CN102643360B - Extraction and separation method of natto polysaccharide - Google Patents
Extraction and separation method of natto polysaccharide Download PDFInfo
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- CN102643360B CN102643360B CN201210115188.5A CN201210115188A CN102643360B CN 102643360 B CN102643360 B CN 102643360B CN 201210115188 A CN201210115188 A CN 201210115188A CN 102643360 B CN102643360 B CN 102643360B
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Abstract
The invention relates to an extraction and separation method of natto polysaccharide, which comprises the following steps: degreasing soybeans, preparing the finished natto product, carrying out microwave acid extraction, deproteinizing by a proteinase method, removing protein polypeptide, depositing with anhydrous alcohol, and drying to obtain the refined natto polysaccharide product. The finished natto product is dissolved in alcohol to be degreased, and is subjected to dilute acid water solution and microwave extraction; the proteinase method is utilized for deproteinization; and concentratation, filtration, deposition with 95% ethanol, drying and the like are carried out to obtain the natto polysaccharide product. The natto polysaccharide produced by the method provided by the invention has the advantages of lower production cost, higher product yield, high safety and reliability, no pollution and wide application range.
Description
Technical field
The invention belongs to probiotics fermention technical field, relate to a kind of simple process, consuming time short, cost is lower, polysaccharide extract rate is high, and product purity, color and luster be good, it is antibacterial, antiviral to have, and strengthens the functions such as immunity, and its biological applications is extraction and the separation method of natto polysaccharide widely.
Background technology
Studies confirm that in a large number, biological polyoses has multiple physiologically active, comprises and reduces body blood sugar and blood lipid level, suppress tumour and strengthen the functions such as immunity, also being important informational molecule in body, is one of developing direction of following physiological regulation agent, has been subject to extensive concern and great attention.At present, scientific worker, for the research of the polysaccharide such as glossy ganoderma, ginseng, the root of large-flowered skullcap, matrimony vine and mushroom, has obtained breakthrough progress both at home and abroad.
Report is pointed out, natto polysaccharide can be served as natural immunopotentiating agent, can enhancing body cellular immunization, bring into play anticancer, antivirus action, in promoting, environment and humoral immunization are stable, also can be used for the daily health caring of man and animal, resisting oxidation and delaying senility, and the field such as antiviral assisting therapy and beauty and skin care, therefore rely on its unique and complex physiological function to become gradually the study hotspot of the subjects such as trophology, physiology, immunology.At present, about the production of natto polysaccharide because of its complicated component, physico-chemical property special, there is no the synthetic maturation method of chemical industry, comparatively desirable approach is to be raw material by finished product natto, extraction with bioactive polysaccharide is realized, this method utilizes water to extract polysaccharide as solvent, and the composition obtaining is mainly neutral Alkali-soluble polysaccharides, and has the defects such as extraction yield is low, purity difference.
Summary of the invention
For overcoming above-mentioned technical disadvantages, the invention provides a kind of simple process, consuming time short, cost is lower, polysaccharide extract rate is high, and product purity, color and luster be good, it is antibacterial, antiviral to have, and strengthens the functions such as immunity, and its biological applications is extraction and the separation method of natto polysaccharide widely.
