CN103333922B - High-yield cinnabarin obtaining method - Google Patents
High-yield cinnabarin obtaining method Download PDFInfo
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- CN103333922B CN103333922B CN201310289889.5A CN201310289889A CN103333922B CN 103333922 B CN103333922 B CN 103333922B CN 201310289889 A CN201310289889 A CN 201310289889A CN 103333922 B CN103333922 B CN 103333922B
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Abstract
The invention relates to a natural pigment obtaining method, and especially relates to a high-yield cinnabarin obtaining method. The obtaining method comprises the following steps: 1, culturing and filtering; 2, carrying out resin pretreatment; 3, adsorbing and eluting; and 4, adjusting the pH value or adding K<+>, Na<+>, Gu<2+>, Zn<2+> or Al<3>. The method is used in the natural fungal pigment extracting and applying fields.
Description
Technical field
The present invention relates to a kind of acquisition methods of natural pigment.
Background technology
Pigment is widely used in the fields such as foodstuffs industry, bionic food, medicine and pharmacology, makeup, high-grade feed, and pigment is divided into synthetic food color and natural pigment.
Synthetic food color refers to take the pigment that coal tar makes as raw material, and it is of a great variety, as Xanthones pyridine based dye, anthraquinone based dye, azo based dye, quinoline based dye etc.The character of synthetic food color is conventionally more stable, has good tone, look valency, solvability and tinting strength, and while production cost is low, product with stable quality, thereby uses comparatively extensive.Yet large quantity research is found in recent years, and synthetic food color not only can not, for human body provides nutritive substance, also can work the mischief to HUMAN HEALTH.
Natural pigment refers to the pigment of natural resource such as deriving from animals and plants, microorganism.According to the difference of its chemical structure, can be divided into: 1. pyrrole derivative, as chlorophyll, protoheme and Choline etc.; 2. isoprene derivatives, as carotenoids etc.; 3. Polyphenols derivative, as anthocyanidin, words flavine, catechin, tannin etc.; 4. ketones derivant, as monascorubin, curcumine etc.; 5. naphthoquinone derivatives, as shellac color, cochineal etc.; According to the difference of its dissolving properties, natural pigment can also be divided into water colo(u)r and fat-soluble pigment.
Comparing natural pigment security with synthetic food color good, also with nutrition and pharmacological action, is that desirable pigment is selected.Fungi pigment is a kind of of natural pigment, the fungi dye compound of finding at present comprises quinones, acids, heterocycle and nitrogenous compound, wherein take quinones as main, this compounds has important pharmacologically active, for example violacein by numerous scholars report there is pain relieving, reduce phlegm and internal heat, anticancer effect.And animal and plant raw material has unrivaled advantage with fungi production pigment relatively, it is with short production cycle, output is high, be not subject to spatio-temporal restriction, is convenient to suitability for industrialized production, simultaneously the not impact of climate and environment.Therefore, fungi has huge potentiality to be exploited as the generation resource of pigment.
Red fungus (Trametes cinnabarina (Jaxq.) Fr.var.sanguine) is the aperture mutation of red bolt bacterium (Trametes cinnabarina (Jacq.:Fr.) Fr.), is under the jurisdiction of Basidiomycotina, Hymenomycetes, Aphyllophorales, polyporaceae, trametes.Red fungus is born on pin, deciduous tree deadwood, often growth in flakes in groups.In China, be mainly distributed in the provinces and cities such as Heilungkiang, Yunnan, Guizhou, Guangdong and Chongqing.Red fungus has the effects such as anti-inflammatory, antipruritic, removing toxic substances, myogenic, pleasant, removing damp-heat, hemostasis; Inhibiting rate to ehrlich carcinoma and small white mouse sarcoma 180 is 90%, cancer cells is also had to restraining effect simultaneously.In addition, red fungus also contains polyporin, thereby Gram-negative and positive bacteria are all had to restraining effect.Red fungus contains red natural pigment---cinnabarin (cinnabarine), cinnabarin also has pharmacological action, can rabies poison (
aJr, Marques CJ, Smania EF, Zanetti CR, Carobrez SG, Tramonte R, Loguercio-Leite C.Toxicity andAntiviral Activity of Cinnabarin Obtained from Pycnoporus sanguineus (Fr.) Murr Phytother Res.2003Nov; 17 (9): 1069-1072.).
