A kind of beta amylase is extracting the application in Radix Notoginseng total arasaponins
technical field:
The present invention relates to a kind of beta amylase and extracting the application in Radix Notoginseng total arasaponins, belong to the field of Chinese medicines.
background technology:
Radix Notoginseng is araliaceae ginseng plant; main product, in Yunnan, begins to be loaded in Compendium of Materia Medica, is China's tradition rare Chinese medicine; there is blood circulation promoting and blood stasis dispelling; subduing swelling and relieving pain, the effects such as hemostasia and dissipation blood stasis, Radix Notoginseng different parts contains more than 20 kind of saponin constituent; its main active is Radix Notoginseng total arasaponins; Radix Notoginseng total arasaponins has increase coronary flow, and protection cerebral tissue, expands the effect such as blood vessel and blood pressure lowering.
Current extraction process for notoginseng saponin mainly contains decoction alcohol precipitation method, ethanol refluxing process and percolation.Wherein decoction alcohol precipitation method in soak by water process because pseudo-ginseng contains a large amount of starch and the easy gelatinizing of polysaccharide; Adsorption saponin loss due to precipitation after precipitate with ethanol is comparatively large, and extraction ratio is lower, and the content less stable of product component, be used for the exploitation of food; Percolation extraction ratio is higher, simple to operate, and extracting solution impurities is few, but solvent consumption is excessive, is mainly used in medicine; Ethanol refluxing process method is easy, and process conditions are easy to control, but extraction ratio is lower, and this several extracting method all requires the rhizomes of Panax notoginseng of raw material choose drying (clip), processing leftover pieces extract, and obviously makes extracting cycle extend.
And emerging enzymatic isolation method and said method such as decoction alcohol precipitation method combine, thus the Radix Notoginseng total arasaponins extracted in fresh Radix Notoginseng effectively shortens the process-cycle, increase the productive rate of arasaponin, improve extraction efficiency, but cellulase or the pectase of adopting carries out enzymolysis processing more in traditional enzymatic isolation method, traditional enzymatic isolation method cellulase or pectase effectively cannot solve the gelatinizing problem of decocting in alcohol process.
Beta amylase generally in food processing, grain processing, ferment, brewage, the industry such as textile has important function, beta amylase is as saccharifying agent, can be applicable to the commercial production such as medicated beer, maltose, beverage, it is the important enzyme source of food processing and brewing industry, beta amylase is usually used in being used as the amylolysis in fruit juice production and the raising rate of filtration, and the outer glycosidase of penicillium oxalicum born of the same parents is usually used in the conversion of glycoside compounds.
Simultaneously, the dry Radix Notoginseng of many employings is that raw material extracts arasaponin now, cycle is long, cost is high, be unfavorable for the intensive of related industry, also the market regulation of Yunnan notoginseng, control ability is unfavorable for, to skill upgrading, the economic benefit growth of enterprise of one's native land, to promoting that the sustainable development of Radix Notoginseng industry has a negative impact.
summary of the invention:
The object of the invention is to the deficiency for existing extracting method, a kind of beta amylase is provided to extract the application in Radix Notoginseng total arasaponins, beta amylase being carried out enzyme introduces in the new technology extracting saponin constituent in fresh Radix Notoginseng, with fresh Radix Notoginseng for raw material, ensure that quality and the extraction ratio of Radix Notoginseng total arasaponins, shorten the traditional mode of production cycle, be conducive to the market regulation of Yunnan notoginseng, control ability, realize the skill upgrading to enterprise of one's native land, economic benefit increases, promote the sustainable development of Radix Notoginseng industry.
