CN101885784B - Method for producing glucomannan by liquid culture of konjac cells - Google Patents
Method for producing glucomannan by liquid culture of konjac cells Download PDFInfo
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- CN101885784B CN101885784B CN2010102315402A CN201010231540A CN101885784B CN 101885784 B CN101885784 B CN 101885784B CN 2010102315402 A CN2010102315402 A CN 2010102315402A CN 201010231540 A CN201010231540 A CN 201010231540A CN 101885784 B CN101885784 B CN 101885784B
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Abstract
The invention provides a method for producing glucomannan by liquid culture. Researches are made on a plurality of main factors which influence the production of a secondary metabolite glucomannan by the liquid culture of konjac cells, and results show that: in the process of producing the glucomannan by the liquid culture of the konjac cells, an MS culture medium is an optimal culture medium; the glucomannan content reaches a maximum value approximately in the 15th day; the synthesis of the glucomannan is facilitated when pH is 6.0; 25g/L lactose is an optimal carbon source; the accumulative effect of the glucomannan is obvious when the concentration of 2,4-D is 1.0mg/L, and the accumulative effect of the glucomannan is unobvious when the 2,4-D and NAA are at other concentrations; cytokinin 6-BA makes the accumulative effect of the glucomannan obviously superior to that of KT, and the accumulative amount of the glucomannan is highest when the concentration of the 6-BA is 1.5mg/L; an optimal culture medium for producing the glucomannan by the liquid culture of the konjac cells consists of the MS, the 2,4-D(1.0mg/L), the 6-BA(1.5mg/L), and the lactose (25mg/L); and the highest accumulative amount of the glucomannan can reach 125.70mg/100mL.
Description
Technical field
The present invention relates to a kind of preparation method of konjac glucomanna, be specifically related to a kind of method that adopts producing glucomannan by liquid culture of konjac cells.
Background technology
The konjaku formal name used at school is Amorphophallus konjac, monocot genus plant guiding principle, and the stem tuber of Araeceae shade tolerance per nnial herb is a big special product of China, is distributed widely in the Yangtze valley.Yunnan, Sichuan output maximum, also there is production in west place in Hubei, the western Hunan, Fujian.Contain konjac glucomanna (Konjac Glucomannan in the konjak corm; Be called for short KGM), starch, fiber, protein and the ash nutritive ingredient of grading; Wherein main effective constituent is konjac glucomanna, is the macromolecule polysaccharide that is passed through β-1,4 glucosidic linkage by D-glucose and D-seminose.It has multiple unique physico-chemical property, like swelling property, rheological, thickening property, gelling and film-forming properties etc.In recent years, research both at home and abroad confirms, KGM is the water-soluble dietary fibre of a kind of good low in calories, lower fat, high-cellulose, and nutritional imbalance is had important regulating effect.Many scholar's research show; That KGM has is hypoglycemic, blood fat and reverse fatty liver; Defaecation fat-reducing, anticancer, raise immunity, anti-ageing peculiar health-care effect and the medical functions of waiting for a long time all have good using value in industry such as food, medicine, chemical industry, weaving, oil drillings.Konjaku is unfavorable for the large-scale production of konjac glucomanna because its source receives environment and seasonal effect bigger, does not appear in the newspapers as yet about konjaku cell liquid culture product konjac glucomanna technology.
Summary of the invention
The technical problem that the present invention will solve provides the method for the producing glucomannan by liquid culture of konjac cells of a kind of material homogeneous, good reproducibility.