Because fungi pigment stability is poor, in addition traditional extraction technology, as milling process, comminuting method, circumfluence method, organic solvent extraction method, there is complicated operation, production cost is high, product purity is low, have dissolvent residual, heat-sensitive component is destroyed etc., cause cinnabarin look valency low, hindered applying of cinnabarin.
Summary of the invention
The present invention is in order to solve the low problem of existing cinnabarin look valency, and the acquisition methods of a kind of high look valency cinnabarin providing.
High look valency cinnabarin obtains according to the following steps:
One, the red fungus mycelia after rejuvenation is transferred in PDA liquid nutrient medium, the condition bottom fermentation that is 140r/min at 28 ± 1 ℃, rotating speed is cultivated 10～15 days, then removes by filter mycelium;
Two, resin pre-treatment: the alcohol immersion resin 24h that is 95% by concentration, then with wet method dress post, resin is packed into chromatography column, adverse current is washed post, the washing with alcohol resin that is 95% by concentration again, flow velocity is 2 times of column volumes per hour, until that elutant is mixed with distilled water 1:5 is not muddy, then uses distilled water washing resin, flow velocity is 2 times of column volumes per hour, until elutant does not have ethanol taste;
Three, will add in step 1 filtrate and adsorb through pretreated resin, after being uniformly mixed 12～14h, resin is packed in chromatography column, with 2 column volumes of 15mL/min speed washing, collect elutriant, and then take the ethanol elution that the speed of 15mL/min is 70% by concentration, to the A of ethanol eluate
416value is less than 0.5 and stops collecting elutriant;
Four, reconciling the elutriant pH value of collecting is 11～12, or in the ratio of 0.25mol/L, adds K in elutriant
+, Na
+, Gu
2+, Zn
2+or Al
3+ion, obtains high look valency cinnabarin.
Step 1 of the present invention transfers the red fungus mycelia after rejuvenation into PDA liquid nutrient medium, early stage mycelium Fast Growth, form a large amount of mycelium pellets, be cultured to 4th～5 days and start substratum rubicundity, mycelia starts to produce cinnabarin, be cultured to about the 10th day, cinnabarin output reaches high value.
The look valency of step 1 filtrate of the present invention is 2.59, and the look valency of the elutriant of collecting through step 3 is 19.06.The look valency of the high look valency cinnabarin that the present invention obtains is greater than 20.5.The measuring method of look valency: determinand is rotated to evaporate to dryness, accurately take pigment sample 0.1g (being accurate to 0.0002), water is settled to 100mL, measures it in λ with the cuvette of light path 1cm
maxabsorbancy, result is used
represent.
(wherein A represents absorbance reading; N represents extension rate; M represents quality).
The inventive method has only been used a kind of solvent of ethanol in the whole process that obtains high look valency cinnabarin, has cost low, and cinnabarin is disturbed to the advantages such as little.And there is not chemical reaction in the inventive method, do not change original composition in red fungus, so can also utilize the waste of the inventive method to continue to extract polysaccharide and the albumen of red fungus, starting material are fully utilized.
The inventive method has with short production cycle, and product purity is high, and look valency is high, simple to operate, without the advantage that has dissolvent residual, composition not to be destroyed.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the high look valency of present embodiment cinnabarin obtains according to the following steps:
One, the red fungus mycelia after rejuvenation is transferred in PDA liquid nutrient medium, the condition bottom fermentation that is 140r/min at 28 ± 1 ℃, rotating speed is cultivated 10～15 days, then removes by filter mycelium;
Two, resin pre-treatment: the alcohol immersion resin 24h that is 95% by concentration, then with wet method dress post, resin is packed into chromatography column, adverse current is washed post, the washing with alcohol resin that is 95% by concentration again, flow velocity is 2 times of column volumes per hour, until that elutant is mixed with distilled water 1:5 is not muddy, then uses distilled water washing resin, flow velocity is 2 times of column volumes per hour, until elutant does not have ethanol taste;
Three, will add in step 1 filtrate and adsorb through pretreated resin, after being uniformly mixed 12～14h, resin is packed in chromatography column, with 2 column volumes of 15mL/min speed washing, collect elutriant, and then take the ethanol elution that the speed of 15mL/min is 70% by concentration, to the A of ethanol eluate
416value is less than 0.5 and stops collecting elutriant;
Four, reconciling the elutriant pH value of collecting is 11, or in the ratio of 0.25mol/L, adds K in elutriant
+, Na
+, Gu
2+, Zn
2+or Al
3+ion, obtains high look valency cinnabarin.
