CN1236067C - Preparation process of rise bran polysaccharose kind active component - Google Patents
Preparation process of rise bran polysaccharose kind active component Download PDFInfo
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- CN1236067C CN1236067C CN 200410014267 CN200410014267A CN1236067C CN 1236067 C CN1236067 C CN 1236067C CN 200410014267 CN200410014267 CN 200410014267 CN 200410014267 A CN200410014267 A CN 200410014267A CN 1236067 C CN1236067 C CN 1236067C
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Abstract
The present invention relates to a preparing method of an active component of rice bran polysaccharide, which belongs to the technical field of rice refining processing. In the present invention, defatted rice bran is used as a raw material; polysaccharide is extracted by hot water, starch and protein are removed by composite enzymolysis, and phytate is removed by calcium hydroxide treatment; then coarse rice bran polysaccharide (RBP) is prepared with total saccharide content of 85.4% and yield of 2.82%; continuously, gel filtration chromatography and ion-exchange chromatography are adopted to separate and purify the coarse rice bran polysaccharide (RBP) to obtain an RBP-M active component. Animal experiments adopted for studying the immunological regulation activity of the rice bran polysaccharide (RBP) and RBP-M find that the rice bran polysaccharide (RBP) and RBP-M have obvious reinforcing functions at the aspects of cell immunity, humoral immunity and nonspecific immunity of mice.
Description
Technical field
A kind of preparation method of rice bran polysaccharide class active ingredient belongs to rice intensive processing technical field.
Background technology
Paddy just obtains brown rice after rice huller paddy is removed rice husk, rice bran then is the mixture that is reached a small amount of rice embryo and crack rice by the cortex under grinding in the brown rice pearling process.The main component of rice bran is carbohydrate, fat and protein, and contains more ash content and VITAMIN.The quality of rice bran and the quantity of each nutritive ingredient depend on the degree of endosperm fragmentation in the precision of husk rice and the rice milling process.
Rice bran generally accounts for 6～8% of rice gross weight, but it includes the important nutritive ingredient and the 90% above necessary element of human body of rice 64%.The United Nations Industrial Development Organization (UNIDO) is referred to as a kind of resource of underusing (an under-utilized raw material) to the rice bran of the third world.Rice bran is described as " nutrition source of being bestowed by heaven " abroad.Along with the progress of modern science and technology, the rice bran whole body all is that precious secret is disclosed.Many in the world major companies are very interested for the extraction enrichment of functional ingredient in the rice bran.According to incompletely statistics, the patent of disclosed up to now relevant rice bran deep processing has more than 50.
Polysaccharide is one of four big base substances that constitute life as from the natural high moleculer eompound in higher plant, animal cell membrane and the microorganism wall.People only notice it as the importance of energy substance and the moiety of cell for the early stage research of carbohydrate, and just people have just had further understanding to the biological activity of polysaccharide in recent decades.Polysaccharide has multiple biological activity, and is closely related with keeping biological function.Glycoprotein, lipopolysaccharides that polysaccharide and protein, lipid form play a part can not be ignored at aspects such as cell recognition, cell growth, differentiation, metabolism, fetal development, cell carcinogenesis, virus infection, immunne responses.Since late 1950s it is found that fungus polysaccharide has anti-tumor activity, STUDY ON POLYSACHAROSE was subjected to attention more and more widely.Over nearly 20 years, because the develop rapidly of subjects such as biology, chemistry, people have had more and more deep understanding to the active function of polysaccharide and mixture thereof.Polysaccharide is being brought into play biological action at aspects such as antitumor, immunostimulant, hypoglycemic, antiviral, anti-ageing, anti-inflammatory, anticoagulations.
