| By employing Pumpkin leaves as studying materials, this project was designed to investigate the separation methods of Pumpkin leaf protein, including water extraction method, low-temperature and high-temperature hydrolysis of proteins by hydrochloric acid, high-temperature hydrolysis of proteins by acetic acid and study on the acute toxicity and the ability to decrease blood lipids level of the high-temperature hydrolyzed products by hydrochloric acid and acetic acid. The main conclusions of this study are as follows:(1) Study on the water extraction of fresh Pumpkin leaves was also carried out. Through serious single factor experiments on extraction temperature, time, and water to material ratio, it was discovered that the water extraction yield of Pumpkin leaf proteins was quite low. This result indicated that the water-solubility of Pumpkin leaf protein is poor. So, orthogonal experiment to optimize water extraction conditions was given up. Although the water extract may have the potentiality of being applied to food products, the limitation of studying time makes me give up the investigation into its toxicity and ability to decrease blood lipids level.(2) In experiment on the hydrolysis of Pumpkin leaf proteins by hydrochloric acid at low-temperature, the results of single factor followed by orthogonal experiments indicated that the optimum conditions were hydrochloride concentration0.5mol/L, water to material ratio1:10, hydrolysis temperature30℃, and hydrolysis time48h. The protein content of the extract obtained by this method was quite low, which was37.38%.The reason for this may be that many other substances, such as polyphenols, alkaloids, pigments, sugars, etc in Pumpkin leaves were also be co-extracted.(3) The removal of polyphenols, alkaloids, etc by100%ethanol followed by hot water to remove water-soluble substances such as sugars in Pumpkin leaves produced a concentrate containing40.34%protein that is much higher than28.19%in original leaf powder. Therefore, this method is good for producing protein concentrate from Pumpkin leaves, which may also be ready to be applicable to other kind of leaf processing. (4) Hydrolysis rate of the proteins in Pumpkin leaves by hydrochloric acid at high temperature was84.53%, and the hydrolysate (or called extract) has a good flavor. The protein content of this hydrolysate was45.22%, which is comparable with that (42.32%) of the product obtained by the same acid hydrolysis at low. temperature. And also, hydrolysis rate of the proteins in Pumpkin leaves by the two methods is similar. This means that hydrolysis of Pumpkin leaf proteins by hydrochloric acid at low temperature is also worth being exploited. Although the extract produced by hydrochloric acid hydrolysis at low temperature may be valuable to be exploited, the limitation of studying time makes me give up the investigation into its toxicity and ability to decrease blood lipids level. However, it is suggested that further study on this aspect be undertaken in the future if somebody has an opportunity to do this.(5) Single factor followed by orthogonal experiments found that the optimum conditions for hydrolyzing proteins in Pumpkin leaves treated by ethanol followed by hot water extraction were acetic acid concentration10mol/L, water to material ratio1:20, hydrolysis time25h, and hydrolysis temperature110℃. Under these conditions, the recovery rate of amino acids content was45.11mg/g, and hydrolysis rate of proteins was74.59%that was slightly lower than that of the product obtained by hydrocholric acid hydrolysis at high temperature. The reason for this may be that the acidity of acetic acid is weak, so that it may not fully hydrolyze proteins into amino acids, but some protein may be just transferred into peptides.The component analysis indicated that the protein content of the hydrolysate by hydrochloric acid at high temperature is slightly lower than that (51.27%) of the product obtained by acetic acid hydrolysis at high temperature. Although the hydrolysate produced by hydrochloric acid at high temperature contained chloropropyl alcohol, its level can be reduced to be<1mg/kg by proper treatment such as weak alkali treatment with distillation at low temperature and meet the industry standard published by related authority. Furthermore, the high content of amino acid in the extract may allow the addition of water for a proper adjustment, which can significantly reduce the chloropropyl alcohol level to that is much lower than that required by the industry standard in the final product.(6) The LD50of the extact produced either by hydrochloric acid or acetic acid was more than15g/(kg.bw) in mice. This showed that these hydrolysates were non-toxic. (7) The study on the ability of the extract obtained by hydrochloric and acetic acid hydrolysis to reduce the blood lipids in mice fed high-fat diet was also undertaken. After intragatric adminsration for4weeks, serum TC, TG, LDL-C, HDL-C and AI levels of experimental mice were determined. The experimental results showed that the serum TC, TG and LDL-C levels of hyperlipidemia mice model were substantially and significantly higher than that of the control group while its HDL-C was significantly lower than that of the control group. The experimental results also showed that certain dose of hydrolysates produced by both hydrochloric acid and acetic acid had a certain effct for reducing blood lipids. |
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