CN104403017B - The preparation method of a kind of low viscosity, high purity yeast mannans - Google Patents
The preparation method of a kind of low viscosity, high purity yeast mannans Download PDFInfo
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- CN104403017B CN104403017B CN201410665920.5A CN201410665920A CN104403017B CN 104403017 B CN104403017 B CN 104403017B CN 201410665920 A CN201410665920 A CN 201410665920A CN 104403017 B CN104403017 B CN 104403017B
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Abstract
The invention provides the preparation method of a kind of low viscosity, high purity yeast mannans, with brewing yeast cell wall for raw material, adopt water extraction and alcohol precipitation method, obtain crude product yeast mannans, centrifugal through neutral protease and PRONASE A enzymolysis, reprocess and get final product.The present invention is by combining secondary enzymolysis, centrifugal, ultrafiltration and spray drying technology, the equipment adopted all can realize the needs of suitability for industrialized production, the yeast mannans purity of gained is greater than 90%, viscosity is low and be Newtonian fuid, can not affect the rheological properties of system in actual applications.
Description
Technical field
The present invention relates to the preparation of yeast mannans, specifically, relate to the preparation method of a kind of low viscosity, high purity yeast mannans.
Background technology
Yeast mannans is the one of yeast cells wall polysaccharide, account for 40% of yeast cells wall dry weight, be made up of main chain and side chain, main chain is connected to form with α-1,6 key by multiple mannose molecules, main chain is connected with α-1,2 or α-1, the seminose side chain that 3 keys connect, it is connected together by O-cardohydrata-peptide linkage and N-cardohydrata-peptide linkage and protein, by 5% ~ 20% protein, 80% ~ 90% seminose form its molecular mass and be about 20KDa ~ 200KDa.Mannosans has panimmunity function, can regulating intestinal canal colony balance, strengthen immunity, to combine or absorbing mycotoxin, anti-oxidant etc., have broad application prospects.
Yeast mannans is connected with protein with covalent linkage form in yeast cells wall, the mannosans that thick extraction obtains contains more protein, purity is lower, adopting appropriate means to reduce protein content is the key preparing high purity yeast mannans, at present, the method of removing protein mainly contains: Sevage method, trichloroacetic acid method and Calcium Chloride Method etc., but, there are the following problems for these methods: albumen clearance is high and polysaccharide retention rate is low, organic solvent is introduced and residual, aqueous phase system is inapplicable, is unfavorable for the lifting of yeast mannans purity.
The yeast mannans of domestic market is mainly the specification of content 20% ~ 50%, price is lower, and low-purity limits the widespread use of yeast mannans, therefore, research and develop a kind of method improving yeast mannans purity, for raising yeast mannans added value of product, meet the need of market and there is important economic benefit and social benefit.
The rheological characteristics of polysaccharide soln, for most important the food be employed, directly can have influence on the organoleptic quality of this food, so when developing food products will select a kind of colloid, the rheological characteristics of this colloid is the factor needing first to consider.The yeast mannans of different purity, its viscosity differences is comparatively large, different on the impact of application system, how to accomplish not change system rheological property, and the functional performance that can play yeast mannans just seems most important.
Summary of the invention
In order to solve problems of the prior art, the object of this invention is to provide a kind of preparation method of high purity yeast mannans.
In order to realize the object of the invention, technical scheme of the present invention is as follows:
The preparation method of a kind of low viscosity, high purity yeast mannans; with brewing yeast cell wall for raw material; adopt water extraction and alcohol precipitation method; obtain crude product yeast mannans; first through neutral protease enzymolysis; and under phosphoric acid buffer protection, carry out PRONASE A enzymolysis repeatedly centrifugal, reprocess and get final product.
The low viscosity of technical solution of the present invention gained, high purity yeast mannans product are the flocculence solid substance of white, fluffy.
Further, described preparation method specifically comprises the steps:
(1) with brewing yeast cell wall for raw material, adopt water extraction and alcohol precipitation method, obtain crude product yeast mannans I (purity is below 80%);
(2) adopt deionized water, step (1) gained crude product yeast mannans is mixed with solution;
(3) in step (2) gained solution, add neutral protease neutrase, carry out enzymolysis, after enzymolysis terminates, carry out going out enzyme, obtain the primary enzymolysis liquid of crude product yeast mannans;
(4) the primary enzymolysis liquid of step (3) gained is carried out centrifugal treating;
(5) get step (4) gained supernatant, add ethanol and carry out alcohol precipitation, centrifugal collecting precipitation, obtain crude product yeast mannans II;
(6) get step (5) gained precipitation, add phosphate buffered saline and become solution, adopt NaOH regulator solution pH value to 7.5 ~ 8.0;
(7) get step (6) gained solution, add PRONASE A, carry out enzymolysis, after enzymolysis terminates, carry out going out enzyme, obtain the secondary enzymolysis liquid of crude product yeast mannans;
(8) step (7) gained secondary enzymolysis liquid is carried out centrifugal treating;
(9) get step (8) gained supernatant, add ethanol and carry out alcohol precipitation, centrifugal collecting precipitation;
(10) adopt deionization to dissolve step (9) gained precipitation, stir complete to dispersion, described solution is adopted ultrafiltration desalination, collects gained trapped fluid;
(11) trapped fluid of step (10) gained is carried out spraying dry, obtain low viscosity, high purity yeast mannans III.