In present embodiment with K
2sO
4, NaCl, CuSO
45H
2o, ZnSO
4or Al
2(SO
4)
3mode adds K
+, Na
+, Gu
2+, Zn
2+or Al
3+ion; Gu especially wherein
2+and Zn
2+look valency increases significantly.
In present embodiment step 2, adverse current is washed post and broken resin and small-particle can be washed away.
Present embodiment is filtered the mycelium in fermented liquid removal in step 1, does not only affect the acquisition of high look valency cinnabarin, has avoided the not segregative problem of macroporous resin and mycelium pellet simultaneously.
The high look valency cinnabarin that present embodiment obtains is under 20 ℃～60 ℃ conditions, absorbancy varies with temperature there was no significant difference, cinnabarin Heat stability is good under 20 ℃～60 ℃ conditions is described, under 60 ℃～100 ℃ conditions, absorbancy raises and significantly reduces with temperature, illustrates that long high temperature makes cinnabarin decomposed.Therefore, in cinnabarin application, should avoid long-time pyroprocessing as far as possible.
Concentration is the Na of 0.02g/L～0.06g/L
2sO
3cinnabarin absorbancy is all had no significant effect.The cinnabarin that present embodiment obtains is for reductive agent Na
2sO
3there is satisfactory stability, can in the fields such as food, makeup, apply, meet the requirement of pigment to reductive agent stability.
Embodiment two: the difference of present embodiment and embodiment one is: the resin described in step 2 is HPD722 macroporous resin.Other step and parameter are identical with embodiment one.
Embodiment three: present embodiment and embodiment one or twos' difference is: add the ratio of 100g pre-treatment resin to add through pretreated resin in step 1 filtrate in 500mL step 1 filtrate in step 3.Other step and parameter are identical with embodiment one or two.
Embodiment four: present embodiment and embodiment one, two or threes' difference is: the high look valency of step 4 cinnabarin is rotated evaporation or lyophilize or the dry high look valency cinnabarin pressed powder that obtains of spraying.Other step and parameter are identical with embodiment one, two or three.
Embodiment five: the high look valency of present embodiment cinnabarin obtains according to the following steps:
One, the red fungus mycelia after rejuvenation is transferred in PDA liquid nutrient medium, the condition bottom fermentation that is 140r/min at 28 ℃, rotating speed is cultivated 15 days, then removes by filter mycelium;
Two, resin pre-treatment: the alcohol immersion resin 24h that is 95% by concentration, then with wet method dress post, resin is packed into chromatography column, adverse current is washed post, the washing with alcohol resin that is 95% by concentration again, flow velocity is 2 times of column volumes per hour, until that elutant is mixed with distilled water 1:5 is not muddy, then uses distilled water washing resin, flow velocity is 2 times of column volumes per hour, until elutant does not have ethanol taste;
Three, will add in step 1 filtrate and adsorb through pretreated resin, after being uniformly mixed 12～14h, resin is packed in chromatography column, with 2 column volumes of 15mL/min speed washing, collect elutriant, and then take the ethanol elution that the speed of 15mL/min is 70% by concentration, to the A of ethanol eluate
416value is less than 0.5 and stops collecting elutriant;
Four, reconciling the elutriant pH value of collecting is 11, obtains high look valency cinnabarin;
Wherein, the resin described in step 2 is HPD722 macroporous resin; In step 3, in 500mL step 1 filtrate, add the ratio of 100g pre-treatment resin to add through pretreated resin in step 1 filtrate.
The measuring method of look valency in present embodiment: determinand is rotated to evaporate to dryness, accurately take pigment sample 0.1g (being accurate to 0.0002), water is settled to 100mL, measures it in λ with the cuvette of light path 1cm
maxabsorbancy, result is used
represent.
(wherein A represents absorbance reading; N represents extension rate; M represents quality).
The look valency of the cinnabarin that present embodiment obtains is 22.7.