The objective of the invention is to realize like this: a kind of method of producing glucomannan by liquid culture of konjac cells; May further comprise the steps: the inducing of (1) callus: water is clean with elephant-foot yam stem tuber surface cleaning; Soak through alcohol-pickled, mercury, at last with the aqua sterilisa washing, the epidermis and being cut into small pieces pruned; The konjaku stem tuber stripping and slicing that disinfects is moved into callus of induce supports in the base; The pH that the control callus of induce is supported base is 6.0, and organizing once more of will expanding after placing dark culturing room to cultivate is cut into small pieces, and the callus of induce substratum that moves into new configuration is further cultivated; (2) cell liquid culture: will pass through the eugonic callus chopping of callus of induce culture medium culturing, and be seeded in the container that fills liquid nutrient medium and cultivate, and obtain nutrient solution; (3) konjac glucomanna is produced: medium centrifugal is handled, and supernatant adds ethanol under whipped state, nutrient solution is made contain alcohol amount 50%; After placing 24h, recentrifuge is handled, obtain throw out; With 50% Ethanol Treatment for several times, the throw out lyophilize gets white mass, i.e. KGM.
Callus of induce substratum in the step (1) is in 1 liter of MS nutrient solution, to add 1.0mg NAA, 1.0mg 6-BA and 25g lactose to mix.
Liquid nutrient medium in the step (2) is in 1 liter of MS nutrient solution, to add 1.0mg 2,4-D, and 0.1mgKT and 30g lactose mix, and control pH value is 6.0.
Step (1) places the cultivation of dark culturing room to be meant under 26 ℃ temperature and cultivates incubation time 2 months.
Being seeded in culture condition in the container that fills liquid nutrient medium and being in illumination, temperature is shaking culture under 25 ± 1 ℃, the shaking table condition of 110r/min.
The method of producing glucomannan by liquid culture of konjac cells provided by the invention; Proposed a kind of new be the method that main raw material extracts konjac glucomanna with konjaku cell liquid; Solved konjaku because its source receives environment and seasonal effect bigger; Be unfavorable for that konjac glucomanna carries out the problem of large-scale production, can realize producing from konjaku the suitability for industrialized production of konjac glucomanna.
Description of drawings
Below in conjunction with accompanying drawing and embodiment the present invention is described further: Fig. 1 suspension liquid substratum is to the influence of konjak glucomannan content; The influence that Fig. 2 different vaccination amount produces konjak glucomannan; Fig. 3 pH is to the influence of konjak glucomannan content; The different carbon sources of Fig. 4 are to the influence of konjak glucomannan content; Fig. 5 different concns lactose is to the influence of konjak glucomannan content.
Embodiment
One, preparation embodiment: the inducing of (1) callus: water is clean with elephant-foot yam stem tuber surface cleaning; Use 75% alcohol-pickled 1min again; 0.1% mercuric chloride soaks 10min, at last with aqua sterilisa washing 4 times, and the epidermis and being cut into small pieces pruned; The konjaku stem tuber stripping and slicing that disinfects is moved into callus of induce supports in the base; The pH that the control callus of induce is supported base is 6.0, places organizing once more that dark culturing room will expand after cultivating 2 months under 26 ℃ the temperature to be cut into small pieces, and the callus inducing medium that moves into new configuration is further cultivated; It is in 1 liter of MS nutrient solution, to add 1.0mg NAA (naphthylacetic acid), 1.0mg 6-BA (6-benzyl aminopurine is a kind of phytokinin) and 30g sucrose to mix that above-mentioned callus of induce is supported base; (2) cell liquid culture: will pass through the eugonic callus chopping of callus of induce culture medium culturing, and be seeded in the container that fills liquid nutrient medium, be shaking culture under 25 ± 1 ℃, the shaking table condition of 110r/min in illumination, temperature, obtains nutrient solution; Liquid nutrient medium is in 1 liter of MS nutrient solution, to add 1.0mg 2,4-D, and 0.1mgKT (6-furfuryl group VITAMIN B4, phytokinin) and 30g sucrose mix, and control pH value is 6.0.
(3) konjac glucomanna is produced: with the centrifugal 5min of nutrient solution 4000r/min, supernatant adds 95% ethanol under whipped state makes the alcohol amount 50% that contains, behind the placement 24h; The centrifugal 5min of 4000r/min gets throw out, uses 50% Ethanol Treatment for several times repeatedly; Lyophilize gets white mass, i.e. KGM.
