Analysis And Control Of Component Distributions And Rancidity Factors In Rice Bran

Rice bran is the main by-product of rice milling,consisting of pericarp,aleurone layer,embryo and some endosperm.Although rice bran only accounts for about 8%of rice grain’s total weight,it possesses rich contents of nutrients and phytochemicals,including protein,lipid,dietary fiber,oryzanol,phytosterol,vitamin E,squalene and polyphenol.Rice bran oil is the main product of rice bran processing.It has suitable fatty acid composition and is evaluated as one of the healthiest edible oils.Andγ-oryzanol is the most valuable phytochemical in rice bran oil.It has numerous health functions like hypocholesterolemic effect,curing cardiovascular disorders,anti-cancers,antioxidant,anti-inflammation,anti-obesity,anti-aging and relieving stress.Despite so many advantages the rice bran has,data showed that over 90%rice bran was wasted or utilized in valueless ways each year due to the rapid rancidity after rice milling caused by its lipase.Although numerous rice bran stabilization methods have been put forward nowadays,these methods are still difficult to change the current situation of low utilization rate for rice bran.The main reasons may be as follows:lack of researches about the component distributions in rice grain,especially for the distributions of lipase and lipid.Lack of detailed researches about the rice bran lipase,such as the characterization,catalytic center,catalytic mechanism,existence forms,etc.Lack of researches about detailed influence factors for rice bran rancidity.This project explored the component distributions in rice grain and possible reasons for rice bran rancidity.The aim of this project was to provide useful guidance for the control of rice bran rancidity and utilization of rice bran.The researches of this project could be divided into the following parts:component distributions in different rice bran fractions,extraction,purification and structural characterization of rice bran lipase,exploration of lipase characterization and catalytic mechanism,influence of microwave treatment on the rice bran rancidity and oil quality,effects of different rice bran stabilization methods on lipase inhibition and oil quality,exploration of bound lipase in rice bran,influence of microbial contamination on rice bran rancidity.The results were shown as follows:1.Rice grains were obtained from two Chinese provinces（Hubei and Guangxi）and then their rice bran was abraded into four equal fractions by weight from outside to inside.The component distributions in these four fractions were then investigated.The contents of total lipid-soluble antioxidant showed no significant difference between rice bran from Hubei and Guangxi.But the content of total water-soluble antioxidant was1690.20±86.40 mg/kg for rice bran from Hubei,significantly higher than those from Guangxi（1014.67±71.09 mg/kg）.Obvious differences between four rice bran fractions or two provinces were proved by principal component analysis（PCA）.Lipase activities decreased significantly from outer fractions to inner fractions,and lipids mainly distributed in three outer fractions.The essential element contents decreased significantly,but a main heavy metal element（Pb）in cereal increased significantly to0.36 mg/kg from outer fractions to inner fractions,which was just slightly below the upper limit of Chinese Standard（0.40 mg/kg）.Although protein contents had no significant difference in four fractions,the amino acid compositions were quite different.In summary,different fractions of rice bran had different compositions,which could provide guidance for the utilization of rice bran.2.The extraction condition of rice bran was optimized by One-Way ANOVA and response surface methodology,and the optimal condition was as follows:extraction temperature was 20℃,extraction time was 2 h and solid-liquid ratio was 6:1.Two lipases with similar molecular weights were obtained after the crude rice bran lipase was purified by ion exchange column（DEAE-Sepharose Fast Flow）and molecular sieve（Sephadex G-75）.The purification fold was 16.31 and the yield was 10.64%.These two lipases were identified by LC-MS.Results showed these two rice bran lipases belonged to Oryza sativa source and their amino acid sequences were similar,which proved they were in high homology degree（97.78%）.The molecular weights of these two rice bran lipases were 34928.23 Da and 34071.18 Da,respectively.This was the first time to report that there were two lipases in one rice bran,3.Rice bran lipase showed highest catalytic activity at 40.00℃,pH 7.50 and held good stability below 50.00℃ and at pH 7.00–7.50.Most metal ions(except Na+and Co²⁺)and organic solvents had significant inhibition effect on the purified lipase activity.Surfactants like Tween 80,Triton 100 and sodium dodecyl sulfonate（SDS）decreased the lipase activities significantly to 