Study On Preparation Of Bioactive Peptide And Cigarette Essence From Discarded Tobacco Leaf Protein

Water-soluble protein was extracted from discarded tobacco leaf in this study. The protein was extracted from tobacco at alkaline condition and precipitated at acidic condition. Enzymatic hydrolysis was taken to modify the physico-chemical properties of tobacco protein to produce bio-active peptides. The membrane ultrafiltration was used to separate the bio-active peptides. The antioxidant activity and bacteria growth inhibition activity of the tobacco protein hydrolysates were measured. Extensive enzymatic hydrolysis technique and combined enzymatic hydrolysis technique were taken to hydrolyze tobacco, and the hydrolysates were used to produce cigarette essence by using Maillard reaction. This research had great significance in improving by-product value of tobacco process, favor to protecting enviroment and benefiting tobacco-planting farmer.The protein extraction process was investigated in this research. Under the optimal conditions for tobacco grinding, protein dissolving and protein precipitation, the protein recovery and the amino acid score were 86.71%, the resulting product included 70.66% of crude protein.Three proteinases (Papain, Protamex and Alcalase) were chosen to hydrolyze tobacco protein. The highest peptide recovery was obtained at 37.42% when tobacco protein was hydrolyzed by means of Papain. During the hydrolysis, free amino acids increased as hydrolysis time went on. The free amino acid content was 15 times higher than that in unhydrolyzed tobacco protein. The kinetic mathematical model of tobacco protein enzymatic hydrolysis by Papain was built as follows:Enzymatic deactivitation kinetic constant k d=0.0077 min^-1;When the hydrolysis time ranged from 0-300 min, the DH value computed by this model had no significant difference from the results determined.In vitro antioxidant activity determination of tobacco protein hydrolysates showed that peptides with 5 kDa of molecular weight had the highest antioxidant activity. The hydrolysates by Papain had the highest superoxide anion scavenging capacity (28.41%), similar to that of VE (28.56%). They also had the highest DPPH radical scavenging capacity (83.82%), lower than that of VE (93.24%). The hydrolysates by Alcalase had the highest hydroxyl radical scavenging capacity (67.18%), similar to that of VE (67.36%). By comparison of in vitro antioxidant activities of the hydrolysates with different molecular weight, it indicated that the molecular weight of tobacco bio-active peptides should be in the range of 3-5 kDa.The amino acid composition in tobacco protein showed that the total content of tryptophan, histosine, methine and cysteine in tobacco protein supernatant hydrolyzed by Papain for 6hr and the filtrate through 5 kDa membrane ultrafiltration were 45.87 mg/mL hydrolysate and 80.44 mg/mL hydrolysate, respectively. The content in tobacco protein supernatant hydrolysed by Alcalase for 4 hr and the filtrate through 5kDa ultra-membrane were 42.03 mg/mL hydrolysate and 76.79 mg/mL hydrolysate. Different hydrolysates had different antoxidant activities for their differences in bio-active amino acid content.The results of bacterial-growth inhibition activity trials showed only the hydrolysate produced by Papain had this activity. This hydrolysate was only effective to E. coli. The hydrolysate produced by Papain with 6 hr hydrolysis had the strongest activity in inhibiting bacterial growth, and the diameter of inhibition circle reached 1.80 cm. After partitioned by ultra-membrane, the hydrolysate with 5 kDa of molecualr weight had the highest inhibition activity with 3.60 cm of the inhibition circle diameter. The lowest effective concentration was 0.40 g/L, little more than that of propyl p-hydroxybenzoate (0.35 g/L); The 5 kDa molecular weight hydrolysate had strong alkaline-endurant and acid-endurant capacity and thermostability. Analysis by high performance liquid chromatography / mass spectrometry suggested ultra-membrane filtration had a good resolution for separation of tobacco protein hydrolysates.Extensive hydrolysis could improve the conversion rate of amino acid. The highest conversion rate of amino acid of the hydrolysate reached 36.94%.The cigarette essence made by Maillard using tobacco protein hydrolysate as material had good effect on improving the flavor of cigarette. The gas chromatography / mass spectrometry profile showed high contents of acid, alcohol and ester in ether-soluble product of Maillard reaction, especially those components having significant roasting-cigarette flavor and sweet flavor existed with relative high content. Therefore, this cigarette essence could be used for bad flavor removal and flavor enhancing.