The present invention solves the technological method that its technical problem adopts: a kind of extraction of natto polysaccharide and separation method, and its processing step is:
The first step is soybean degreasing: high quality soybean is cleaned removal of impurities, eluriates post-drying, fragmentation, is soybean by volume: sherwood oil is that 1:3 adds sherwood oil, and room temperature degreasing 24h, to remove lipid component, crosses leaching filter residue, retains for subsequent use after the volatilization of unrecovered oil ether;
Second step is the preparation of finished product natto: soak 3～6 hours through the first step gained filter residue water purification, 110～120 DEG C of temperature, under vapor pressure 0.05～0.11Mpa condition, carry out cooking disinfection 20～60min, then be cooled to 38～45 DEG C, mother's kind amount inoculation Bacillus natto that concentration is 1% by mass percentage, and be placed in 36～45 DEG C, relative air humidity is greater than in 72% incubator and ferments after 16～24h, then it is stand-by to put under 4 DEG C of conditions passivation refrigeration;
The 3rd step is microwave acid formulation: after the making beating of finished product natto, add the HCl solution of 0.08mol/L taking volume ratio as 1:8, be placed under the microwave condition of 280W, low fire and extract 15min, cooled and filtered, obtains Crude polysaccharides concentrated solution;
The 4th step is protease method deproteinated: regulate pH=7.0, adopt the water-soluble foreign protein in protease hydrolysis Crude polysaccharides concentrated solution, proteolytic enzyme consumption is mass percent 1%, press after 50 DEG C of Water Under solution 1h of constant temperature water bath, bath temperature is risen to 90 DEG C, maintain 8min and make proteolytic enzyme inactivation, stop enzyme reaction;
The 5th step is to remove protein polypeptide: adopting volume ratio is 80% ethanolic soln of 5 times of amounts, at 50 DEG C, extract the protein polypeptide 30min that upper step reaction produces, then temperature is soaked and is spent the night, and next day is the centrifugal 10min of speed with 4000r/min by sample, and polysaccharide is fully precipitated;
The 6th step is dehydrated alcohol precipitation, dry: in the precipitation obtaining through the 5th step, add 2 dehydrations of dehydrated alcohol of 4 times of volumes, cross filter cake and wash 2 times with acetone, ether, spread out on plate, 50 DEG C of negative pressure are dried, and obtain refining natto polysaccharide product.
The invention has the beneficial effects as follows: applied microwave acid formulation technology of the present invention, slough in advance soybean oil, avoid it in subsequent handling, to separate out the increase that hinders polysaccharide extract rate; Dilute acid solution and microwave treatment are conducive to release and the extraction of natto polysaccharide; And improved impurity removal process, and reduce the subtractive process of natto pigment, greatly shortened extraction time compared with traditional formulation, reduced production cost, and products obtained therefrom purity is high, color and luster good; Agents useful for same of the present invention is all within safe dose scope, and extraction effect is comparatively desirable, can not cause the secondary pollution in natto polysaccharide leaching process, introduces ectogenic objectionable impurities to product; In addition, alcohol solvent used can reuse, and greatly reduces productive expense; Technical process of the present invention is simple, and equipment is easy to get, workable, is convenient to realize production practice; The natto slag that natto polysaccharide produces in producing, is rich in the nutritive ingredient such as albumen and trace element, and safety non-pollution, can be used as livestock biological feed to be used, and is convenient to cost recovery; Gained natto polysaccharide can add other function ingredients, as VITAMIN, amino acid or flavonoid etc., to improve the compound action of its health care and treatment, also can capsule or the form such as tablet be developed to multiple physiology and adjust preparation.
Adopt finished product natto after alcohol solution-off fat, with dilute acid solution and microwave method extract, again through protease method deproteinated, thickening filtration, 95% ethanol precipitation, drying and other steps, obtain natto polysaccharide goods, produce natto polysaccharide with the present invention, not only production cost is slightly low, product yield is higher, and safe and reliable, green non-pollution, its purposes is very extensive.
Brief description of the drawings
Fig. 1 is technological process block-diagram of the present invention.
embodiment
Below in conjunction with embodiment, the present invention is further described.
