Study on Preparation and Properties of Protein Isolates from Tilapia
|School||Guangdong Ocean University|
|Course||Aquatic Products Processing and Storage Engineering|
|Keywords||Tilapia Protein isolate pH-shifting Nutritional properties Protein structure|
For the existence of low protein extraction yield, high impurity content, poor functional properties and flavor in processing and utilization of aquatic protein, fish protein isolates (FPI) was prepared from Tilapia muscle and by-products by pH-shifting in this study. Fish muscle proteins were extracted at either acidic (pH<3.5) or alkaline (pH>10.5) at low temperature to obtain the maximum solubility and remove fat and insoluble impurities via high-speed centrifugation followed by the recovery of precipitated proteins at their isoelectric point. The optimum processing conditions of protein isolates from Tilapia muscle via pH-shifting was determined. And the nutritional properties, functional characteristics, structural features, as well as the changes of molecular structure and properties in protein extraction processing were investigated. The main purpose of this study was preparing fish protein isolates with excellent functional and nutritional properties, and providing theoretical basis for high-efficient utilization of low-valuable aquatic protein and new protein resources development. The main results are as follows:1. Effect of the wide range of pH, solid to liquid ratio and dissolution time on the yield of soluble protein from Tilapia muscle was determined. And then effect of pH value on precipitated protein yield from acid-soluble and alkali-soluble was determined as well. Results of soluble protein yield showed that the optimum conditions of protein extracting from Tilapia muscle using pH-shifting were as follows: dissolve conditions was pH 2.0, 3.0, 11.0, and 12.0, ratio of solid to liquid of 1:9 (w/v), dissolving time of 10 min, and the temperature of 4℃. Studies of protein yield of precipitation from soluble protein showed that the optimum condition was pH 5.5. SDS-PAGE electrophoresis analysis showed that, under the extreme acidic (pH≤3) and alkaline (pH≥11) conditions, the soluble proteins strips included myosin heavy chain, actin, myosin light chain, sarcoplasmic proteins and small water-soluble protein band, which was in line with the typical fish protein electrophoresis pattern. When the acid-soluble and alkali-soluble was precipitated at pH 5.5, no protein strips of resulting supernatant displayed at SDS-PAGE profile, and so a higher protein recovery from precipitation process was obtained. Comparatively, the precipitation yield of alkali-soluble protein was relatively lower.2. Fish protein isolates was prepared from Tilapia muscle by solving at pH 2.0, 3.0, 11.0 and 12.0 and then precipitated at pH 5.5, in which protein yield was from 56.06% to 64.95%. The protein powder with white color and no smell was obtained by freeze drying, in which the content of crude protein in dry basis was more than 95%,fat content was 1% approximately, ash content was below 2.03%. And fat and ash content of alkali-made protein were lower than that of acid-made protein. These results indicated that for removing fat and ash impurities, alkali-soluble processing was more effective than acid alkali-soluble processing. Protein isolates contained a full range of amino acid with balanced composition, especially with high lysine content, and the total essential amino acids account for about 49 percent of total amino acids, which was in line with FAO/WHO recommended model. Therefore, these fish protein isolates could be used as protein food additive.3. Studies of solubility of four kinds of protein isolates showed that effect of pH value on solubility of protein isolate from Tilapia muscle was obvious, and the general trend was similar to fish protein pH solving curve. Solubility of alkali-made protein was higher, and its water-soluble and salt-soluble protein content were higher. Studies of SDS-PAGE showed that the salt-soluble content of protein isolate was high, and myosin heavy chain, actin, myosin light chain and small molecule water-soluble protein bands were displayed. Comparatively, the more composition and higher total sulfhydryl content of alkali-made protein were observed, and so did better gel strength and water holding capacity However, and a loss of small amount of sarcoplasmic protein was observed in acid-made protein, and the higher relative surface hydrophobicity and red shifting of tryptophan fluorescence emission peaks was detected. These results indicated a greater degree of protein denaturation in acid-extracted process of fish protein isolate. Compared with Tilapia muscle protein, water-soluble protein content of four protein isolates decreased, this may be related to the degradation in acid solving process and the protein loss during precipitation process. Therefore, alkali solubilization and isoelectric precipitation was more suitable for extracting fish protein isolates.4. Effect of acid and alkali dissolution process on structure and properties of protein isolates was further studied. Results showed that, in extreme acidic (pH 2.0 and 3.0) and alkaline (pH 11.0 and 12.0) conditions, the solubility of fish protein and total thiol content of soluble protein was better, relative surface hydrophobicity was lower, and red shifting of tryptophan fluorescence emission peak was observed, indicating occurrence of protein unfolding and denaturation in extreme pH conditions. Especially in acidic conditions, part of degradation of protein molecules resulted to disappearance of some soluble protein bands on SDS-PAGE profile. 5. Studies on isoelectric precipitation process showed that the solubility of acid-soluble and alkali-soluble protein decreased by precipitation at pH 5.5, and some sarcoplasmic protein bands on SDS-PAGE profile disappeared and the active thiol reduction in the proportion of total sulfhydryl decreased. Comparatively, effect of isoelectric precipitation on soluble protein was more obvious. Soluble proteins solved at pH 2.0, 3.0 and 12.0 followed by the recovery of precipitated proteins at their isoelectric point resulted in significantly increasing surface hydrophobicity and obviously shifting of tryptophan emission peak of water-soluble fractions. And soluble proteins solved at pH 11.0 followed by the recovery of precipitated proteins at their isoelectric point resulted to significantly decreasing surface hydrophobicity, but no significant effect on salt-soluble proteins was observed. The overall analysis showed that, in the range of experiment, molecular structure and properties for alkali-soluble protein extracted at pH 11.0 were well maintained.6. Fish protein isolates was prepared by pH-shifting from Tilapia by-products. Fish by-products was extracted at pH 2.0, 3.0 and pH 12.0, 13.0, and then was precipitated at pH 5.5, in which protein yield was more than 52%. Protein isolates powder was obtained by freeze-drying, and the content of crude protein in dry basis was more than 85% and ash content was below 4%. Comparatively, mineral elements such as Ca and Mg of acid-made protein isolates were higher. Besides, fish protein isolates from Tilapia by-products contained a full range of amino acid with balanced composition and high lysine content, which accorded with FAO/WHO recommended model, and was a kind of high quality protein. Studies of functional properties showed that protein isolates extracted at pH 12.0 had the best solubility and emulsifying activity. Studies of SDS-PAGE showed that several protein isolates from different extracting conditions had similar and complex molecular weight distribution, and typical fish protein strips was displayed on SDS-PAGE profile. In addition, no protein strips displayed at 66.4~44.3kDa for acid-made protein on SDS-PAGE profile, this may be connected with some degradation of protein during acid-extracted process, which was similar to the result of protein isolates from Tilapia muscle.