CN101974589B - Method for preparing immunocompetent soybean peptide by enzymolysis and membrane separation - Google Patents
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Abstract
The invention relates to a method for preparing an immunocompetent soybean peptide by enzymolysis and membrane separation, belonging to the technical field of soybean protein deep processing. The method comprises the following steps of: by taking defatted soybean protein powder as a raw material, firstly, carrying out acid cleaning on the defatted soybean protein powder to obtain acid washed soybean protein; then carrying out enzyme hydrolysis by using an Alcalase protease under certain conditions to obtain coarse soybean peptide liquid; clarifying the prepared coarse soybean peptide liquid by adopting microfiltration; then carrying out enrichment and desalination treatment by adopting ultrafiltration and nanofiltration; and finally, drying to obtain the powdery immunocompetent soybean peptide. The activity of the soybean peptide is determined by adopting a neutral red phagocytosis test and a lymphopoiesis method, which proves that the prepared soybean peptide has high immunocompetence.
Description
Technical field
The present invention relates to a kind of method of utilizing protease hydrolyzed and film separating preparing soybean protein immunization bioactive peptide, belong to Sunlover 10 deep process technology field.
Background technology
The immunity system of healthy human body need be kept a rational balance.If a little less than the immunizing power, the people infects, causes diseases such as tumour easily.Along with the going deep into of cell and the research of molecular level mechanism, and to the understanding of the complicated network structure in immune system inside, people recognize that gradually immunomodulatory is more much bigger than in the past in the effect of keeping aspect healthy.It is found that in recent years much and can respond the little peptide of regulating that be called as immune-active peptides, they possibly play a role as the chemical mediator in antigenicity substance or the immunocyte network to the natural immunity.Immune-active peptides has profound significance for strengthen the diseases prevention ability, delay senility etc.; They can through diet come enhances human body immunizing power, improve body and resist extraneous pathogenic infection ability, therefore study in the different food proteins potential immune-active peptides and be one for keeping the research work highly significant of HUMAN HEALTH, enhancing human immune.
About 1,450 ten thousand tons of China's soybean yields in 2009 accounts for 7% of Gross World Product, occupies the 4th.To the more and more deep research of soybean function property composition, soybean peptides will further be developed as a kind of novel soybean deep processed product, and is containing huge market potential along with domestic.
Mainly be synthetic peptide in the immune-active peptides of research report, and the research of wherein a fraction of food source immune-active peptides also mainly concentrate on milk protein sources, less about the research of the immune-active peptides in Sunlover 10 source.Yoshikawa and Takahashi use high-efficient liquid phase chromatogram technology and from the trypsin digestion thing of Sunlover 10, obtain six peptides that a peptide chain sequence is His-Cys-Gln-Arg-Pro-Arg; This Toplink stimulates the phagolysis of scavenger cell and multinuclear leucocyte, has immunoloregulation function.The people such as Yang Xiaojun of the biochemical emphasis of the Ministry of Agriculture of Agricultural University Of Nanjing animal physiological open laboratory discover; The solution that Sunlover 10 obtains with pepsin hydrolysis can significantly promote rat abdominal cavity macrophage phagocytic ability, stimulate peripheral blood lymphocyte to transform, improve enteric cavity sIgA level, and the effect of soybean protein hydrolysate is superior to the mucedin zymolyte.
Soybean peptides is meant that Sunlover 10 handles the hydrolysate that obtains through enzymolysis or microbial technique, and its multiple peptide molecule after by soybean protein hydrolysis mixes to be formed, comprising compositions such as small-amount free amino acid, soybean oligosaccharide, inorganic salt.There are problems such as purity and activity are not high in soybean peptides product in the market, and the present invention prepares soybean peptides through enzymolysis, further adopts membrane separation technique enriching and purifying soybean peptides, finally obtains the soybean peptides product of high peptide content, high immunological activity.
Summary of the invention
The object of the invention provides and a kind ofly prepares the method for immunocompetence soybean peptides through enzymic hydrolysis and membrane sepn, and the peptide content of acquisition is high, and immunocompetence is high, has improved the comprehensive value of Sunlover 10.