Wherein, in described step (1), Hot water extraction is adopted to extract crude product yeast mannans.
Wherein, in described step (2), adopt deionized water, step (1) gained crude product yeast mannans is mixed with the solution of 15%w/v.
Wherein, in described step (3), the addition of neutral protease is 0.05% of protein content in crude product yeast mannans, and hydrolysis temperature is 45 DEG C, and enzymolysis time is 2h.
Wherein, in described step (4), centrifugal rotational speed is 4000 ~ 5000rpm, and centrifugation time is 5 ~ 10min.
Wherein, in described step (5), the consumption of ethanol is that the diploid of gained supernatant amasss, and centrifugal rotational speed is 4000 ~ 5000rpm, and centrifugation time is 10 ~ 20min.
Wherein, in described step (6), described strength of solution is 15%w/v, and the concentration of phosphate buffered saline buffer is 0.02M ~ 0.2M, pH is 7.0; NaOH concentration is 0.01M.
Wherein, in described step (7), the addition of PRONASE A is 0.05% of protein content in crude product yeast mannans, and hydrolysis temperature is 40 DEG C, and enzymolysis time is 8h.
Wherein, in described step (8), centrifugal rotational speed is 4000 ~ 5000rpm, and centrifugation time is 5 ~ 10min.
Wherein, in described step (9), the consumption of ethanol is that the triploid of gained supernatant amasss, and centrifugal rotational speed is 4000 ~ 5000rpm, and centrifugation time is 10 ~ 20min.
Wherein, in described step (10), the condition that ultrafiltration adopts be respectively molecular weight retain for the ultra-filtration membrane of 10kDa, working pressure be 20 ~ 25psi, feed concentration is 4% ~ 6%, service temperature is normal temperature.
Wherein, in described step (11), spray drying treatment is carried out to trapped fluid.
Beneficial effect of the present invention is:
1, technological operation is easy, and by the method for gel filtration chromatography column purification before contrast, centrifugal by enzymolysis coupling, can improve the purity of yeast mannans fast, reduce the content of its protein, required time is short and a gained sample size is large.
2, the method that the present invention improves yeast mannans purity adopts phosphate buffered saline buffer to carry out the preparation of solution, ensures that the original structure of polysaccharide is not damaged in the process regulating pH to the full extent.
3, the present invention improves the centrifugal coupling of method employing enzymolysis of yeast mannans purity; Its albumen decreasing ratio is high, and polysaccharide loss is few.The impurity in original in solution and twice enzymolysis solution can be removed by centrifugal treating; improve the purity of sample to a greater extent; and PRONASE A enzymolysis required time can be reduced; relative to traditional Sevage method etc.; there is the advantages such as environmental protection, efficient, safety, be applicable to the requirement of large-scale production.
4, the present invention improves yeast mannans purity method application secondary enzymolysis, repeatedly centrifugal, ultrafiltration and spray drying technology combine, and the equipment adopted all can realize the needs of suitability for industrialized production.
5, in process of the present invention, relate to the yeast mannans of three kinds of purity altogether, higher with yeast mannans I and II viscosity, and be pseudoplastic fluid, and final gained yeast mannans viscosity is low, purity is tall and big in 90%, and be Newtonian fuid, can apply with food system, improve its functional property and do not affect the original rheological properties of system.
Accompanying drawing explanation
Fig. 1 is the schematic flow sheet of the preparation method of a kind of high purity yeast mannans of the present invention.
Fig. 2 is the rheological properties contrast of different purity yeast mannans solution in the embodiment of the present invention.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
The experimental technique used in following examples if no special instructions, is ordinary method.
Material used in following examples, reagent etc., if no special instructions, all can obtain from commercial channels.
Yeast cells wall in following examples is purchased from Zhejiang Senyo Biotech Co., Ltd..
The data processing of the every step of following examples all adopts DPS software to complete.
