The Study Of Process On Pilot-Plant Scale And Ingredients Analysis Of Alliin Substances

Garlic is considered as a medicinal plant and especially one of the best disease-preventive food against some forms of cancer and cardiovascular disorders. Central Asia included Xinjiang of China was hypothesized to be the primary center of garlic evolution and diversity. Its beneficial widespread effect on health is attributed to sulphur compounds and particularly to thiosulfinates. Odorless and stable, alliin is the most abundant sulfur compound in whole, unbruised Garlic. When the clove of garlic is cut or crushed, the enzyme alliinase is released from its compartment and transforms S-allyl-L-cysteine sulfoxide or alliin into diallyl thiosulfinate allicin, the characteristic compound of garlic flavor. This very unstable thiosulfinate is rapidly degraded to related organosulfur compounds. Allicin is often thought of as the "active ingredient" in Garlic, and has been the subject of numerous scientific studies, but it's a fairly unstable compound that likes to react with itself. According to the thought that alliin is the original active ingredient and the predominant flavour precursor, alliin can be separately extracted and purified, and garlic polysaccharide can be collected also. In order to realize the pilot-scale production and ingredients analysis of alliin substances, the major contributions of this paper are accomplished as follows:Firstly, simple, sensitive, and highly throughput assay methods with low cost for allicin and alliin were developed. Among the six kinds of garlic enzyme deactivated method, microwave is most effective. Without derivatization, the content of alliin in fresh garlic was determined by HPLC with internal standard substance. Compared to the external standard method and USP method with derivatizing reaction, it's more accurate, simple and convenient. In addition, we proved that the USP method which has obvious failures need to be improved. This method is also appropriated for substances and preparations of alliin. On the other side, a simple 4-mercaptopyridine-UV method was set up for the determination of the content of allicin in fresh garlic. The results are in good agreement with BP method and this method can be also applied to alliin and enzyme activity of alliinase. Through investigation on the quality of fresh garlic in different area of Xinjiang from more than 50 samples, the contents of alliin were universally high, but the differences were relatively remarkable. Because the quality of garlic is mainly effected by ecological environment, climatic factors, soil, water and fertilizer, the standardization planting technique of garlic is imperative. Second, in the processing conditions for the extraction and purification of alliin in the lab, the time that enzyme deactivated needed by microwave heating was ensured; 20% alcohol was not only helpful to the promotion of extraction efficiency, but also in favor of deposition for a long time for the sample solution. Based on orthogonal design, the times to extract was optimized to be 3 by means of ultrasonic surge; the ratio of substance and liquid was 1:3. The type of 001×7(732) was selected from two candidate ion-exchange resins because of its larger adsorption amount. The influencing factors were studied and the optimal process route and conditions were suggested: at room temperature and acidic medium (pH=2) under the moderate concentration of sample. The volume of water to wash polysaccharide was 6 times of column volume. And the concentration of ammonia eluent was 0.5mol/L. At the same time, garlic polysaccharide were 90% collected. The alliin recovery from fresh garlic reached above 50%. The purity quotient of crystallization alliin achieved above 90% under temperature 40℃in 70% alcohol.Thirdly, under the situation of that the processing in lab was feasible, stable and the assay methods have been developed to control the quality of crude material, intermediate product and final product, the amplified process was bring into effect in pilot-scale matched the practical condition in factory. In the course of establishing standard operating procedure(SOP), The temperature, time , the feasibility without decorticating and efficiency were investigated when alliinase was deactivated used a dry heating apparatus in tunnel type, but the efficiency can be achieved less than 70%, need to be improved; The reformative cribriform plate has been proved to be good in practice result in pulping enzyme-deactivated garlic without dehulling; The extraction efficiency achieved more than 90% when volumic ratio of 20% alcohol and water is 3 I 1 and 2:1 was adopted to extract alliin two times respectively; During exact filtration, the ratio of diatomite in precoating and filtration was 3% and 1% respectively, the losses of alliin adsorbed by diatomite in extraction solution were not remarkable. In the stage of alliin isolation by ion exchange resin, three simple and sensitive detected measures were successfully adopted to control the whole process; the efficiency of alliin isolation was similar to laboratory results; The alliin yield was over 50% in acidic fractions and the purity quotient of alliin achieved above 50% after drying; Furthermore, the phenomenon was found that losses of alliin were seriously affected by the temperature and time periods in concentration. As a result, the yield and quality of alliin attained the requirement of predesign.At last, in order to comprehend thoroughly the components of alliin substances, alliin in different impurities were systematically analyzed by non- derivatizing with NP-HPLC-MS-MS and derivatizing with RP-HPLC-MS-MS. It has been proved that alliin substances are consisted of not only isomers and homologens of alliin, including isoalliin, cycloalliin, methiin and deoxyalliin, but also other nature amino acids, such as arginine, cystine, asparagine, proline, glutamine, etc. Except some individuals, the total ion current mass spectrogram(TIC) was more sensitive than ultraviolet spectrum of mass spectrogram; The same results with NP-HPLC-MS-MS, five other nature amino acids were detected in RP-HPLC-MS-MS while cycloalliin and deoxyalliin were not found in RP-HPLC-MS-MS. By mass spectrogram of refined product and pilot-scale product of alliin, with the purity going up, the species and quantities of impurity were decreased obviously. And these research results would make solid base for the establishing the quality standard of alliin substance and applying for new drug.