Referring to Fig. 1, a kind of extraction of natto polysaccharide and separation method, its processing step is:
The first step is soybean degreasing: high quality soybean is cleaned removal of impurities, eluriates post-drying, fragmentation, is soybean by volume: sherwood oil is that 1:3 adds sherwood oil, and room temperature degreasing 24h, to remove lipid component, crosses leaching filter residue, retains for subsequent use after the volatilization of unrecovered oil ether;
Second step is the preparation of finished product natto: soak 3～6 hours through the first step gained filter residue water purification, 110～120 DEG C of temperature, under vapor pressure 0.05～0.11Mpa condition, carry out cooking disinfection 20～60min, then be cooled to 38～45 DEG C, mother's kind amount inoculation Bacillus natto that concentration is 1% by mass percentage, and be placed in 36～45 DEG C, relative air humidity is greater than in 72% incubator and ferments after 16～24h, then it is stand-by to put under 4 DEG C of conditions passivation refrigeration;
The 3rd step is microwave acid formulation: after the making beating of finished product natto, add the HCl solution of 0.08mol/L taking volume ratio as 1:8, be placed under the microwave condition of 280W, low fire and extract 15min, cooled and filtered, obtains Crude polysaccharides concentrated solution;
The 4th step is protease method deproteinated: regulate pH=7.0, adopt the water-soluble foreign protein in protease hydrolysis Crude polysaccharides concentrated solution, proteolytic enzyme consumption is mass percent 1%, press after 50 DEG C of Water Under solution 1h of constant temperature water bath, bath temperature is risen to 90 DEG C, maintain 8min and make proteolytic enzyme inactivation, stop enzyme reaction;
The 5th step is to remove protein polypeptide: adopting volume ratio is 80% ethanolic soln of 5 times of amounts, at 50 DEG C, extract the protein polypeptide 30min that upper step reaction produces, then temperature is soaked and is spent the night, and next day is the centrifugal 10min of speed with 4000r/min by sample, and polysaccharide is fully precipitated;
The 6th step is dehydrated alcohol precipitation, dry: in the precipitation obtaining through the 5th step, add 2 dehydrations of dehydrated alcohol of 4 times of volumes, cross filter cake and wash 2 times with acetone, ether, spread out on plate, 50 DEG C of negative pressure are dried, and obtain refining natto polysaccharide product.
Embodiment mono-
The first step is soybean degreasing: high quality soybean 500g is cleaned to removal of impurities, clear water elutriation post-drying, fragmentation, inject the sherwood oil of 1.5 liters, room temperature lixiviate 24h, to extract lipid component, filters and obtains filter residue, treats that sherwood oil volatilization retention is for subsequent use;
Second step is to prepare finished product natto: filter residue soaks 4h, with 110 DEG C of temperature, vapor pressure 0.08 Mpa, carry out cooking disinfection 30min, then be cooled to 40 DEG C, female amount inoculation Bacillus natto of planting by 1%, and be placed in 40 DEG C, in the incubator of relative air humidity 80%, ferment after 20h, be placed in 4 DEG C of refrigerator passivation and refrigerate stand-by;
The 3rd step is that microwave acid is carried after filter residue making beating homogeneous, adds 4.0 liters of the diluted hydrochloric acid aqueous solutions of 0.08mol/L, is placed in the low fire of 280W() microwave method extract 15min, cooled and filtered, is evaporated to 1/5 volume, obtains Crude polysaccharides concentrated solution; Filter residue is for the preparation of biological fodder;
The 4th step is that protease method deproteinated regulates pH=7.0, adopts the water-soluble foreign protein in papain hydrolysis Crude polysaccharides concentrated solution, proteolytic enzyme consumption 1%, be placed in after 50 DEG C of Water Under solution 1h of constant temperature water bath, bath temperature is risen to 90 DEG C, maintain 8min and make proteolytic enzyme inactivation, stop enzyme reaction;
The 5th step is to remove protein polypeptide to add dehydrated alcohol, reach 80% ethanolic soln to its final concentration, at 50 DEG C, extract the protein polypeptide 30min that upper step reaction produces, then temperature is soaked hold over night, make polysaccharide precipitation complete, be placed in again the centrifugal 10min of speed with 4000r/min in whizzer, filter to obtain polysaccharide precipitation;
The 6th step is to add 2.0 liters of dehydrated alcohols to divide 2 dehydrations in ethanol precipitation, dry upper step precipitation, crosses filter cake and washes 2 times with acetone, ether, spreads out on plate, and 50 DEG C of negative pressure are dried, and obtain refining natto polysaccharide product.