70.88%,80.08%and 73.74%respectively and cetyltrimethyl ammonium bromide（CTAB）increased the catalytic activity significantly to 109.00%.The Ser-His-Asp residues were speculated to be the catalytic center of purified lipases by using the inhibitors phenylmethanesulfonyl fluoride（PMSF）,diethylpyrocarbonate（DEPC）and Pepstatin which aimed at specific amino acids and then verified by molecular docking.Therefore,blocking or destroying the Ser-His-Asp catalytic triad might be new choices to restrain the rancidity of rice bran.4.Microwave treatment was used to stabilize fresh rice bran.Then a 56 days’storage experiment was carried out to prove the good stabilization effect of microwave treatment.During the storage,the acid value of control group increased from21.00±0.22 mg/g to 156.96±1.79 mg/g.The increase amount of acid values for microwave 4 min group and microwave 6 min group only accounts for about 28.34%and 7.04%in comparison to those for control group,which proved the microwave treatment inhibit the rancidity of rice bran significantly.Furthermore,microwave had no significant influence on the quality of oil.Therefore,microwave treatment was proved to be an effective,efficient,low-cost and convenient stabilization method for rice bran.5.Eleven stabilization treatments including 6 heating treatments and 5 non-heating treatments were used to stabilize rice bran.Lipase activities as well as oil colors,oil compositions and contents of vitamin E andγ-oryzanol after different treatments were investigated to evaluate the stabilization effects of these treatments.Results showed that all the treatments had no significant influence on the oil compositions andγ-oryzanol content.Among 6 heating treatments,autoclaving had the best stabilization effect for 10.73%lipase activity remained and other heating treatments held about30%-35%lipase activities residual except dry heating（68.88%）.Heating treatments except steam heating resulted in the significant decreases of oil acid values and significant increases of peroxide values,meanwhile six heating treatments increased oil colors.Among 5 non-heating treatments,ultraviolet irradiation for 18 h and extreme low temperature（-80 o C）for 72 h were found 57.08%and 58.85%lipase activities residual,respectively.Peroxide value was significantly increased only by ultrasound.All non-heating treatments had little effect on oil acid values and color.Ultraviolet irradiation might be a potential stabilization method for rice bran.6.Rice bran residue possessed a steady lipase activity（26.68±3.69%）after being extracted its endogenous lipase continuously for 24 h,which proved that there might be bound lipase in rice bran.Three physical treatments（grinding,homogenizing and ultrasound crush）and six enzymatic treatments（cellulase,hemicellulase,pectinase,complex cellulase,glucoamylase andα-amylase）were applied to rice bran in order to investigate this bound lipase.The relative catalytic activities of extraction supernatant and residue for pectinase group were 437.63±22.54%and 159.26±2.12%respectively,which were significantly higher than other groups.This phenomenon demonstrated that lipase was the most possible to combine with pectin.Molecular simulation proved that pectin could combine with two rice bran lipases（lipase 315 and lipase 308）and cover the catalytic centers so as to prevent the lipases from encountering the substrate and therefore inhibiting their catalytic activities.In the bound lipase,pectin could make the lipases more compact and reduce the solvent accessible surface area of lipases,which would make the lipases inactive to molecular interaction.This was the first report that part of rice bran lipase existed in bound form with the pectin.7.Five strains with lipid hydrolysis abilities were isolated and identified by 16S r DNA sequence analysis.Results showed all of them were Bacillus.During the storage test,fresh rice bran was divided into four groups including control group（CG）,ultraviolet group（UG）,ultraviolet-inoculated group（UIG）and ultraviolet-uninoculated group（UUG）and the final microbial amounts for these four groups were11973.58±331.07 CFU/g,1647.21±56.48 CFU/g,407846.20±10276.76 CFU/g and19673.97±568.32 CFU/g,respectively.The acid values of UG increased from21.75±0.69 mg/g to 94.25±0.29 mg/g,in comparison with 18.72±0.30 mg/g to112.40±1.04 mg/g for CG.And the rancidity of rice bran was accelerated significantly after being inoculated with the five isolated strains（UIG）.The increase ratio of acid values between CG,UG,UUG and UIG was about 8:6:9:15 during storage.The lipase activities performed no significant difference between CG and UG,but the UIG and UUG only held about 40%and 80%of the lipase activities in comparison to CG.In summary,rice bran lipase was still the main reason for rice bran rancidity,but microbial contamination also played a non-negligible acceleration effect on the rancidity.