Two, index of correlation is measured: 1, Content of Glucomannan 1-1 drawing standard working curve pipettes the glucose standardized solution 0.40,0.80,1.20 that concentration is 1mg/mL successively; 1.60 2.0mL is in 5 10mL volumetric flasks, adding distil water is mended to 2mL; Add 1.5mL3 then respectively, the 5-edlefsen's reagent is behind heating 5min on the boiling water bath; Be settled to 10mL with zero(ppm) water, measure its absorbancy (A) in the 550nm place.
The mensuration of KGM is got nutrient solution 4ml in the 2-2 culture, and the centrifugal 5min of 4000r/min, supernatant add 95% ethanol under whipped state makes the alcohol amount 50% that contains; After placing 24h, the centrifugal 5min of 4000r/min gets throw out; Use 50% Ethanol Treatment for several times repeatedly; Lyophilize gets white mass, and promptly KGM is measured content.
KGM assay: lyophilize is got white mass add 0.5mLddH
2The O dissolving adds 0.5mL3mol/LH
2SO
4Hydrolysis 2h on boiling water bath, cooling adds 1mL3mol/LNaOH, and adds water and be settled to 10mL, gets this hydrolyzed solution 2.0mL, by standard glucose working curve step measurements KGM absorbance A (OD
550).With absorbance A substitution regression equation A=-0.019+0.0392T, r=0.99958 calculates the corresponding glucose in milligrams of each absorbance and counts T.Is KGM content used computes: KGM content=ε? T? In the extension rate formula: ε=0.9; For the glucose that obtains after glucose and the residue relative molecular mass of seminose in KGM and the KGM hydrolysis and the ratio T of seminose relative molecular mass are KGM hydrolyzed solution colorimetric estimation value, look into typical curve gained milligram number.
2, different substratum are adding 2, MS, the N of 4-D1.0mg/L+KT0.1mg/L+ sucrose 30g/L to the konjac glucomanna effect of accumulation
6, in the B5 nutrient solution; Inoculum size with 4g/100mL inserts the konjaku callus; In illumination, temperature is shaking culture under 25 ± 1 ℃, rotating speed 110r/min condition; Can know that by Fig. 1 the MS substratum is beneficial to the synthetic of KGM, so select for use the MS substratum as obtaining the basic suspension culture base of high yield secondary metabolite KGM.
3, the different vaccination amount to the konjac glucomanna effect of accumulation at nutrient solution MS+2; 4-D1.0mg/L+KT0.1mg/L+ among the sucrose 30g/L, add 2,4 respectively, the fresh konjak callus of 6g/100mL, be shaking culture under (25 ± 1) ℃, the rotating speed 110r/min condition in illumination, temperature; Every separated 3d gets once appearance and measures konjac glucomanna content curve; As shown in Figure 2, when inoculum size was 4.0g/100mL, KGM reached maximum level during 15d.
4, the initial pH of substratum regulates substratum MS+2 to the influence of KGM content, 4-D1.0mg/L+KT0.1mg/L+ sucrose 30g/L original ph, and it is 5.6,5.8,6.0,6.24 gradients that original ph is set; The callus inoculum size is 4g/100mL, is shaking culture under 25 ± 1 ℃, rotating speed 110r/min condition in illumination, temperature, measures konjac glucomanna content behind the 15d; As shown in Figure 3; 15d, the pH value is 6.0 o'clock, KGM content is up to 54.82mg/100mL.
5, different carbon sources to the konjac glucomanna effect of accumulation at substratum MS+2; 4-D1.0mg/L+KT0.1mg/L the different carbon sources of middle adding are respectively sucrose, glucose, SANMALT-S, lactose, the concentration of different carbon sources is 30g/L; The callus inoculum size is 4g/100mL; In illumination, temperature is shaking culture under (25 ± 1) ℃, the rotating speed 110r/min condition, measures konjac glucomanna content behind the 15d, can be known by Fig. 4; Lactose is the righttest carbon source of KGM accumulation, and its KGM accumulation volume can reach 86.1mg/100mL.