The 7th step is that finished product is preserved: dried natto polysaccharide pulverized, obtained Powdered natto polysaccharide products 5.1g, pack the moistureproof preservation of lucifuge in encloses container into, and for subsequent use.
Embodiment bis-
The first step is soybean degreasing: high quality soybean 500g is cleaned to removal of impurities, clear water elutriation post-drying, fragmentation, inject the sherwood oil of 1.8 liters, room temperature lixiviate 24h, to extract lipid component, filters and obtains filter residue, treats that sherwood oil volatilization retention is for subsequent use;
Second step is to prepare finished product natto: filter residue soaks 5h, with 110 DEG C of temperature, vapor pressure 0.09 Mpa, carry out cooking disinfection 25min, then be cooled to 38 DEG C, female amount inoculation Bacillus natto of planting by 1%, and be placed in 38 DEG C, in the incubator of relative air humidity 80%, ferment after 24h, be placed in 4 DEG C of refrigerator passivation and refrigerate stand-by;
The 3rd step is that microwave acid is carried: in filter residue, add 4.0 liters of the diluted hydrochloric acid aqueous solutions of 0.08mol/L, be placed in the low fire of 280W() microwave method extract 15min, cooled and filtered, filter residue repeats microwave acid and carries, merging filtrate, is evaporated to 1/5 volume, obtains Crude polysaccharides concentrated solution; Filter residue is for the preparation of biological fodder;
The 4th step is protease method deproteinated: regulate pH=7.0, adopt the water-soluble foreign protein in papain hydrolysis Crude polysaccharides concentrated solution, proteolytic enzyme consumption 1%, be placed in after 50 DEG C of Water Under solution 1h of constant temperature water bath, bath temperature is risen to 90 DEG C, maintain 8min and make proteolytic enzyme inactivation, stop enzyme reaction;
The 5th step is to remove protein polypeptide: add dehydrated alcohol, reach 80% ethanolic soln to its final concentration, at 50 DEG C, extract the protein polypeptide 30min that upper step reaction produces, then temperature is soaked hold over night, make polysaccharide precipitation complete, be placed in again the centrifugal 10min of speed with 4000r/min in whizzer, filter to obtain polysaccharide precipitation;
The 6th step is ethanol precipitation, dry: in upper step precipitation, add 2.0 liters of dehydrated alcohols to divide 2 dehydrations, cross filter cake and wash 2 times with acetone, ether, spread out on plate, 50 DEG C of negative pressure are dried, and obtain refining natto polysaccharide product.
The 7th step is that finished product is preserved: dried natto polysaccharide pulverized, obtained Powdered natto polysaccharide products 5.3g, pack the moistureproof preservation of lucifuge in encloses container into, and for subsequent use.
Applied microwave acid formulation technology of the present invention, sloughs soybean oil in advance, avoids it in subsequent handling, to separate out the increase that hinders polysaccharide extract rate; Dilute acid solution and microwave treatment are conducive to release and the extraction of natto polysaccharide; And improved impurity removal process, and reduce the subtractive process of natto pigment, greatly shortened extraction time compared with traditional formulation, reduced production cost, and products obtained therefrom purity is high, color and luster good; Agents useful for same of the present invention is all within safe dose scope, and extraction effect is comparatively desirable, can not cause the secondary pollution in natto polysaccharide leaching process, introduces ectogenic objectionable impurities to product; In addition, alcohol solvent used can reuse, and greatly reduces productive expense; Technical process of the present invention is simple, and equipment is easy to get, workable, is convenient to realize production practice; The natto slag that natto polysaccharide produces in producing, is rich in the nutritive ingredient such as albumen and trace element, and safety non-pollution, can be used as livestock biological feed to be used, and is convenient to cost recovery; Gained natto polysaccharide can add other function ingredients, as VITAMIN, amino acid or flavonoid etc., to improve the compound action of its health care and treatment, also can capsule or the form such as tablet be developed to multiple physiology and adjust preparation.
