Study On The Preparation, Purification, And Stability Of Schizochytrium Limacinum Antioxidant Peptide

Schizochytrium limacinum is a kind of single cell marine fungi microalgae of Thraustochytrium Department. It has rich content of fat and polyunsaturated fatty acid, and was approved as a new kind of food resource in application by Ministry of health of China in 2007, which is mainly used for the production of health care products with high purity DHA, infant dairy additives and other products at present. However, Schizochytrium limacinum also contains rich components of vitamins and essential amino acids. For example, the protein content of its meals after oil extracting is 40% or above, which were mostly treat as feed or fertilizer, means that the protein resources have not been fully developed and utilized.In this paper, the Schizochytrium limacinum antioxidant peptide is prepared by Protamex and Alcalase 2.4L complex method after oil extraction. We investigate the effects of temperature, pH, food materials, preservative, metal ions and digestion environment in vitro on the antioxidation stability, isolated and purified by ultrafiltration, gel filtration chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC), and finally identified through analysis of electrospray ionization (ESI) mass spectrometer. This study will provide new ideas and methods for high value utilization of meals of Schizochytrium limacinum, and provide a theoretical basis and important reference for its products development.Protein recovery and product antioxidant activity are as index to determine the best complex enzymatic processes. The results showed that the optimum conditions were as follows:pH 7.5, temperature 50℃, ratio of material to water 1:12, time 6h, Protamex and Alcalase2.4L complex method, total amount of enzyme dosage 0.5%(E/S, w/w,1:1), which is significantly better than the effect of single enzymatic hydrolysis. In this condition, the hydrolysates have the degree of hydrolysis of 11.15%, protein recovery 70.82%. In addition, IC50 of hydroxyl radicals was 1.656 mg/mL and reducing power was 1.417, which shows great antioxidant activity.The scavenging activity on DPPH radical of enzymatic hydrolysates and reducing power from Schizochytrium limacinum meal was selected as the index to investigate the effects of temperature, pH, food materials, preservative, metal ions and digestion environment in vitro on the antioxidation stability. The results showed that the enzymatic hydrolysates from Schizochytrium limacinum meal has a strong heat-resistant ability from 30℃ to 100℃; It can keep high activity in the acidity environment; Some food materials, such as sucrose and NaCl has little effects on the antioxdation stability while glucose has increasing effects with increase in concentration from 2% to 10%; Effect of potassium sorbate on the activity of product is slightly stronger than that of sodium benzoate; The order of metal ions effect is Zn2+>Cu2+>K+> Ca2+; The activity can be maintained above 65% after simulated gastrointestinal environment treatment in vitro. It means that the hydrolysates has good stability and practical value in production and application.The hydrolysates were isolated and purified by ultrafiltration, gel filtration chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). We find that the part of SH-4-I-D with molecular weight less than 5 kDa has the strongest antioxidant activity, and the scavenging rate of DPPH radicals was 83.8%(0.5mg/mL) while the reducing power was 0.937(1 mg/mL). Finally, through analysis of electrospray ionization (ESI) mass spectrometer, the purified antioxidant peptide was identified as Pro-Tyr-Lys (406 Da).