The Preparation Of Bromelain And Its Application In Beef Tenderization

China has rich resources of pineapple,and its pulp is mainly used.However,its residues are discarded as garbage.Therefore,extracting bromelain from pineapple residues could reduce the environmental pollution and improve the additional value of pineapple by-products.In this paper,the distribution of bromelain in pineapple tissues(pulp,peel,stem and leaf)was studied,and the effects of salt precipitation of ammonium sulfate method solely and the combination method of salt precipitation of ammonium sulfate with ultrafiltration on the extraction of bromelain were compared.Then,preliminary extracted bromelain was purified by the CM Sepharose FF column chromatography,and enzymatic properties of the purified bromelain were investigated.On the basis,the effects of Fe2+ and Fe3+ on the properties of bromelain as well as its mechanism of action were studied.Besides,the iron ion removal effect of EDTA-2Na combined with ultrafiltration was studied.In the end,the competitiveness of the bromelain in the commercial application of the pineapple residue was investigated on the basis of the tender effect of the meat.The results showed that the sequence of protease content and enzyme activity of different parts of pineapple was: stem > pulp > peel > leaf.The enzyme activity recoveries of salt precipitation of ammonium sulfate solely and the combination method of salt precipitation of ammonium sulfate with ultrafiltration were 73.28% and 67.56%,respectively.The purification multiples were 1.29 and 1.52,respectively.The specific activities were 153.50 and 181.43 U/mg,respectively.In general,the bromelain extraction effect of the combination method of salt precipitation of ammonium sulfate with ultrafiltration was significantly higher than salt precipitation solely.After purified by CM Sepharose FF cation exchange column chromatography,the relative molecular mass of the purified bromelain was about 25.0 kD.The enzymatic properties of the purified bromelain showed that the optimum pH value was 7.0,and the optimal reaction temperature was 60 ?.EDTA-2Na and Ca2+,Mg2+ and Mn2+ at the concentration of 2 mmol/L could activate the enzyme activity,while Zn2+,Cu2+,Fe3+ and Fe2+ at the same concentration could inhibit the enzyme activity.The effects of Fe2+ and Fe3+ on bromelain activity,stability and half-life were assessed.The results showed that Fe2+ activated the enzyme activity at the concentration lower than 0.75 mmol/L,while inhibited the enzyme activity higher than 0.75 mmol/L.The optimal activating concentration was 0.5 mmol/L.Fe3+ had a negative effect on the enzyme activity,and the inhibition was higher than that of Fe2+.Fe2+ with the concentration of 0.5 mmol/L enhanced the thermostability of bromelain at 60 ?,and the half-life of the enzyme was extended for 10.93 min.Fe3+ and Fe2+ at the concentration higher than 0.5 mmol/L had a negative effect on the thermostability of bromelain.The inhibition effect and concentration showed a positive correlation.According to CD spectroscopy,Fe2+ and Fe3+ could inhibit the enzyme activity by decreasing the content of ?-Helix,?-Sheet,?-Turn,while increasing the content of the random curl.The method that EDTA-2Na combined with ultrafiltration was effective in iron ion removal with a reduction of iron content from 291.63 mg/kg to 142.99 mg/kg(dry weight),and the elimination rate of iron ion was 50.97%.Besides,the growth rate of the activity was 26.13%.Bromelain extracted by the combination method of salt precipitation of ammonium sulfate with ultrafiltration was subsequently applied in beef tenderization.The orthogonal test showed that the optimum enzyme dosage,treatment temperature,time and pH were 20 U/g,50 ?,60 min,and 7.0,repectively.The sensory evaluation with the four indicators including color,viscosity,elasticity and organization status,showed the quality sequence were: extracted enzyme tenderization beef > commodity bromelain beef > beef without enzyme tenderness.