There is polytype polysaccharide in the rice bran, rice bran polysaccharide (RBP) is the heteroglycan of a class formation complexity, be made up of rhamnosyl, pectinose, wood sugar, seminose, glucose, semi-lactosi etc., main polysaccharide has lipopolysaccharides, araboxylan and dextran etc.The rice bran active polysaccharide not only has the physiological function of general polysaccharides, but also having the function of control old cardiovascular diseases such as stronger raising blood serum tumor necrosin level, enhancing body immunologic function and hypoglycemic, hypotensive, decreasing cholesterol, this is confirmed by scientific circles and medical circle.The rice bran active polysaccharide is considered to have and the similar effects of herbal medicine polysaccharide such as genseng, Radix Angelicae Sinensis, and rice bran aboundresources and cheap is fit to development and use very much.
Summary of the invention
The purpose of this invention is to provide a kind of preparation method of rice bran polysaccharide class active ingredient, defatted rice bran is fully utilized, improve the agricultural byproducts added value.
Technical scheme of the present invention: with the byproduct defatted rice bran in the Rice pollard oil production is raw material, with hot water extraction water-soluble polysaccharide wherein, remove starch with amylase and saccharifying enzyme enzymolysis, remove protein with the protease hydrolyzed reaction, with calcium hydroxide precipitate and separate phytate, make rough rice bran polysaccharide RBP product through ultrafiltration and concentration, ethanol sedimentation again; Or RBP proceeded Sepharose CL-6B gel filtration chromatography, and get the RBP-a component, again RBP-a is carried out DEAE Sepharose Fast Flow ion-exchange chromatography, obtain the main immunocompetence component of RBP-M.
(1) extruding puffing is to the influence of rice bran polysaccharide extraction
Rice bran is carried out extruding puffing handle effectively passivation lipase wherein, thus the physico-chemical property of stabilising rice bran.Particularly adopt extruding-puffing technique also can improve the content of soluble-carbohydrate in the rice bran, can improve the yield of rice bran polysaccharide thus.So the defatted rice bran that we adopt the process extruding puffing to handle is a raw material.
(2) material-water ratio is to the influence of polysaccharide yield
Polysaccharide yield increases along with the increase of material-water ratio, but its increase degree is very little; But less than 1: 6 o'clock, thicker being difficult to of the mixed system of rice bran and water stirred at material-water ratio, was 1: 6～1: 20 so adopt material quality ratio.
(3) optimization of hot water extraction processing condition
Hot water extraction: material quality ratio is 1: 6～1: 20, extracts 60～95 ℃ of temperature, 1～5 hour extraction time, pH value 5.0.Adopt response surface analysis method (RAS) to seek optimal processing parameter, composite design makes the yield of rice bran polysaccharide for best.Optimizing processing condition is 80 ℃ of temperature, 1.5 hours time, pH value 5.0.Amplify 5 times under these processing condition, revision test 2 times is carried out the extraction of rice bran polysaccharide, and average yield is 2.82%.
(4) protein is removed in the protease hydrolyzed reaction
Except that containing a large amount of polysaccharide (67.2%), also contain a certain amount of protein (17.4%) and other non-sugared impurity (mainly being the phytic acid salt) among the RBP.Being used for polysaccharide at present has except that proteic main method: Sevage method, Freon 113 method, phynol method, isoelectric point precipitation, trichloroacetic acid method, propyl carbinol method and enzyme process.Above relatively several method, it is then better relatively that enzyme process is removed albumen technology, can finish the reservation of proteinic removal and glucide preferably.Choose enzyme dosage, temperature, time, four factors of pH value and carry out optimization of process conditions: enzyme dosage is the 0.5-1.5% of concentration of substrate, temperature is 40-60 ℃, water-bath 1-2.5h is an appropriate processing condition, and this moment, protein percentage was 2.75%, and proteinic removal percentage reaches 85%.