Embodiment 1
A preparation method for high purity yeast mannans, through following processing step (as shown in Figure 1):
(1) with brewing yeast cell wall for raw material, adopt water extraction and alcohol precipitation method, prepare crude product yeast mannans I (purity is 76.60%);
(2) to the crude product yeast mannans that step (1) obtains, add deionized water dissolving, be stirred to and be uniformly dispersed, make crude product yeast mannans solution ultimate density be 15% (w/v);
(3) enzymolysis processing is carried out with the solution of neutral protease neutrase to step (2) gained, the consumption of neutral protease neutrase is 0.05% of protein content in step (1) gained crude product yeast mannans, and hydrolysis temperature and time are respectively 45 DEG C, 2h;
(4) carry out centrifugal to gained enzymolysis solution in step (3), centrifugal rotational speed and time are respectively 4000rpm, 10min;
(5) get step (4) gained supernatant, add diploid and amass ethanol and carry out alcohol precipitation, the centrifugal 20min collecting precipitation of 4000rpm, obtains crude product yeast mannans II;
(6) get step (5) gained precipitation, the phosphate buffered saline adding 0.02M, pH7.0 becomes the solution of 15% (w/v), regulates pH to 7.8 with the NaOH of 0.01M;
(7) step (6) gained solution is got, add PRONASE A and carry out enzymolysis processing, the consumption of PRONASE A is 0.05% of protein content in the crude product yeast mannans of step (1) gained, and hydrolysis temperature and time are respectively 40 DEG C, 8h;
(8) carry out centrifugal to gained enzymolysis solution in step (7), centrifugal rotational speed and time are respectively 4000rpm, 20min;
(9) collect the supernatant in step (8) after centrifugal, the ethanol adding triploid long-pending carries out alcohol precipitation, centrifugal collecting precipitation, and centrifugal rotating speed and time are respectively 4000rpm, 20min;
(10) by the precipitation that deionized water dissolving step (9) is collected, adopt ultra-filtration technique desalination, working pressure is 20psi, and feed concentration is 4%, and service temperature is normal temperature.
(11) collect step (10) gained trapped fluid, carry out spray drying treatment, obtain the higher yeast mannans of purity III.
The yeast mannans that the present embodiment obtains is the flocculence solid substance of white, fluffy, after the centrifugal coupling processing of enzymolysis, the purity of the yeast mannans obtained can reach 90.20%, protein content 6.33%, compared with yeast mannans I, II, the purity of yeast mannans III obtains significant lifting, viscosity significantly declines (p < 0.05), and becomes Newtonian fuid (see Fig. 2) from pseudoplastic fluid.
Embodiment 2
A preparation method for high purity yeast mannans, through following processing step (as shown in Figure 1):
(1) with brewing yeast cell wall for raw material, adopt water extraction and alcohol precipitation method, prepare crude product yeast mannans I (purity is 77.75%);
(2) to the crude product yeast mannans that step (1) obtains, add deionized water dissolving, be stirred to and be uniformly dispersed, make crude product yeast mannans solution ultimate density be 15% (w/v);
(3) enzymolysis processing is carried out with the solution of neutral protease neutrase to step (2) gained, the consumption of neutral protease neutrase is 0.05% of protein content in step (1) gained crude product yeast mannans, and hydrolysis temperature and time are respectively 45 DEG C, 2h;
(4) carry out centrifugal to gained enzymolysis solution in step (3), centrifugal rotational speed and time are respectively 5000rpm, 5min;
(5) get step (4) gained supernatant, add diploid and amass ethanol and carry out alcohol precipitation, the centrifugal 10min collecting precipitation of 5000rpm, obtains crude product yeast mannans II;
(6) get step (5) gained precipitation, the phosphate buffered saline adding 0.2M, pH7.0 becomes the solution of 15% (w/v), regulates pH to 8.0 with the NaOH of 0.01M;
(7) step (6) gained solution is got, add PRONASE A and carry out enzymolysis processing, the consumption of PRONASE A is 0.05% of protein content in the crude product yeast mannans of step (1) gained, and hydrolysis temperature and time are respectively 40 DEG C, 8h;
(8) carry out centrifugal to gained enzymolysis solution in step (7), centrifugal rotational speed and time are respectively 5000rpm, 10min;
(9) collect the supernatant in step (8) after centrifugal, the ethanol adding triploid long-pending carries out alcohol precipitation, centrifugal collecting precipitation, and centrifugal rotating speed and time are respectively 5000rpm, 10min;
(10) by the precipitation that deionized water dissolving step (9) is collected, adopt ultra-filtration technique desalination, working pressure is 25psi, and feed concentration is 6%, and service temperature is normal temperature.
