve in water and keep red when itwas diluted in 200 to 300 times. It could be used in food manufacture as red pigments. Theamount suggested was 0.5 to 1.0 percent.2.The structure of glycosylated NO-Hb was confirmed. The glycosylated NO-Hb waspurified by select heating method and Sephadex G-75 chromatogram. Then its purity wasalmost 96.2% detected by Sephdex G-150 chromatogram and the HPLC. Theultraviolet-visible absorption of the pigment was different from NO-Hb. Its soret regionred-moved to 433nm,the visible region became a big slowly ascend 530～570nm strip. Thecurve model showed that the heme was six ligand state. The glycosylated NO-Hb displayedtwo distinct apexes at 1650 cm-1 and 1530 cm-1 of imine absorption and a character apex at1000~1300cm-1 of C-OH absorption of amylase. It indicated that aldehyde of chitosan reactedwith amino of NO-Hb to form conjugation complex. The circular dichroism (CD) displayedthat the secondary-structure of hemoglobin changed notably. The content of Helix and Betawas reduced and content of Turn and Random was increased. It indicated that peptides on thehemoglobin stretched and combined with chitosan to form stable structure. The enzymichydrolysis and the analysis of number of aldehyde and amino before and after glycosylation,all showed that glycoslated NO-Hb was covalent complex.3 . The physics and chemical properties of glycosylated NO-Hb were studiedsystematically. The denaturalization temperature of glycosylated NO-Hb was 97.69℃detected by DSC, higher than 74.13℃ of NO-Hb and 79.19℃ of encapsulated NO-Hb. Theglycosylated NO-Hb was weak red and few cloudy after exposed to sunlight for 6d, butencapsulated NO-Hb decolored and sedimented entirely Moreover the stability ofglycosylated NO-Hb under the conditions of heat,oxygen and metal ion was improveddistinctly. The diameter distribution of glycosylated NO-Hb was around 0.1um, and that ofencapsulated NO-Hb was 10～20um. With the dissolve experiment it showed the dispersionof the new pigment was better.4.For preparation of nitrite-free hemoglobin curing pigment, the coordinated reaction ofhistidine and hemoglobin was studied. The UV absorption and the infrared (IR) spectra ofproduct demonstrated that the six-coordinated complex of hemoglobin-histidine was created.The equilibrium constants β was 3.66×103 (at 543nm), higher than the ligands of pyridineand organic amines. It indicated that histidine was ideal substitute for nitrite. The standardmolecular enthalpy change △rHθm was -97.0 KJ.mol-1 and standard entropy change △rSθm was -0.257 KJ.mol-1 of coordinated reaction. It showed the reaction was reducing courseof △rHθm,△rSθm and easily progressed. After the nitrite-free hemoglobin complex treatedby glycosylation, the denaturation temperature of nitrite-free pigment was 91.23℃. Itsstability on the conditions of light and heat was higher than encapsulated NO-Hb. There were81.5 percent hemoglobin,2860mg/kg iron, and no nitrite in the spray-drying product.5.The nitrite-free curing pigment was prepared through coordinated reaction ofhistidine and ferrous-heme obtained by enzyme hydrolysis of hemoglobin. The neutrase wasselected as first-step hydrolysis. The most suitable hydrolysis conditions were theconcentration of hemoglobin 5.0 percent, tea polyphenols 0.04 percent, temperature 45℃,pH 8.0, enzyme amount 5000u/g and hydrolysis time 4h. The flavour enzyme (exopeptidase)was added at secondly hydrolysis for increase the degree of hydrolysis and content of aminoacides. The hydrolysis conditions were that substract 5.0 percent, temperature 55℃, pH6.0, enzyme amount 1.0 percent, and hydrolysis time 5h. The hydrolysate was ultrafiltratedwith 3 or 10 kDa molecular weight cut-off (MWCO) of the membrane. Ferrousheme-enriched peptide was obtained, which the ratio of heme and peptide reached 9.92percent. Red product was stable after buried with microcapsule. The UV spectra andchemical analysis indicated that the histidine was reacted with ferrous heme, and came intostable six coordinated complex. The hard red powder product which consist of protein 30.85percent, moisture 5.10 percent, ash 6.03 percent and ferrous 2517mg/kg was obtained afterencapsulated.6.The application of the curing pigments was discussed in production of sausage. Thepoly-composition system built up with glycosylated NO-Hb, tea polyphenols, nisin et al wasapplied in sausages product. The detection of color detector,physics character apparatu andsensory valuation made clear that meat product added with 0.5% glycosylated NO-Hb getideal and stable color, the texture and flavor better than the samples cured by nitrite or addedwith 0.5 percent encapsulated NO-Hb. The detection by GC-MS and TBARS value indicatedthat the content of aldehyde of product used with the poly-composition system was lower thancontraposition. The lower degree fat was oxidated, the better was flavor. The nitrite remnantof sausage added the new pigment was only tenth of sample cured by nitrite, but content ofiron was enhanced in large scale.
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