The present invention adopts finished product natto after alcohol solution-off fat, with dilute acid solution and microwave method extract, again through protease method deproteinated, thickening filtration, 95% ethanol precipitation, drying and other steps, obtain natto polysaccharide goods, produce natto polysaccharide with the present invention, not only production cost is slightly low, product yield is higher, and safe and reliable, green non-pollution, its purposes is very extensive.
Claims (1)
1. the extraction of natto polysaccharide and a separation method, is characterized in that processing step is:
The first step is soybean degreasing: high quality soybean is cleaned removal of impurities, eluriates post-drying, fragmentation, is soybean by volume: sherwood oil is that 1:3 adds sherwood oil, and room temperature degreasing 24h, to remove lipid component, crosses leaching filter residue, retains for subsequent use after the volatilization of unrecovered oil ether;
Second step is the preparation of finished product natto: soak 3～6 hours through the first step gained filter residue water purification, 110～120 DEG C of temperature, under vapor pressure 0.05～0.11Mpa condition, carry out cooking disinfection 20～60min, then be cooled to 38～45 DEG C, mother's kind amount inoculation Bacillus natto that concentration is 1% by mass percentage, and be placed in 36～45 DEG C, relative air humidity is greater than in 72% incubator and ferments after 16～24h, then it is stand-by to put under 4 DEG C of conditions passivation refrigeration;
The 3rd step is microwave acid formulation: after the making beating of finished product natto, add the HCl solution of 0.08mol/L taking volume ratio as 1:8, be placed under the microwave condition of 280W, low fire and extract 15min, cooled and filtered, obtains Crude polysaccharides concentrated solution;
The 4th step is protease method deproteinated: regulate pH=7.0, adopt the water-soluble foreign protein in protease hydrolysis Crude polysaccharides concentrated solution, proteolytic enzyme consumption is mass percent 1%, press after 50 DEG C of Water Under solution 1h of constant temperature water bath, bath temperature is risen to 90 DEG C, maintain 8min and make proteolytic enzyme inactivation, stop enzyme reaction;
The 5th step is to remove protein polypeptide: adopting volume ratio is 80% ethanolic soln of 5 times of amounts, at 50 DEG C, extract the protein polypeptide 30min that upper step reaction produces, then temperature is soaked and is spent the night, and next day is the centrifugal 10min of speed with 4000r/min by sample, and polysaccharide is fully precipitated;
The 6th step is dehydrated alcohol precipitation, dry: in the precipitation obtaining through the 5th step, add 2 dehydrations of dehydrated alcohol of 4 times of volumes, cross filter cake and wash 2 times with acetone, ether, spread out on plate, 50 DEG C of negative pressure are dried, and obtain refining natto polysaccharide product.
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| CN106387676A (en) * | 2016-09-19 | 2017-02-15 | 江苏师范大学 | Preparation method of active natto powder |
| CN106721047A (en) * | 2016-11-25 | 2017-05-31 | 南宁学院 | A kind of method that edible fungi leftovers prepare multifunctional feed |
| TW201822778A (en) * | 2016-12-30 | 2018-07-01 | 天明製藥股份有限公司 | A method of extracting polysaccharides from inonotus obliquus |
| CN108056174A (en) * | 2018-01-02 | 2018-05-22 | 中国农业科学院农产品加工研究所 | Health-care instant soybean milk powder and preparation method thereof |
| CN108410767B (en) * | 2018-03-19 | 2021-02-23 | 青岛大学 | Bacillus natto and application thereof in preparation of active polysaccharide by fermenting Ruditapes philippinarum |
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