Beta amylase is extracting the application in Radix Notoginseng total arasaponins, and in extraction Radix Notoginseng total arasaponins, use beta amylase, its concrete grammar step is as follows:
The first step: pulverize and granulate, fresh for raw material Radix Notoginseng material is cleaned, cleaning, is broken into the fresh granule of 10-20 object with pulverizer after surperficial moisture content is dried at shady and cool place;
Second step: enzymolysis processing, puts the fresh particulate samples of fresh Radix Notoginseng 10-20 order crushed and extracts in molten device, add beta amylase (0.1ml-0.2ml/200g) and carry out enzymolysis processing 1-2h, the 50%-90% ethanol adding 6-10 times amount again carries out reflux, extract, filter, collect filtrate, for subsequent use;
3rd step: merging filtrate, concentrated, vacuum 0.075-0.08 handkerchief decompression recycling ethanol, temperature 70 C, obtains fresh Radix Notoginseng extracting solution;
4th step: precipitate with ethanol process, carries out precipitate with ethanol process by fresh Radix Notoginseng extraction flocculating agent and precipitate with ethanol ethanol, and the precipitate with ethanol time is 12-36h, precipitate with ethanol concentration of alcohol is 60%-90%, precipitate with ethanol number of times is 1-3 time, and alcohol precipitation concentration and sample liquid ratio are 1:1-1:3, and flocculant concentration is 4%-7%;
5th step: eluting process, the fresh Radix Notoginseng extracting solution handled well by precipitate with ethanol is by D101 macroporous adsorptive resins, and resin demand is 1.2-1.8 times of raw material, first uses distilled water eluting, rear 60%-90% ethanol elution, collects ethanol elution, for subsequent use;
6th step: desolventing technology, reclaims ethanol by ethanol elution, is concentrated into dry, is decoloured, use 65%-80% ethanol elution after water dissolution by anion exchange resin D-941 pillar, collects effluent, is concentrated into dry, obtains Radix Notoginseng total arasaponins.
Further, in second step enzymolysis processing, add beta amylase (0.1-0.2ml/200g) simultaneously, the outer glycosidase (0.05-0.1ml/200g) of penicillium oxalicum born of the same parents can be added.
Further, as optimum proportioning consumption, in second step enzymolysis processing, add beta amylase (0.1ml/200g) simultaneously, the outer glycosidase (0.05ml/200g) of penicillium oxalicum born of the same parents can be added.
Further, in the crushing process of the first step, fresh Radix Notoginseng material fragmentation becomes the fresh granule of 10 object for best.
The present invention compares with other extraction process for notoginseng saponin:
1, the present invention with fresh Radix Notoginseng for raw material, extraction ratio is not less than corresponding dry Radix Notoginseng raw material, integrated cost lower than with dry Radix Notoginseng for raw material extracts arasaponin, cycle is short, cost is low, be applicable to suitability for industrialized production, simultaneously, exploitation is with the deep process technology of fresh Radix Notoginseng for raw material extraction saponin, Neng Shi local enterprise gives full play to and utilizes place of production advantage, directly get involved the middle reaches of Radix Notoginseng industrial chain, improve added value of product, with fresh Radix Notoginseng for raw material carries out processing in production place, the intensive of related industry will be contributed to, contribute to the market regulation improving Yunnan notoginseng, control ability, to the skill upgrading of enterprise of one's native land, economic benefit increases, to the sustainable development promoting Radix Notoginseng industry, there is apparent effect.
2, utilize beta amylase to process, introduced by beta amylase in extraction process, ensure that quality and the extraction ratio of Radix Notoginseng total arasaponins, the extraction for Radix Notoginseng total arasaponins provides a kind of enzymolysis material, effectively solves gelatinizing problem simultaneously.
3, two kinds of adsorption columns carry out adsorption treatment, and effectively improve the purity of Radix Notoginseng total arasaponins, the present invention's three monomer (R1+Rg1+Rb1) content can reach more than 65%, higher than existing national standards (more than 60%).
4, the present invention is simple to operate, is conducive to quality control and the industrial implementation of product.
detailed description of the invention:
Below in conjunction with specific embodiment, the present invention is described in further details, to facilitate the technical staff to understand.But the present invention is not limited to these embodiments.