6, lactose the konjac glucomanna effect of accumulation at substratum MS+2, is added 20,25,30 among the 4-D1.0mg/L KT0.1mg/L, the lactose of 35g/L5 kind different concns, the callus inoculum size is 4g/100mL; In illumination, temperature is shaking culture under (25 ± 1) ℃, the rotating speed 110r/min condition; Measure konjac glucomanna content behind the 15d, can know by Fig. 5, when lactose concn is 25g/L; 15d konjac glucomanna accumulation volume is maximum, reaches 103.89mg/100mL.
7, plant-growth regulator concentration concentration that the konjac glucomanna effect of accumulation is regulated liquid nutrient medium phytokinin and growth hormone respectively, it is 0.5,1.0,1.5 that phytokinin (KT and 6-BA) is provided with concentration, 2.0mg/L; Growth hormone (2,4-D and NAA) concentration is 0.5,1.0,2.0,4.0mg/L; The subculture amount is 4g/100mL.3 repetitions of every processing.To be illumination, temperature be shaking culture under (25 ± 1) ℃, the rotating speed 110r/min condition to culture condition; Measure konjac glucomanna content behind the 15d, can know 2 by table 1; 4-D is obvious to the KGM accumulation effect when 1.0mg/L, and KGM content reaches 76.34mg/100mL; Phytokinin 6-BA makes the KGM accumulation apparently higher than KT; And 6-BA KGM accumulation volume when 1.5mg/L is the highest; Reach 90.69mg/100mL; Still select 2 of 1.0mg/L, the 6-BA of 4-D and 1.5mg/L produces the righttest exogenous hormone concentration of KGM as liquid culture.
The different exogenous hormones of table 1 are to the influence of content of konjak glucomannan
Claims (3)
1. the method for a producing glucomannan by liquid culture of konjac cells is characterized in that: may further comprise the steps:
(1) inducing of callus:
Water is clean with elephant-foot yam stem tuber surface cleaning; Soak through alcohol-pickled, mercury, at last with the aqua sterilisa washing, the epidermis and being cut into small pieces pruned; The konjaku stem tuber stripping and slicing that disinfects is moved in the callus of induce substratum; The pH of control callus of induce substratum is 6.0, and organizing once more of will expanding after placing dark culturing room to cultivate is cut into small pieces, and the callus of induce substratum that moves into new configuration is further cultivated; The callus of induce substratum is in 1 liter of MS nutrient solution, to add 1.0mg NAA, 1.0 mg 6-BA and 25g lactose to mix;
(2) cell liquid culture:
To pass through the eugonic callus chopping of callus of induce culture medium culturing, and be seeded in the container that fills liquid nutrient medium and cultivate, obtain nutrient solution; Liquid nutrient medium is in 1 liter of MS nutrient solution, to add 1.0mg 2,4-D, and 0.1mgKT and 25g lactose mix, and control pH value is 6.0;
(3) konjac glucomanna is produced:
Medium centrifugal is handled, and supernatant adds ethanol under whipped state, nutrient solution is made contain alcohol amount 50%, and behind the placement 24h, recentrifuge is handled, and obtains throw out, and with 50% Ethanol Treatment for several times, the throw out lyophilize gets white mass, i.e. KGM.
2. the method for producing glucomannan by liquid culture of konjac cells according to claim 1 is characterized in that: step (1) places dark culturing room to cultivate to be meant under 26 ℃ temperature cultivates incubation time 2 months.
3. the method for producing glucomannan by liquid culture of konjac cells according to claim 1 is characterized in that: being seeded in culture condition in the container that fills liquid nutrient medium and being in illumination, temperature is shaking culture under 25 ± 1 ℃, the shaking table condition of 110 r/min.
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