| Licorice has many pharmacological effects, and glycyrrhizic acid is more important in the effective components of Licorice, its content is higher and its physiological activity is strong, glycyrrhetinic acid is one of a series of derivatives of glycyrrhizic acid. The study shows that glycyrrhizic acid exert their pharmacological effects that include detoxification, inhibit liver fibrosis, anti-inflammatory, anti-tumor effect through turning to glycyrrhetinic acid in vivo, it has the wide application value and prospect in food, medicine, cosmetics and other industries. At present, glycyrrhetinic acid was made by acid hydrolysis in the industrial production. It takes serious pollution to the environment. And another way to gain it in small scale was the way of bio-transformation under the condition of laboratory, although the conversion rate is high, the relative environmental protection, but it was not applied in industrial production because of its high cost. The biological transformation method has become the research hot spots of domestic research ideas, which is likely applied on industrial production in the future because of its environmental protection and efficient style. Choose strains that own good properties which contain short production cycle and not harsh conditions and less vice metabolites is worthy. This thesis hopes to screen strains that own good properties. Combine application of biotechnology with the production and research of traditional Chinese medicine active substance to solve the present problem in the production of glycyrrhetinic acid.Objective:A preparation method of compound liquorice buccal tablets was provided in the patent named Compound Liquorice Buccal Tablets and preparation method by Liu Cuizhe et al. Its biggest feature is the use of glycyrrhetinic acid extract instead of the original extractum glycyrrhizae powder. Advantages of the tablets are no smell, taste suitable for taking orally, can play a better efficacy, no tide variation for easy storage. This paper established the patent on basic research hope to be able to screen the strains that can turn glycyrrhizic acid to glycyrrhetinic acid. The second screening strains had preliminary morphological identification and optimal fermentation conditions to obtain excellent strains that is suitable for pharmaceutical production.Method:1 In this study, the strains were screened from culture liquid of licorice pieces and glycyrrhizic acid crude product. The strains that can use glycyrrhizic acid as nutrients could have higher utilization of using glycyrrhizic acid. Glycyrrhizic acid was used to be the carbon source in the screening model.2 Simultaneous determination the glycyrrhizic acid and glycyrrhe- tinic acid content of screening culture using HPLC method. The target strains have high conversion rate by liquid fermentation rescreening.3 The screened strains were preliminary morphological identification, including colony morphology, color feature, characteristics of odor, and the morphological features of the microscope.4 We carry on the corncob induced test for the strain that own the highest conversion rate to investigate whether the beta glucosidase were from the strains can be induced or inhibited. Each level of a combination of four factors process of strains were obtained by orthogonal design value. They contain the initial p H, amount of inoculation, fermentation temperature and time.Result:1 The modified Martin culture medium and PDA medium that were inoculated of licorice pieces powder bacterial liquid appeared of the colony, 19 strains named YGL-B-0~YGL-B-18 were obtained after the separation by "a method of purification of the three".2 The HPLC method chromatographic conditions: The chromatographic column ZORBAX SB-C18(2.1×150mm, 3.5μm); Pre-column: ZORBAX SB-CB(2.1×12.5mm, 5μm); Column temperature: 28℃; The mobile phase: 0.5% Formic acid water solution-acetonitrile-methanol(20:40:40); velocity of flow: 0.2m L·min-1; The wavelength of detection: 250 nm. Glycyrrhizic acid had a good linear relation in the range of 0.06667~1.06667 g, glycyrrhetinic acid too.The specificity is good and the instrument has good precision. The average recovery of Glycyrrhizic acid and glycyrrhetinic acid were 97.81% and 98.40%, RSD were 2.09% and 1.72%. There were 14 strains can transform glycyrrhizic acid to glycyrrhetinic acid different level were screened, the screening rate reached 73.68%. There are three strains were named YGL-B-0/1/12 with high conversion rate, the conversion rate of 72 h were 18.92%, 21.29%, 37.75%.3 The morphological observation results shows that the bacteria colonies of three strains were round with a convex surface, moist and shiny, neat edge, the color was milk white, and the medium have a fermented aroma after a period of time. Cells were round under the microscope eyepiece 40 times observed morphology, a few cells end budding reproduction. Through these characteristics we can initially determine the strains YGL-B-0/1/12 were yeast.4 In the experiment of corn cob induced: the conversion rate of the experimental group and the ordinary group turn glycyrrhizic acid to glycyrrhetinic acid respectively were 37.40% and 37.38%, with no significant differences. The corncob did not show induction and inhibition on the enzyme. The orthogonal design experiment showed that the fermentation conditions of YGL-B-12 were p H 5.5, 2%, 25℃, 60 h, and the order of the four factors to affect the conversion rate were: initial p H, inoculation amount, temperature, fermentation time. The conversion rate of strain YGL-B-12 reached 40.83% under the best fermentation condition and compared with before the rate increased by 8.16%.Conclusions:The study screened strains that can transform glycyrrhizic acid to glycyrrhetinic acid from culture liquid of licorice pieces powder and glycyrrhizic acid crude products. The method used glycyrrhizic acid as carbon source for screening model and the HPLC method was used to determinate glycyrrhizic acid and glycyrrhetinic acid content in the medium were feasible. The yeast were selected from liquid of licorice pieces powder in this paper, that was the first, and yeast is the preferred species in industrial production, through orthogonal test can establish a relatively optimal fermentation conditions, which makes the original conversion increased. |
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