In an experiment, by beta amylase is carried out contrast orthogonal test, test the proportioning of beta amylase used, wherein, the concrete numerical value of beta amylase (A), concentration of alcohol (C), hydrolysis temperature (B) is as shown in the table,
Table 1: the enzymolysis factor level table of beta amylase
Table 2: the enzymolysis Orthogonal experiment results table of beta amylase
From experimental data, we find that beta amylase dosage, hydrolysis temperature, the concentration of alcohol size that affects on Radix Notoginseng total arasaponins yield is followed successively by: beta amylase (A) > concentration of alcohol (C) > hydrolysis temperature (B); When carrying out beta amylase process in procedure of processing, in extract, Radix Notoginseng total arasaponins yield increases significantly, and the preferred plan of four factors is A
1b
2c
2time, when the dosage in beta amylase namely used is 0.1ml/200g, hydrolysis temperature 70 DEG C time, concentration of alcohol is 70%, Radix Notoginseng total arasaponins yield is the highest, is optimum proportion scheme.
Table 3: beta amylase and the outer glycosidase enzymolysis factor level table of penicillium oxalicum born of the same parents
Table 4: beta amylase and the outer glycosidase enzymolysis Orthogonal experiment results table of penicillium oxalicum born of the same parents
As shown in Table 3 and Table 4, test the proportioning of beta amylase used, beta amylase dosage, the outer glycosidase enzyme dosage of penicillium oxalicum born of the same parents, hydrolysis temperature, the affect size of concentration of alcohol on Radix Notoginseng total arasaponins yield are followed successively by: outer glycosidase (B) > hydrolysis temperature (C) of beta amylase (A) > concentration of alcohol (D) > penicillium oxalicum born of the same parents; When carrying out beta amylase process in procedure of processing, in extract, Radix Notoginseng total arasaponins yield increases significantly, and the preferred plan of four factors is A
2b
2c
3d
1time, when the dosage in beta amylase namely used is 0.1ml/200g, during the dosage 0.075ml/200g of the outer glycosidase of penicillium oxalicum born of the same parents, and hydrolysis temperature about 70 DEG C time, concentration of alcohol is 70%, Radix Notoginseng total arasaponins yield is the highest, is optimum proportion scheme.
Table 5: extract of panax notoginseng saponins yield comparative result in Different Extraction Method
As shown in Table 5, respectively with fresh Radix Notoginseng material 200g for raw material carries out the extraction of distinct methods, relative to traditional ethanol refluxing process or with the enzymatic isolation method of cellulase or pectase, in beta amylase enzymatic isolation method or beta amylase and the outer glycosidase composite algorithm of penicillium oxalicum born of the same parents, extract of panax notoginseng saponins yield is significantly improved.
Simultaneously, traditional enzymatic isolation method cellulase or pectase cannot effectively solve the gelatinizing problem in alcohol process of decocting mainly due to Radix Notoginseng total arasaponins extract in content of starch too high, occur in the process of high temperature, adding of beta amylase, accelerate the decomposition of starch, can effectively avoid decocting the gelatinizing in alcohol process.
specific embodiment:
Embodiment 1
Get fresh Radix Notoginseng material 200g, pulverizing is granulation into 10 object granules and carries out ferment treatment, adds beta amylase (0.1ml/200g) and carries out enzymolysis processing 1-2h; The ethanol after ferment treatment, Radix Notoginseng material being added to the 50%-90% of 6 times amount carries out reflux, extract, merging filtrate after filtering, concentrated, vacuum 0.075 handkerchief decompression recycling ethanol, and temperature 60 C, obtains fresh Radix Notoginseng extracting solution; The fresh Radix Notoginseng extracting solution of gained is carried out precipitate with ethanol process, and the precipitate with ethanol time is 12h, and precipitate with ethanol concentration of alcohol is 60%, and precipitate with ethanol number of times is 1 time, and alcohol precipitation concentration and sample liquid ratio are 1:1, and flocculant concentration is 4%; The fresh Radix Notoginseng extracting solution handled well by precipitate with ethanol is by D101 macroporous adsorptive resins, and resin demand is 1.2 times of raw material, first uses distilled water eluting, rear 60%-90% ethanol elution, collects ethanol elution; Ethanol elution is reclaimed ethanol, is concentrated into dry, decoloured by anion exchange resin D-941 pillar after water dissolution, use 65%-80% ethanol elution, collect effluent, be concentrated into dry, obtain extract of panax notoginseng saponins 26.6g, now the extraction ratio of Radix Notoginseng total arasaponins is 13.3%.
