| Maillard reaction is one of the most important reactions in forming the special aroma of tobacco. The Maillard reaction products (MRPs) are the important fragrant precursors and the prepared MRPs are harmonious to the odour of the cigarette when they are smoked, so Maillard reaction has been known as one critical technique to reduce the harm and enhance the aroma of the cigarettes. D-limonene can inhibit the toxicity of the substance in cigarette smoke, which can induce the cancer of the smoker, but D-limonene is easily to be oxidized and vaporized, and is confined to be used in tobacco. MRPs can enhance the aroma of the tobacco and stabilize the D-limonene when they are encapsulated and added into cigarette, and D-limonene can be released into the main stream smoke, while MRPs improve the flavor of the smoke during the combusting course of the tobacco. The problem of reducing harm and ameliorating aroma of cigarette can be settled to a certain extent.In this study, MRPs were prepared from casein-glucose, and the composition, properties and effect on tobacco were analyzed and researched. The main contens are as follows:Analyzing the effect of processing parameters on the main properties (emulsifying activity and antioxidant activity) of the MRPs, heating temperature, heating time and initial pH were regarded to be the main factors influcing the MRPs, and response surface methodology (RSM) was applied to optimize the processing parameters of casein-glucose Maillard reaction. Substrate concentration 20%, casein-glucose ratio 1:2, heating time 130min, temperature 102°C and initial pH 12.0 were optimum conditions under refluxing for the improved emulsifying ability and antioxidant activity of the MRPs. The analysis of reverse phase high-performance liquid chromatography (RP-HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) indicated that MRPs with molecular weight (MW) from ten thousand Dalton to several million Dalton were the main fraction, and the products with medium MW were the least and the fraction with low MW was medium. Casein was covalently attached to glucose and glucose degradation products (GDPs), which results in high MW MRPs (melanoproteins). Results obtained from the high MW fraction, analysed by Fourier transform infrared spectroscopy (FT-IR) indicated that the amide I, II and III bands of casein were changed by the Maillard reaction and high molecular weight conjugated proteins were generated from native casein and sugars and sugars degradation products and were casein and carbonyls cross-linking conjugates, which includes multiple chromophores and a markedly browning can be observed.Rheological properties and solubility, interaction with flavor and antioxidant activity of MRPs were analyzed, the results showed that Maillard reaction could improve the dissolution property of casein and decrease the isoelectric point of the protein to lower pH. The solution of MRPs was a typical non-Newton fluid at the protein concentration of 200g/l, and MRPs exhibited shear thinning behavior. MRPs interacted with most flavor substances and delayed the release of aroma.Amino acid percentage composition of MRPs showed that MR caused a significant loss in lysine and arginine compared to native casein. Analysis indicated that the free amino acids were generated during the partial hydrolysis of casein and took part in the formation of volatile compounds. Hydrocarbons, acids, esters, ketones and furans were the major products and were distinctly formed at the different stages of heating. Strecker aldehydes and their corresponding substituted pyrazines were also identified at low concentrations in Maillard reaction products (MRPs).The antioxidant results showed that MRPs own strong reducing power and metal chelating ability, inhibition of lipid oxidation and free radical scavenging ability, and indicated the dose-effect relationship at low concentrations. Ultrafiltration results showed that the fraction of molecular weight (MW) higher than 50kDa was major MRPs, and antioxidant results showd that the molecular weight higher than 50kDa component exhibited the strongest reducing power and metal chelating ability, but the product of small molecules possessed the best DPPH free radical scavenging ability. Inhibition of lipid oxidation is provided by synergy with scavenging free radicals and chelating metal ions. The fractions of MW 5-10kDa and 30-50kDa component possess strong inhibition of lipid oxidation.The components of molecular weight greater than 50kDa were separated by gel filtration chromatography (Sephadex G-75), and two components were got with absorbance at 220nm, 280nm and 420nm, and the higher molecular weight component had stronger reducing power and metal ion chelating ability. The analysis of infrared spectroscopy (FT-IR) combined with acid hydrolysis determining monosaccharide showed that covalent bonding between low molecular weight colourants and proteins might, therefore, be involved in melanoidin formation, i.e. upon crosslinking of the N-terminal amino acid or an e-amino function of a lysine residue with carbohydrate-derived intermediates. Melanoprotein hydrolysates still possessed reducing power and scavenging ability and peptic hydrolysates owned higher DPPH radical-scavenging activity than tryptic hydrolysates. The molecular weight distribution with SDS-PAGE and HPLC showed that the melanoprotein had a certain resistance to trypsin hydrolysis. Analysis of RP-HPLC and UPLC-MALDI-TOF-MS showed that melanoprotein and its enzymatic hydrolysis products could not be well separated, and the product is a series of components, meanwhile, chromophore is not a single material, and the results combined with pyrolysis-gas chromatography- mass spectrometry (Pyrolysis-GC-MS) analysis show that the black pigment is mainly casein and sugar degradation products (such as pyrrole, furan and carbonyl compounds, etc.) conjugated products, in which the the benzene ring structure exists, and the derivatives of phenol and benzene, indole, etc.are produced by thermal cracking.Maillard products were used as wall material to encapsulate the D-limonene, and the results show that D-limonene encapsulated with MRPs added to tobacco can improve the retention rate and release in burning smoke. On the other hand, the stability of D-limonene can be improved by microencapsulation with MRPs.The optimal wall material concentration and the core-wall ratio were 20% and 0.125, respectively. Other optimum conditions determined were mixing speed of 12,000rpm for 2 minutes, homogenizing pressure 45MPa, inlet and outlet temperature of 185°C and 80°C, respectively. Microcapsules produced by spray drying were stable and had spherical structure with about 90% of d-limonene encapsulation yield. Results showed that MRPs could be used as an encapsulant for the protection of volatile flavors.The heterocyclic compounds were more in pyrolysates of MRPs than that of casein-glucose mixture, and the pyrolysis of MRPs generated high molecular weight volatile products, the majority of these products are tobacco flavors, such as furanone with fruit, and indole increases the aroma of burley tobacco, while o-benzene dicarboxylate with sweety flavor, benzaldehyde with bitter almond special aroma, and all these products can enhance the natural aroma of tobacco, at the same time, nitrogen-containing heterocyclic compounds can improve the sweet toast flavor of tobacco. The decomposition of MRPs in the pyrolysis process not only produces a large number of basic aroma substances, such as pyrazine and pyridine-type substances, but also generates a lot of alcohol, aldehydes, ketones, esters and acids, the composition and ingredients of MRPs are similar to tobacco pyrolysates, and they are the key materials to tobacco smoke in the aroma components or tobacco spice commonly used in perfumery. From the point of chemical composition of tobacco pyrolysis a view, adding Maillard products to tobacco can reduce aldehydes and increase burley tobacco aroma, and enhance the sweet roasted flavor of tobacco, meantime, and increase the smoke concentration and components containing nitrogen, and the free nicotine concentration can be improved in smoke, increasing momentum of smoke, and the smoke is more plentiful and harmonious. D-limonene added with MRPs can be released into the cigarette smoke so that to ease the irritation and increase the aroma on one hand, more importantly, the role of harm reduction can be achieved. Sensory assessment results prove the above conclusion, and adding casein-glucose MRPs into the tobacco expansion stem can significantly improve the aroma of tobacco and increase the value of tobacco expanding stem.
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