Embodiment six: the difference of present embodiment and embodiment five is:
Step 1, the red fungus mycelia after rejuvenation is transferred in PDA liquid nutrient medium, the condition bottom fermentation that is 140r/min at 28 ℃, rotating speed is cultivated 10 days, then removes by filter mycelium;
The elutriant pH value that step 4, conciliation are collected is 12, obtains high look valency cinnabarin.Other step and parameter are identical with embodiment five.
The look valency of the cinnabarin that present embodiment obtains is 27.60.
Embodiment seven: the high look valency of present embodiment cinnabarin obtains according to the following steps:
One, the red fungus mycelia after rejuvenation is transferred in PDA liquid nutrient medium, the condition bottom fermentation that is 140r/min at 28 ℃, rotating speed is cultivated 12 days, then removes by filter mycelium;
Two, resin pre-treatment: the alcohol immersion resin 24h that is 95% by concentration, then with wet method dress post, resin is packed into chromatography column, adverse current is washed post, the washing with alcohol resin that is 95% by concentration again, flow velocity is 2 times of column volumes per hour, until that elutant is mixed with distilled water 1:5 is not muddy, then uses distilled water washing resin, flow velocity is 2 times of column volumes per hour, until elutant does not have ethanol taste;
Three, will add in step 1 filtrate and adsorb through pretreated resin, after being uniformly mixed 12～14h, resin is packed in chromatography column, with 2 column volumes of 15mL/min speed washing, collect elutriant, and then take the ethanol elution that the speed of 15mL/min is 70% by concentration, to the A of ethanol eluate
416value is less than 0.5 and stops collecting elutriant;
Four, in elutriant, in the ratio of 0.25mol/L, add K
+ion, obtains high look valency cinnabarin;
Wherein, the resin described in step 2 is HPD722 macroporous resin; In step 3, in 500mL step 1 filtrate, add the ratio of 100g pre-treatment resin to add through pretreated resin in step 1 filtrate.
The look valency of the cinnabarin that present embodiment obtains is 20.64.
Embodiment eight: the difference of present embodiment and embodiment seven is: four, add Na in the ratio of 0.25mol/L in elutriant
+ion, obtains high look valency cinnabarin.Other step and parameter are identical with embodiment seven.
The look valency of the cinnabarin that present embodiment obtains is 20.63.
Embodiment nine: the difference of present embodiment and embodiment seven is: four, add Gu in the ratio of 0.25mol/L in elutriant
2+ion, obtains high look valency cinnabarin.Other step and parameter are identical with embodiment seven.
The look valency of the cinnabarin that present embodiment obtains is 22.24.
Embodiment ten: the difference of present embodiment and embodiment seven is: four, add Zn in the ratio of 0.25mol/L in elutriant
2+ion, obtains high look valency cinnabarin.Other step and parameter are identical with embodiment seven.
The look valency of the cinnabarin that present embodiment obtains is 22.00.
Embodiment 11: the difference of present embodiment and embodiment seven is: four, add Al in the ratio of 0.25mol/L in elutriant
3+ion, obtains high look valency cinnabarin.Other step and parameter are identical with embodiment seven.
The look valency of the cinnabarin that present embodiment obtains is 20.62.
Embodiment 12: present embodiment and embodiment one, two or threes' difference is: step 4 is to the dehydrated alcohol that adds 1～3 times of volume of high look valency cinnabarin in high look valency cinnabarin, then water and alcohol mixed solution layer are discharged, obtain liquid high look valency cinnabarin.Other step and parameter are identical with embodiment one, two or three.
The cinnabarin that present embodiment obtains is soluble in water, is insoluble to the organic solvents such as dehydrated alcohol, methyl alcohol, ethyl acetate, chloroform.In the high look valency cinnabarin obtaining in step 4, add a large amount of dehydrated alcohols, high look valency cinnabarin can be precipitated, thereby obtains liquid high look valency cinnabarin.
It is high that the high look valency of the liquid state cinnabarin that present embodiment obtains has purity, and output is high, recyclable recycling, the advantages such as environmental protection, health of repeating of water and ethanol.And have and can directly use, look valency is high, the feature that brightness is high.
Embodiment 13: present embodiment and embodiment one, two or threes' difference is: step 4 is to the high purity ethanol that adds 1～3 times of volume of high look valency cinnabarin in high look valency cinnabarin, in high purity ethanol, the concentration of ethanol is greater than 96.2%, then water and alcohol mixed solution layer are discharged, obtain liquid high look valency cinnabarin.Other step and parameter are identical with embodiment one, two or three.