Embodiment 2
Get fresh Radix Notoginseng material 200g, pulverizing is granulation into 20 object granules and carries out ferment treatment, adds beta amylase (0.2ml/200g) and carries out enzymolysis processing 1-2h; The ethanol after ferment treatment, Radix Notoginseng material being added to the 50%-90% of 10 times amount carries out reflux, extract, merging filtrate after filtering, concentrated, vacuum 0.08 handkerchief decompression recycling ethanol, and temperature 80 DEG C, obtains fresh Radix Notoginseng extracting solution; The fresh Radix Notoginseng extracting solution of gained is carried out precipitate with ethanol process, and the precipitate with ethanol time is 36h, and precipitate with ethanol concentration of alcohol is 90%, and precipitate with ethanol number of times is 3 times, and alcohol precipitation concentration and sample liquid ratio are 1:3, and flocculant concentration is 4%; The fresh Radix Notoginseng extracting solution handled well by precipitate with ethanol is by D101 macroporous adsorptive resins, and resin demand is 1.2 times of raw material, first uses distilled water eluting, rear 50%-90% ethanol elution, collects ethanol elution; Ethanol elution is reclaimed ethanol, is concentrated into dry, decoloured by anion exchange resin D-941 pillar after water dissolution, use 65%-80% ethanol elution, collect effluent, be concentrated into dry, obtain extract of panax notoginseng saponins 26.2g, now the extraction ratio of Radix Notoginseng total arasaponins is 13.1%.
Embodiment 3
Get fresh Radix Notoginseng material 200g, pulverizing is granulation into 10 object granules and carries out ferment treatment, and outside adding beta amylase (0.1ml/200g) and penicillium oxalicum born of the same parents, glycosidase (0.05ml/200g) carries out enzymolysis processing 1-2h; The ethanol after ferment treatment, Radix Notoginseng material being added to the 50%-90% of 6 times amount carries out reflux, extract, merging filtrate after filtering, concentrated, vacuum 0.075 handkerchief decompression recycling ethanol, and temperature 70 C, obtains fresh Radix Notoginseng extracting solution; The fresh Radix Notoginseng extracting solution of gained is carried out precipitate with ethanol process, and the precipitate with ethanol time is 12h, and precipitate with ethanol concentration of alcohol is 90%, and precipitate with ethanol number of times is 1 time, and alcohol precipitation concentration and sample liquid ratio are 1:1, and flocculant concentration is 7%; The fresh Radix Notoginseng extracting solution handled well by precipitate with ethanol is by D101 macroporous adsorptive resins, and resin demand is 1.8 times of raw material, first uses distilled water eluting, rear 60%-90% ethanol elution, collects ethanol elution; Ethanol elution is reclaimed ethanol, is concentrated into dry, decoloured by anion exchange resin D-941 pillar after water dissolution, use 65%-80% ethanol elution, collect effluent, be concentrated into dry, obtain extract of panax notoginseng saponins 26.6g, now the extraction ratio of Radix Notoginseng total arasaponins is 13.3%.