(5) Ca (OH)
2The precipitate and separate phytate
With 10%Ca (OH)
2The primary sedimentation method is removed the phytic acid in the extracting solution, constantly stirs, and constantly adds 10%Ca (OH)
2Solution is regulated extracting liquid pH value to neutral, stirs 0.5-1 hour.Because the pH value of extracting solution system is about 5, so with 10%Ca (OH)
2Regulate pH to neutral.Since milk of lime be dissolved with a process, its alkalescence discharges slowly, often makes the pH value that has just mixed up moments later change again (rising), cause the technology instability, difficult control, for this reason, we have investigated churning time to the sedimentary influence of phytic acid with the variation of (phytic acid) phosphorus content in the extracting solution.Churning time increases to 50min from 10min, and total phosphorous reduces significantly in the extracting solution, and it is remarkable that promptly phytic acid is precipitated as the effect of phytic acid ca, after the 50min, the phytic acid precipitation fully, control was stirred 0.5-1 hour, was the best to select 50min especially.Through the depositing technology of milk of lime, ash oontent reduces to 1.3% by 14% in the rough rice bran polysaccharide (RBP), and it is about 92% that the ash content clearance reaches, and RBP purity is improved.
(6) precipitating liquid ethanol mass concentration is to the influence of rice bran polysaccharide yield
The general trend of polysaccharide yield is to increase along with the increase of precipitating liquid ethanol massfraction, to gradually slow more than 60%, this is relevant with the polysaccharide molecular weight size, the large molecular weight polysaccharides solvability is relatively poor, promptly precipitable in the ethanol of lower concentration, the small molecular weight polysaccharide then will could precipitate under higher alcohol concn, and part monose and oligosaccharides can not precipitate, and also can also be dissolved in wherein even alcohol concn reaches 90%.In extraction process, we control the precipitating final concentration is the precipitation that the ethanol of massfraction 60-80% carries out polysaccharide.
(7) RBP gel filtration chromatography
Adopt Sepharose CL-6B gel-filtration column that the RBP concentrated solution is carried out grading purification, NaCl solution with 0.2mmol/L carries out wash-out, automatically part is collected, and can fractional separation become three main ingredient: RBP-a, RBP-b and RBP-c, and wherein the RBP-a activity is the most remarkable.
(8) RBP-a ion-exchange chromatography
The concentrated solution of RBP-a is added to DEAE Sepharose Fast Flow ion-exchange chromatography, the ionic strength linear gradient elution, automatically part is collected, can fractional separation become four main ingredient: RBP-L, RBP-M, RBP-N and RBP-O, the external activity detected result shows that content higher composition RBP-M activity is the most remarkable.
Beneficial effect of the present invention: have both at home and abroad with full-cream fresh rice bran and make raw material, soak the open source literature report that extracts rice bran polysaccharide with boiling water, the existing open source literature report that organic solvent deposits such as ammonium sulfate or ethanol extract rice bran polysaccharide, the open source literature report of hypoglycemic activity, hypolipidemic activity and the antitumor activity of also existing research rice bran polysaccharide of adding; But the present invention is raw material with the defatted rice bran, adopts hot water extraction (water temperature is lower than boiling point), complex enzyme hydrolysis, calcium hydroxide treatment process, and the protein removal rate reaches 85%, and the ash content clearance reaches about 92%; Adopt gel filtration chromatography and ion-exchange chromatography separation and purification, obtain active ingredient RBP-M.The main monose of RBP-M consists of glucose, rhamnosyl, pectinose etc.; Structure activity study shows that the activity of RBP-M has nothing to do with protein, lipopolysaccharides, phenolic acid mainly by the polysaccharide partial contribution; Adopt the immunoregulatory activity of animal experiment study RBP and RBP-M, prove cellular immunization, humoral immunization and the non-specific immunity activity of the low mouse of its department's raise immunity.Do not see identical therewith open source literature, achievement or patent report both at home and abroad.
Description of drawings
Accompanying drawing is a rice bran polysaccharide class active ingredient preparation technology schema.