Claims (5)
1. an acquisition methods for high look valency cinnabarin, is characterized in that high look valency cinnabarin obtains according to the following steps:
One, the red fungus mycelia after rejuvenation is transferred in PDA liquid nutrient medium, the condition bottom fermentation that is 140r/min at 28 ± 1 ℃, rotating speed is cultivated 10～15 days, then removes by filter mycelium;
Two, resin pre-treatment: the alcohol immersion resin 24h that is 95% by concentration, then with wet method dress post, resin is packed into chromatography column, adverse current is washed post, the washing with alcohol resin that is 95% by concentration again, flow velocity is 2 times of column volumes per hour, until that elutant is mixed with distilled water 1:5 is not muddy, then uses distilled water washing resin, flow velocity is 2 times of column volumes per hour, until elutant does not have ethanol taste;
Three, the pretreated resin of process step 2 being obtained adds step 1 to filter in the filtrate obtaining and adsorbs, after being uniformly mixed 12～14h, resin is packed in chromatography column, with 2 column volumes of 15mL/min speed washing, collect elutriant, and then take the ethanol elution that the speed of 15mL/min is 70% by concentration, to the A of ethanol eluate
416value is less than 0.5 and stops collecting elutriant;
Four, the elutriant pH value that regulating step three is collected is 11～12, or in the ratio of 0.25mol/L, adds K in elutriant
+, Na
+, Gu
2+, Zn
2+or Al
3+ion, obtains high look valency cinnabarin;
Wherein, the resin described in step 2 is HPD722 macroporous resin; Mode with solid reagent in step 4 adds K
+, Na
+, Gu
2+, Zn
2+or Al
3+ion.
2. the acquisition methods of high look valency cinnabarin according to claim 1, is characterized in that the filtrate obtaining in the filtration of 500mL step 1 in step 3 adds the ratio of the pre-treatment resin of 100g step 2 acquisition to step 1, to filter the pretreated resin of process that adds step 2 acquisition in the filtrate obtaining.
3. the acquisition methods of high look valency cinnabarin according to claim 1, is characterized in that the high look valency of step 4 cinnabarin to be rotated evaporation or lyophilize or the dry high look valency cinnabarin pressed powder that obtains of spraying.
4. the acquisition methods of high look valency cinnabarin according to claim 1, it is characterized in that in step 4 to the dehydrated alcohol that adds 1～3 times of volume of high look valency cinnabarin in high look valency cinnabarin, then water and alcohol mixed solution layer are discharged, obtain liquid high look valency cinnabarin.
5. the acquisition methods of high look valency cinnabarin according to claim 1, it is characterized in that in step 4 to the high purity ethanol that adds 1～3 times of volume of high look valency cinnabarin in high look valency cinnabarin, in high purity ethanol, the concentration of ethanol is greater than 96.2%, then water and alcohol mixed solution layer are discharged, obtain liquid high look valency cinnabarin.
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| CN110771428B (en) * | 2019-11-23 | 2022-02-08 | 辽宁省微生物科学研究院 | Cultivation method of chromium-rich medicinal fungus trametes cinnabarina |
| CN111567655A (en) * | 2020-06-03 | 2020-08-25 | 山东省农业科学院农业资源与环境研究所 | Preparation method of ganoderma lucidum berry concentrated tea |
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| CN101032251A (en) * | 2007-04-29 | 2007-09-12 | 西北农林科技大学 | Fungicides using Zuelaemycin as bioactive substance |
| CN101182485A (en) * | 2007-09-29 | 2008-05-21 | 北京市农林科学院 | Streptomyces lydicus producing natamycin and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101032251A (en) * | 2007-04-29 | 2007-09-12 | 西北农林科技大学 | Fungicides using Zuelaemycin as bioactive substance |
| CN101182485A (en) * | 2007-09-29 | 2008-05-21 | 北京市农林科学院 | Streptomyces lydicus producing natamycin and uses thereof |
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| Optimal Parameters for Cinnabarin Synthesis by Pycnoporus sanguineus;Elza F. A. Smaü nia et.al;《J. Chem. T ech. Biotechnol.》;19971231;第70卷;57-59 * |
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