| With the development of abalone aquaculture and the deep processing,a large number about 20 percent of the by-product of abalone in weight was abandoned or processed into fishmeal which not only caused environmental pollution,but also a great waste of resources,it is also violated the principle of the rule by sustainable development and scientific development view.Previous studies showed that,abalone gonad contain a lot of nutrient and have various nutritional components,especially the content of protein was closed to 40%,which possess a very good development and utilization prospect.Therefore,the research object of this paper was abalone gonad.Extraction,isolation and purification of sterols compounds from the abalone gonad were discussed.The extraction condition of abalone gonad crude sterols was studied and determined,and the effects of different experimental conditions on the yield of sterol were discussed.The processes of saponification and separation of saponification resultants were studied.And the optimal aponification conditions were determined.The method of selective solvent crystallization was applied to purify the crude sterol after saponification,effects of factors related in recrystallization process on the the yield and quality of sterol were explored.Meanwhile,the steps and the optimum experimental condition of recrystallization were determined.GC-MS was used to analyze and identify the purified sterol samples.The structure and categories of sterols were determined and the content of sterols was measured.The main research results were as follows:1.Preliminary extraction condition studies of abalone gonad sterolsMethanol,anhydrous ethanol,85%ethanol and 75%ethanol were used as extractants for extracting abalone gonad sterols,and their extraction yield were compared.Results show that methanol group has the best extraction yield,next was the 85%ethanol group and 75%ethanol group showed the lowest extraction yield.Comprehensive consideration for saponification followed and economic benefit,anhydrous ethanol was selected as optimal extractants.Male and female abalone gonad were studied for sterol extraction,it turned out that,extraction yield of male gonad was slightly higher than female gonad.,considering the practicability and maneuverability,the gonad was not divided into male and female to study.Yield of PEF treatment group and control group were compared,it is shown that,the yield of sterols after PEF treatment was improved 30.63%compare to that of control group,which is verified the feasibility of PEF technology used in the extraction of abalone gonad sterols.2.Process optimization for extraction and isolation of abalone gonad sterolsSaponification method was used to isolate the sterols from abalone gonad.KOH-ethanol was used as saponifier,unsaponifiable matters were extracting by Ethyl acetate,then evaporation for concentration,and then the crude sterol was obtained.Box-Behnken Response Surface methodology was designed to optimum technological conditions of saponification,the optimum conditions were:saponifier of 1:2,saponification temperature of 60? and saponification time of 7h.Verification test was conduct under the optimum conditions,results showed that the average saponification rate was 62.21%(RSD=0.46%),experimental result was close to the predictive value,relative error with the theoretical prediction of 62.35%was 0.22%,the content of total sterol in crude sterol was 14.08%,which explain the model reliability for optimizing abalone gonad sterols extraction.3.Purification of abalone gonad crude sterolsThe method of selective solvent crystallization was applied to purify the crude sterol.Petroleum ether was selected as crystallization solvent.Crystal form of sterol recrystallized by petroleum ether was flaky,colorless and transparent,optimum material/solvent was 1:16(g/ml).The optimum parameters of crystallization process was:cooling rate of 1?/min to 40 ? then 0.1 ?/min to 0? crystallization temperature of-4?,crystallization time of 6h,recrystallizing 3 times.Three times of parallel test was conduct under the optimum conditions,yield and purity of sterol were 16.38%and 92.01%,respectively.4.Analysis and identification of abalone gonad sterolsThe purified abalone gonad sterol was identified by chemical reagent coloration method,the results accord with the color reaction of sterol.UV spectrum analysis showed that,the sample and standard cholesterol were all had a strong absorption near 280 nm which further determine the existing of sterol.GC-MS was used to analyze and identify the purified sterol samples,impurity was removing by decompression and sublimation,results indicated that,the abalone gonad obtained through the experiment mainly contain 5 kinds of compound,that is 3a-Hydroxy-17-androstanone,Cholesta-3-5-diene,Cholest-5-en-3?-ol,24-Methylcholesta-Cholest-7-en-3?-ol and Stigmasta-5,22-dien-3?-ol,the content were 6.255%,3.036%,81.867%,1.563%and 4.492%,respectively. |
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