(11) collect step (10) gained trapped fluid, carry out spray drying treatment, obtain the higher yeast mannans of purity III.
The yeast mannans that the present embodiment obtains is the flocculence solid substance of white, fluffy, and after the centrifugal coupling processing of enzymolysis, the purity of the yeast mannans obtained can reach 93.65%, and protein content 5.07%, in table 1.Compared with yeast mannans I, II, the purity of yeast mannans III obtains significant lifting, viscosity significantly declines (p < 0.05), and becomes Newtonian fuid from pseudoplastic fluid.
The main component of table 1 yeast mannans
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (7)
1. a preparation method for low viscosity, high purity yeast mannans, is characterized in that, described preparation method specifically comprises the steps:
(1) with brewing yeast cell wall for raw material, adopt water extraction and alcohol precipitation method, obtain crude product yeast mannans;
(2) adopt deionized water, step (1) gained crude product yeast mannans is mixed with solution;
(3) in step (2) gained solution, add neutral protease, carry out enzymolysis, after enzymolysis terminates, carry out going out enzyme, obtain the primary enzymolysis liquid of crude product yeast mannans;
(4) the primary enzymolysis liquid of step (3) gained is carried out centrifugal treating;
(5) get step (4) gained supernatant, add ethanol and carry out alcohol precipitation, centrifugal collecting precipitation;
(6) get step (5) gained precipitation, add phosphate buffered saline and become solution, adopt NaOH regulator solution pH value to 7.5 ~ 8.0;
(7) get step (6) gained solution, add PRONASE A, carry out enzymolysis, after enzymolysis terminates, carry out going out enzyme, obtain the secondary enzymolysis liquid of crude product yeast mannans;
(8) step (7) gained secondary enzymolysis liquid is carried out centrifugal treating;
(9) get step (8) gained supernatant, add ethanol and carry out alcohol precipitation, centrifugal collecting precipitation;
(10) adopt deionization to dissolve step (9) gained precipitation, stir complete to dispersion, described solution is adopted ultrafiltration desalination, collects gained trapped fluid;
(11) trapped fluid of step (10) gained is carried out spraying dry, obtain low viscosity, high purity yeast mannans;
Wherein, in described step (3), the addition of neutral protease is 0.05% of protein content in crude product yeast mannans, and hydrolysis temperature is 45 DEG C, and enzymolysis time is 2h;
In described step (6), described strength of solution is 15%w/v, and the concentration of phosphate buffered saline buffer is 0.02M ~ 0.2M, pH is 7.0; NaOH concentration is 0.01M;
In described step (7), the addition of PRONASE A is 0.05% of protein content in crude product yeast mannans, and hydrolysis temperature is 40 DEG C, and enzymolysis time is 8h.
2. preparation method according to claim 1, is characterized in that, in described step (2), adopts deionized water, step (1) gained crude product yeast mannans is mixed with the solution of 15%w/v.
3. preparation method according to claim 1, is characterized in that, in described step (4), centrifugal rotational speed is 4000 ~ 5000rpm, and centrifugation time is 5 ~ 10min.
4. preparation method according to claim 1, is characterized in that, in described step (5), the consumption of ethanol is that the diploid of gained supernatant amasss, and centrifugal rotational speed is 4000 ~ 5000rpm, and centrifugation time is 10 ~ 20min.
5. preparation method according to claim 1, is characterized in that, in described step (8), centrifugal rotational speed is 4000 ~ 5000rpm, and centrifugation time is 5 ~ 10min.
6. preparation method according to claim 1 or 5, is characterized in that, in described step (9), the consumption of ethanol is that the triploid of gained supernatant amasss, and centrifugal rotational speed is 4000 ~ 5000rpm, and centrifugation time is 10 ~ 20min.
7. preparation method according to claim 6, it is characterized in that, in described step (10), the condition that ultrafiltration adopts be respectively molecular weight retain for the ultra-filtration membrane of 10kDa, working pressure be 20 ~ 25psi, feed concentration is 4% ~ 6%, service temperature is normal temperature.
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| CN103570840A (en) * | 2012-07-26 | 2014-02-12 | 宜兴市江山生物科技有限公司 | Yeast polysaccharide separation and purification method |
| CN103613682A (en) * | 2013-11-19 | 2014-03-05 | 济南大学 | Method for preparing yeast glucan and coproducing mannan and trehalose |
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| CN103613682A (en) * | 2013-11-19 | 2014-03-05 | 济南大学 | Method for preparing yeast glucan and coproducing mannan and trehalose |
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