Embodiment
Embodiment 1
Get the 1000g defatted rice bran, add 10L distilled water, be heated to 85 ℃, stir to extract 2 hours, centrifugal 20 minutes of 2500rpm adds amylase (0.5%) enzyme digestion reaction half an hour under 85 ℃ of conditions, be cooled to 65 ℃, transfer pH to 4.0 to add the saccharifying enzyme enzymolysis and remove starch, be cooled to 45 ℃ again and add proteolytic enzyme (1.5%) enzyme digestion reaction removal in 1 hour protein, add 10%Ca (OH)
2Readjustment pH is extremely neutral, stirs 1 hour, and centrifugation separates phytate, and supernatant liquor is through ultrafiltration and concentration, and 4 times of alcohol precipitations leave standstill, and are centrifugal, concentrates after precipitation is redissolved or through rotary evaporation, and vacuum or spraying drying obtain RBP; Or the further separation and purification in precipitation redissolution back obtains RBP-M.
Embodiment 2
Get the 1000g defatted rice bran, add 20L distilled water, be heated to 95 ℃, stir and extracted 1.5 hours, centrifugal 20 minutes of 2500rpm adds amylase (0.5%) enzyme digestion reaction 1 hour under 85 ℃ of conditions, be cooled to 65 ℃, transfer pH to 4.0 to add the saccharifying enzyme enzymolysis and remove starch, be cooled to 40 ℃ again and add proteolytic enzyme (1.5%) enzyme digestion reaction removal in 1.5 hours protein, add 10%Ca (OH)
2Readjustment pH is extremely neutral, stirs 0.5 hour, and centrifugation separates phytate, and supernatant liquor is through ultrafiltration and concentration, and 4 times of alcohol precipitations leave standstill, and are centrifugal, concentrates after precipitation is redissolved or through rotary evaporation, and vacuum or spraying drying obtain RBP; Or the further separation and purification in precipitation redissolution back obtains RBP-M.
Embodiment 3
Get RBP concentrated solution (8mg/ml) 10ml, add Sepharose CL-6B gel-filtration column,
NaCl solution with 0.2mmol/L carries out wash-out, and part is collected automatically, obtains three component: RBP-a (38mg), RBP-b (18mg) and RBP-c (20mg), and wherein the RBP-a activity is the most remarkable.Concentrated solution (20mg/ml) 20ml of RBP-a is added to DEAE Sepharose Fast Flow ion-exchange chromatography, the ionic strength linear gradient elution, automatically part is collected, can fractional separation become four main ingredient: RBP-L (58mg), RBP-M (219), RBP-N (88mg) and RBP-O (16.5mg), the external activity detected result shows that content higher composition RBP-M activity is the most remarkable.
Claims (4)
1. the preparation method of a rice bran polysaccharide class active ingredient, it is characterized in that with the byproduct defatted rice bran in the Rice pollard oil production be raw material, with hot water extraction water-soluble polysaccharide wherein, remove starch with amylase and saccharifying enzyme enzymolysis, remove protein with the protease hydrolyzed reaction, with calcium hydroxide precipitate and separate phytate, make rough rice bran polysaccharide RBP product through ultrafiltration and concentration, ethanol sedimentation again; Or RBP proceeded Sepharose CL-6B gel filtration chromatography, and get the RBP-a component, again RBP-a is carried out DEAE Sepharose Fast Flow ion-exchange chromatography, obtain the main immunocompetence component of RBP-M;
Hot water extraction, material quality ratio is 1: 6～1: 20, extracts 60～95 ℃ of temperature, 1～5 hour extraction time, pH value 5.0;
The protease hydrolyzed reaction, enzyme dosage is 0.5%～1.5% of a concentration of substrate, 40～60 ℃ of hydrolysis temperatures, enzymolysis time 1～2.5 hour;
Separate phytate, with 10%Ca (OH)
2The precipitator method are removed the phytic acid in the extracting solution, constantly stir, and constantly add 10%Ca (OH)
2Solution is regulated extracting liquid pH value to neutral, stirs 0.5～1 hour;
The ethanol sedimentation rice bran polysaccharide, control precipitating final concentration is the ethanol of massfraction 60%～80%.