Embodiment 4
Get fresh Radix Notoginseng material 200g, pulverizing is granulation into 20 object granules and carries out ferment treatment, and outside adding beta amylase (0.2ml/200g) and penicillium oxalicum born of the same parents, glycosidase (0.1ml/200g) carries out enzymolysis processing 1-2h; The ethanol after ferment treatment, Radix Notoginseng material being added to the 50%-90% of 10 times amount carries out reflux, extract, merging filtrate after filtering, concentrated, vacuum 0.08 handkerchief decompression recycling ethanol, and temperature 70 C, obtains fresh Radix Notoginseng extracting solution; The fresh Radix Notoginseng extracting solution of gained is carried out precipitate with ethanol process, and the precipitate with ethanol time is 36h, and precipitate with ethanol concentration of alcohol is 90%, and precipitate with ethanol number of times is 3 times, and alcohol precipitation concentration and sample liquid ratio are 1:3, and flocculant concentration is 4%; The fresh Radix Notoginseng extracting solution handled well by precipitate with ethanol is by D101 macroporous adsorptive resins, and resin demand is 1.2 times of raw material, first uses distilled water eluting, rear 60%-90% ethanol elution, collects ethanol elution; Ethanol elution is reclaimed ethanol, is concentrated into dry, decoloured by anion exchange resin D-941 pillar after water dissolution, use 65%-80% ethanol elution, collect effluent, be concentrated into dry, obtain extract of panax notoginseng saponins 28.6g, now the extraction ratio of Radix Notoginseng total arasaponins is 14.3%.
Embodiment 5
Get fresh Radix Notoginseng material 200g, pulverizing is granulation into 20 object granules and carries out ferment treatment, and outside adding beta amylase (0.1ml/200g) and penicillium oxalicum born of the same parents, glycosidase (0.05ml/200g) carries out enzymolysis processing 1-2h; After ferment treatment, reflux, extract, is carried out to the ethanol that Radix Notoginseng material adds 70% of 10 times amount, merging filtrate after filtering, concentrated, vacuum 0.08 handkerchief decompression recycling ethanol, temperature 70 C, obtains fresh Radix Notoginseng extracting solution; The fresh Radix Notoginseng extracting solution of gained is carried out precipitate with ethanol process, and the precipitate with ethanol time is 36h, and precipitate with ethanol concentration of alcohol is 90%, and precipitate with ethanol number of times is 3 times, and alcohol precipitation concentration and sample liquid ratio are 1:3, and flocculant concentration is 4%; The fresh Radix Notoginseng extracting solution handled well by precipitate with ethanol is by D101 macroporous adsorptive resins, and resin demand is 1.2 times of raw material, first uses distilled water eluting, rear 60%-90% ethanol elution, collects ethanol elution; Ethanol elution is reclaimed ethanol, is concentrated into dry, decoloured by anion exchange resin D-941 pillar after water dissolution, use 65%-80% ethanol elution, collect effluent, be concentrated into dry, obtain extract of panax notoginseng saponins 29.0g, now the extraction ratio of Radix Notoginseng total arasaponins is 14.5%.
The present invention with fresh Radix Notoginseng for raw material, extraction ratio is not less than corresponding dry Radix Notoginseng raw material, integrated cost lower than with dry Radix Notoginseng for raw material extracts arasaponin, cycle is short, cost is low, be applicable to suitability for industrialized production, simultaneously, exploitation is with the deep process technology of fresh Radix Notoginseng for raw material extraction saponin, Neng Shi local enterprise gives full play to and utilizes place of production advantage, directly get involved the middle reaches of Radix Notoginseng industrial chain, improve added value of product, with fresh Radix Notoginseng for raw material carries out processing in production place, the intensive of related industry will be contributed to, contribute to the market regulation improving Yunnan notoginseng, control ability, to the skill upgrading of enterprise of one's native land, economic benefit increases, to the sustainable development promoting Radix Notoginseng industry, there is apparent effect, utilize beta amylase to process, introduced by beta amylase in extraction process, ensure that quality and the extraction ratio of Radix Notoginseng total arasaponins, the extraction for Radix Notoginseng total arasaponins provides a kind of enzymolysis material, two kinds of adsorption columns carry out adsorption treatment, and effectively improve the purity of Radix Notoginseng total arasaponins, the present invention's three monomer (R1+Rg1+Rb1) content can reach more than 65%, higher than existing national standards (more than 60%), the present invention is simple to operate, is conducive to quality control and the industrial implementation of product.
The content that the present invention is described by embodiment, without departing from the present invention, various conversion and equivalent replacement can also be carried out to patent of the present invention, therefore, patent of the present invention is not limited to disclosed specific implementation process, and should comprise the whole embodiments fallen within the scope of Patent right requirement of the present invention.