Extract of Rhus typhina fruit and preparation method thereof, and application of extract to fermentation production of genetic engineering products

A technology of extracts and fruits, applied in the field of agricultural product processing, can solve problems such as high cost, difficult quality control of genetic engineering products, high energy consumption induced by temperature, etc., and achieve the effects of reducing toxicity, saving fermentation costs, and efficient expression

Active Publication Date: 2017-10-24
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to overcome the adverse effects of IPTG, methods such as temperature-induced lac system and lactose-induced lac system have been developed and applied by different research groups, but temperature induction requires large energy consumption and high cost for industrial large-scale fermentation (MurrayNE.et al.J Molec Biol, 1979 132:493~505.); when lactose is induced, lactose has many influences on the proliferation and physiological behavior of the host as the carbon source of bacteria, and the quality control of genetic engineering products is difficult (Beckwith JR.1987.Cellularand Molecular Biology. American Society for Microbioloty, Washington, DC. Vol 2, pp1444~1452.)

Method used

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  • Extract of Rhus typhina fruit and preparation method thereof, and application of extract to fermentation production of genetic engineering products
  • Extract of Rhus typhina fruit and preparation method thereof, and application of extract to fermentation production of genetic engineering products
  • Extract of Rhus typhina fruit and preparation method thereof, and application of extract to fermentation production of genetic engineering products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Preparation of RtSE

[0021] Put the fresh torch tree fruit in a cool and ventilated place at room temperature to dry in the shade, then air-dry at 60°C to a water content of 5% by weight, and mix the dried torch tree fruit with 20% ethanol aqueous solution by volume percentage, relative to 1kg of dry torch Tree fruit, the consumption of ethanol aqueous solution is 8L, obtains the material after mixing, and the material after described mixing is put in the round-bottomed flask and heats, and keeps refluxing 2 hours under slightly boiling state, after the material after reflux is cooled, hollow fiber Membrane filtration, the filtered material was centrifuged at 5000rpm for 30 minutes to obtain the clear liquid, and the clear liquid was placed in a rotary evaporator at 45°C to remove ethanol under reduced pressure and concentrated according to the ratio of 10:1, then passed through a 0.22μm Sterilize by filtration to obtain RtSE.

[0022] The obtained RtSE adopts Folin's...

Embodiment 2

[0026] Effects of RtSE on the ability of LacUV5-T7 system and LacI Q system to express foreign substances

[0027] In this example, Escherichia coli BL21 (DE3) was used as the host, and the LacUV5-T7 system used the pET28a plasmid; the LacI Q system used the pUC19 plasmid. The coding gene of exogenous target gene arginase was respectively inserted behind the Lac promoter of the two plasmids.

[0028] Plasmid pUC-ARG(LacI Q), pET28a-ARG(LacUV5-T7); strain E.coli BL21(DE3)[F-ompThsdS(r- B m- B )gal dcm(DE3)] was purchased from TransGen Biotech.

[0029] The RtSE prepared in Example 1 was used and pre-diluted with deionized water, so that the final concentrations of RtSE in each experimental system were 74.7, 18.7, 3.7 and 0.7 mg / L, respectively.

[0030] 2.1 Effect of RtSE on BL21(DE3)-pUC-ARG

[0031] Pick a single colony from the BL21(DE3)-pUC-ARG transformant and place it in LB liquid medium containing 100 μg / mL ampicillin, and place it overnight at 32° C. with a constan...

Embodiment 3

[0045] Effects of different concentrations of RtSE on the expression of exogenous protein activity induced by IPTG in LacUV5-T7 system.

[0046] The effects of different RtSE concentrations on the ability of LacUV5-T7 to express active arginase were investigated when 0.05mM IPTG was added to the fermentation broth.

[0047] Using the RtSE prepared in Example 1, the concentrations of RtSE in the fermentation system were 74.7mg / L, 18.7mg / L, 3.7mg / L and 0.7mg / L respectively.

[0048] The pET28a-ARG plasmid in Example 2 was used, and Escherichia coli BL21(DE3) was used as a host.

[0049] The biomass, protein amount, enzyme activity assay method and data statistical analysis method in Example 2 were used for statistics and analysis of data.

[0050] 3.1 Interaction between RtSE and IPTG

[0051] Pick a single colony from the BL21(DE3)-pET28a-ARG transformant and culture it in LB liquid medium containing 35μg / mL kanamycin 100mL at 37°C with a constant temperature shaker at 200rpm...

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Abstract

The invention provides the extract of Rhus typhina fruit and a preparation method thereof, and application of the extract to fermentation production of genetic engineering products, belonging to the technical field of processing of agricultural products. The method comprises the following steps: mixing dried Rhus typhina fruit with an extraction solution so as to obtain a mixed material, wherein the extraction solution is an aqueous solution of ethanol; subjecting the mixed material to heating reflux under a slight boiling state, then carrying out cooling to room temperature and carrying out filtering and centrifugation so as to obtain clear liquid; and subjecting the clear liquid to pressure-reduced concentration according to a volume ratio of 8-12: 1 and carrying out degerming so as to obtain the extract of Rhus typhina fruit, i.e., RtSE. The extract RtSE can increase the amount of host bacteria in a genetic engineering fermentation system; when only RtSE is used in the fermentation system, the biomass of host cells in the fermentation system is increased by 5% or above, and a total protein amount and the active expression quantity of exogenous proteins are both increased by 20% or above; and when RtSE and IPTG are both used in the fermentation system, the biomass of the host cells in the fermentation system is increased by 12% or above, the total protein amount is increased by 30% or above, and the active expression quantity of the exogenous proteins are increased by 35% or above.

Description

technical field [0001] The invention belongs to the technical field of agricultural product processing, and in particular relates to a torch tree fruit extract (extraction from Rhus Typhina, RtSE), a preparation method and the application of the extract in the fermentation production of genetic engineering products. Background technique [0002] Torch tree (Rhus hirta L.), a Anacardiaceae plant, is a deciduous shrub native to North America, mainly distributed in tropical and subtropical regions. It was introduced to my country by the Institute of Botany, Chinese Academy of Sciences in 1959, and has been promoted to all provinces and regions of the country since 1974. It is more cultivated in the provinces (regions) north of the Yellow River Basin. Torch tree fruit can be used not only as a dietary condiment, but also made into beverages or other medicines. It has been reported that the extract of torch tree fruit can be anti-cancer, anti-inflammatory, and even has the effects...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12N15/70C12N9/78C12R1/19
CPCC12N9/78C12N15/70C12P21/02C12Y305/03001
Inventor 周杰辛鑫杨少林吕明袁俊杰刘保勤宋彦君
Owner JILIN UNIV
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