2. the preparation method of rice bran polysaccharide class active ingredient according to claim 1 is characterized in that raw materials used defatted rice bran, is the defatted rice bran of handling through extruding puffing.
3. the preparation method of rice bran polysaccharide class active ingredient according to claim 1, it is characterized in that gel filtration chromatography, adopt Sepharose CL-6B gel-filtration column that the RBP concentrated solution is carried out grading purification, NaCl solution with 0.2mmol/L carries out wash-out, automatically part is collected, can fractional separation become three main ingredient: RBP-a, RBP-b and RBP-c, get the RBP-a component.
4. the preparation method of rice bran polysaccharide class active ingredient according to claim 1, it is characterized in that ion-exchange chromatography, the concentrated solution of RBP-a is added to DEAE Sepharose Fast Flow ion-exchange chromatography, the ionic strength linear gradient elution, automatically part is collected, can fractional separation become four main ingredient: RBP-L, RBP-M, RBP-N and RBP-O, obtain the main immunocompetence component of RBP-M.
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| CN101360762B (en) * | 2006-01-26 | 2011-07-13 | 不二制油株式会社 | Water-soluble polysaccharides derived from rice bran, process for producing the same, and emulsifier using the same |
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| CN100447250C (en) * | 2006-12-26 | 2008-12-31 | 江南大学 | Separation and refining process for rice bran polysaccharide |
| CN102225161A (en) * | 2011-05-12 | 2011-10-26 | 江南大学 | Method for preparing functional components of antioxidation, blood sugar reduction and cholesterol reduction by using rice bran |
| CN102239931B (en) * | 2011-05-19 | 2013-04-24 | 湖南省中科农业有限公司 | Comprehensively deep processing technology for rice bran |
| CN102807629A (en) * | 2011-06-01 | 2012-12-05 | 鹤岗市三江平原米业集团有限公司 | Method for extracting rice bran polysaccharides by using continuous countercurrent ultrasonic equipment |
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| CN102911287B (en) * | 2012-11-21 | 2014-07-23 | 福建科宏生物工程有限公司 | Method for preparing bamboo leaf polysaccharides from bamboo pulp |
| CN103250973A (en) * | 2013-04-22 | 2013-08-21 | 江南大学 | Industrialized pretreatment method of high-temperature rice bran meal |
| CN104306391A (en) * | 2014-09-18 | 2015-01-28 | 河北科星药业有限公司 | Rice bran polysaccharide oral liquid, injection and soluble powder and corresponding preparation method |
| CN108546305A (en) * | 2018-04-23 | 2018-09-18 | 哈尔滨商业大学 | A kind of sprouted unpolished rice polysaccharide composition and its preparation method and application |
| CN112300298A (en) * | 2020-12-02 | 2021-02-02 | 铜仁学院 | Extraction method of akebia trifoliata peel polysaccharide |
| CN112602932B (en) * | 2020-12-17 | 2022-09-16 | 武汉轻工大学 | Method for processing defatted rice bran |
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Assignee: Xiangxiang Hualong Rice Industry Co., Ltd. Assignor: Jiangnan University Contract fulfillment period: 2009.8.6 to 2019.8.5 contract change Contract record no.: 2009430000175 Denomination of invention: Preparation process of rise bran polysaccharose kind active component Granted publication date: 20060111 License type: Exclusive license Record date: 20091027 |
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Free format text: EXCLUSIVE LICENSE; TIME LIMIT OF IMPLEMENTING CONTACT: 2009.8.6 TO 2019.8.5; CHANGE OF CONTRACT Name of requester: XIANGXIANG CITY HUALONG RICE CO., LTD. Effective date: 20091027 |