Study On The Preparation Of Rubber Seed Oil And Protein

Rubber seeds are planted in Guangdong,Hainan,Yunnan,Guangxi,Fujian,Taiwan and other provinces in China.The production of rubber seeds is about 80~100 million ton per year.According to traditional habits,rubber plantation is only the source of rubber and rubber trees.A lot of valuable rubber seeds rot in rubber plantation,resulting in serious waste of resources.Rubber seed kernel contains crude fat 50.2±0.4%,crude protein21.5±0.6%,crude fiber 6.5%±0.5%,carbohydrate 18.2%±0.9%and vitamins.The concentration of copper,iron,zinc,manganese,selenium and other trace elements in rubber seed kernel are as high as 260 mg/kg,which is nearly 3 times of that in soybean meal.The rubber seed kernel is rich in nutrition and can be used as a new oil and protein resource.This paper used Xishuangbanna rubber seeds as raw and four kinds of methods were applied to prepare rubber seed oil.The oil yield,fatty acid composition,physicochemical and sensory properties were compared for different methods.UF membrane was used to degumming the rubber seed oil,and relative physicochemical indexes were detected before and after the degumming study.The process for extracting rubber seed protein was optimized.Electrophoresis,amino acid analysis and scanning electron microscope observation were introduced to analysis the rubber seed protein.Rubber seed protein was separated into four different kinds of proteins according to the modified Osborne method.The four kinds of proteins were separated and concentrated by ultrafiltration membrane filtration.SDS-PAGE gel electrophoresis was applied to detecte the molecular weight distribution of rubber seed proteins and scanning electron microscope was used to observe the microstructure and distribution of each protein.The main conclusions of this study were as follows:(1)The yield,fatty acid composition,physicochemical properties and sensory properties of rubber seed oil prepared by pressing,leaching,ultrasonic assisted solvent and enzymatic method were studied.Rubber seed had high kernel content,and kernel contain high amoumt of fat and protein,accounting for more than 70%of the whole kernel.The content in rubber seed was 1.5×10~2 mg/Kg.Ultrasonic assisted solvent method yielded the highest oil rate of 98.41%;The oil rate of aqueous enzymatic extraction was the lowest to be 68.74%.The acid value of the oil extracted by leaching was as high as 0.58 mgKOH/g and the acid value of the oil by pressing was the lowest(0.12 mgKOH/g).When it came to the peroxide value,oil obtained by pressing showed the highest peroxide value(0.65mmol/Kg),oil obtained by aqueous enzymatic extraction had the lowest value(0.46mmol/Kg).Cyanide was not deteceted in the oils obtained by all the four kinds of method.Unsaturated fatty acids in the rubber seed oil was about 84%.(2)The degumming of rubber seed oil by ultrafiltration membrane method was studied.The rubber seed oil was used as raw material.On the basis of single factor experiments,Plackeet-Burman screening test of single factor analysis and Box-Behnken regression model by response surface method were applied.The results were analyzed by variance,regression model and the regression model.The optimum process conditions for degumming were achieved:the membrane aperture 10 kDa,in 37%oil concent in the mixed liquid and operating pressure 0.23 MPa.Under these conditions,phospholipid retention rate of membrane degumming was 96.51%and the membrane flux was 46.22Kg/m~2·h.Basic indexes before and after degumming:for crude rubber seed oil,the phosphorus content 691 mg/kg,peroxide value 6.3 mmol/kg,acid value 3.32 mgKOH/g,moisture and volatile matter 0.54%,iodine value 92 g/100g for rubber seed oil after membrane degumming,phosphorus content 16.54 mg/kg,peroxide value 2.7 mmol/kg,acid value 1.28 mgKOH/g,moisture and volatile matter 0.24%,iodine value 108 g/100g.The phospholipid content in rubber seed oil was extremely reduced after being degummed by membrane,and the peroxidation,acid value,moisture and volatile matter and iodine were also decreased generally.The smell of rubber seed oil became better and the color became clear and more transparent.(3)The preparation process and characteristics of rubber seed protein were studied.The optimized process for extracting rubber seed crude protein:liquid feed ratio of 15:1,pH 10.2,time of alkali soluble 60 min,extraction temperature 50?,ultrasonic power 274W,pH of acid precipitation 3.8,time of acid precipitation 120 min,the extraction rate of rubber seed crude protein was as high as 80.62%.The optimum purification process of rubber seed protein:enzym atic hydrolysis temperature 45?,hydrolysis pH 4.5,the ratio of material to liquid 8:1,adding amount of enzyme 0.4%,hydrolysis time was 4.5h,the protein content of rubber seed protein reached 94.67%.Amino acid analysis of rubber seed protein showed that the total amino acid content of rubber seed protein purified by glucoamylase was up to 84.72%,and glutamic acid had the highest content of 24.47%.The content of essential amino acid was rich and accounting for 27.14%of the total amino acid content.Scanning electron microscopy of rubber seed protein indicated that the glucoamylase-treated protein shows a flaky appearance with a slightly rough surface.(4)The preparation and characteristics of four kinds of protein of rubber seed were studied.The optimized conditions for modified Osborne sequential extraction method and ultrafiltration membrane preparation of rubber seed protein were as follow:for albumin,ultrafiltration membrane 50 kDa,ultrafiltration time 30 min,pressure 0.39 MPa,ultrafiltration temperature of 39?,the concentration of protein 0.69g/L,the retention rate was 97.16%and the membrane flux was 17.33 Kg/(m~2·h);for globulin,ultrafiltration membrane 50 kDa,ultrafiltration time 38min,pressure 0.34 MPa,ultrafiltration temperature of 31?,the concentration of protein 0.72 g/L,the retention rate was 99.09%and the membrane flux was 18.01 Kg/(m~2·h);for gliadin,ultrafiltration membrane 30 kDa,ultra filtration time 31min,pressure 0.33 MPa,temperature 32?,concentration of protein0.51 g/L,the retention rate was 97.58%and the membrane flux was 18.54 Kg/(m~2·h);for glutenin:ultrafiltration membrane 50 kDa,ultrafiltration time 28 min,pressure 0.37 MPa,ultrafiltration temperature of 38?,concentration of protein 0.69 g/L,the retention rate was 97.01%and the membrane flux 26.42 Kg/(m~2·h).SDS-PAGE gel electrophoresis was introduced to predict the molecular weigh of four proteins:albumin distribution was about49.8 kDa,41.4 kDa,24.3~16.4 kDa,globulin distribution was about 49.5 kDa and 25.1~24.3 kDa,gliadin distribution was about 26.2 kDa and 17.1~12.6 kDa,36.4 kDa,the distribution of Glutenin was 24.2 kDa,20.4 kDa,16.8~15.3 kDa and smaller molecular weight.Scanning electron microscope on the microstructure of four kinds of rubber seed protein observed that:the connection between the rubber seed albumin and rubber seed globulin molecules closely,the surface of rubber seed albumin carefully,with a ridge shape part with a hole;the surface of the loose rubber seed globulin convex surface,fewer pores.Rubber seed gliadin and glutenin with albumin and globulin is different,the loose connection between protein molecules and soluble protein of alcohol,alcohol soluble protein showed spherical shape irregular parts,local relatively detailed,interconnected large molecular groups;surface smooth glutenin molecules more.