Technical scheme of the present invention: a kind of enzymolysis and membrane sepn prepare the method for immunocompetence soybean peptides, and said method steps is:
(1) defatted soybean protein powder is carried out pickling and obtain the pickling Sunlover 10: being raw material with the defatted soybean protein powder, is that 1:10 ~ 1:20 (w/v) adds pickle solution by g/mL, and pickle solution adopts the Na of mass concentration 2g/L
2SO
3The aqueous solution is regulated pH4.0 ~ 5.0,50 ~ 60 ℃ of temperature, and washing time 10 ~ 20min, pickling 2 ~ 4 times, the centrifugal supernatant of removing obtains the pickling Sunlover 10 after the pickling;
(2) use Alcalase proteolytic enzyme to carry out enzymic hydrolysis under certain condition and obtain thick soybean peptides liquid: with the pickling Sunlover 10 of step (1) gained, be that 1:10 ~ 1:20 (w/v) adds water, regulate pH to 8.0 by g/mL; Transfer in the pH8.0 process, protein viscosity increases gradually, stir; Attemperation is 55 ~ 65 ℃, adds Alcalase proteolytic enzyme, E/S=1.0% ~ 3.0%; Keep pH value constant in the reaction process, when degree of hydrolysis DH=20%, adjusting pH to 4.0 enzyme that goes out; The centrifugal 15min of 3000r/min, gained supernatant are thick soybean peptides liquid;
(3) adopt micro-filtration, ultrafiltration and nanofiltration to the prepared thick soybean peptides liquid that obtains clarify, enrichment, desalting treatment;
Adopting micro-filtration that the prepared thick soybean peptides liquid that obtains is clarified removal of impurities handles: at room temperature soybean peptides liquid is carried out the operation of total reflux micro-filtration, the microfiltration membrane of employing is the PS membrane of aperture 0.10 ~ 0.45 μ m; In treating processes, micro-filtration is a cycle of operation with 180min, collects the thick soybean peptides liquid that obtains clear; The cycle of operation finishes back shutdown the carrying out cleaning and the regeneration of membrane module;
Thick soybean peptides liquid after adopting ultrafiltration to micro-filtration carries out enrichment to be handled: at room temperature the thick soybean peptides liquid behind the micro-filtration is carried out the total reflux ultrafiltration; The molecular weight cut-off (MWCO) of the ultra-filtration membrane that adopts is 5000 ~ 8000; The soybean peptides starting point concentration of feed liquid is 6.0% ~ 8.0%, and pH is 4.0, and pressure is between 0.35～0.8MPa; Collect filtrate, realize enrichment active soybean peptides liquid;
Thick soybean peptides liquid after adopting nanofiltration to ultrafiltration carries out desalting treatment: at room temperature the thick soybean peptides liquid after the ultrafiltration is carried out total reflux nanofiltration operation; The molecular weight cut-off (MWCO) of the nf membrane that adopts is 150 ~ 300; Operation pH is 4.0, and pressure is collected filtrate between 0.4 ~ 0.7MPa;
(4) filtrate of collecting after the nanofiltration is obtained powdered immunocompetence soybean peptides through spraying drying;
(5) neutral red test or lymphopoiesis method (MTT) mensuration soybean peptides activity are engulfed in employing, show that the soybean peptides for preparing gained has good immunocompetence.
Adopt following two kinds of methods that the immunocompetence of soybean peptides is measured:
1. lymphopoiesis method (mtt assay)
Mtt assay is according to being rich in succinodehydrogenase in the viable cell plastosome, and this enzyme can make MTT decompose and produce blue crystalloid first the part between the ribs and the hips, and is deposited in the cell around the viable cell, forms first the part between the ribs and the hips amount and cell increment degree and is proportionate.Measure the OD value with ELIASA after the dissolving first the part between the ribs and the hips, just can obtain the propagation situation of cell.This method is simple comparatively speaking, is widely used in estimating the effect of xenobiotics to lymphocyte or body's immunity now.
Concrete grammar is following: the disconnected neck of mouse is put to death the aseptic spleen of getting in back, places the little plate that fills an amount of aseptic PBS liquid, gently spleen is torn to shreds with tweezers; Process the individual cells suspension, filter, wash 3 times with PBS liquid through 200 eye mesh screens; Each centrifugal rotational speed 1500 r/min, time 8min.Then with cell suspension in complete culture solution, the adjustment cell density be 4 * 10
9Individual/L.Cell suspension is added in 24 well culture plates, and every hole 0.5 mL is again by the following peptide liquid that adds complete culture solution or 400mg/L respectively that is provided with; Make every hole TV reach 1 mL; Every sample is established 8 multiple holes, and wherein 4 holes add ConA solution, and making it final concentration is 7 mg/L; Other 4 holes do not add ConA solution, put 37 ℃, 50 mL/L CO
2Cultivate 72 h in the incubator.Cultivate and finish preceding 4 h, supernatant is inhaled in every hole gently, adds the RPMI RPMI-1640 that 0.7 mL does not contain calf serum, adds MTT (5 g/L) 50 μ L/ holes simultaneously, continues to cultivate 4 h.After cultivating end, every hole adds 1 mL acid isopropyl alcohol, and the piping and druming mixing dissolves purple crystal fully.Transfer to then in 96 well culture plates, use enzyme-linked immunosorbent assay instrument, measure absorbance in wavelength 570nm.In the experiment with the nutrient solution group as blank.The immunoregulatory activity of soybean peptides is with SI (SI) expression, and the SI value is big more, explains that the immunoregulatory activity of enzymolysis product is strong more, and calculation formula is: the positive OD value of SI=/negative OD value.
2. toluylene red is engulfed experiment
Scavenger cell is a kind of important immunocyte of body immune system; Have and engulf and kill and wound several diseases pathogenic microorganism, processing and antigen-presenting, secrete cytokines effects such as (IL21, IL26, IL28, IL212, TNF2 α); In numerous disease therapeutic processes such as inflammation, infection and tumour, all brought into play vital role; Activated scavenger cell can be engulfed toluylene red dyeing, the size of OD value size reflection cytophagy ability.
Concrete grammar is following: gets healthy mice, cuts off abdominal skin, and the soft 5min of abdominal injection 5mL PBS, slowly the suction of cells suspension adds in the centrifuge tube, and centrifuge washing 2 times transfers to 1.8 * 10 with 1640 complete culture solutions with cell concn
6/ mL adds the medicine of 100 μ L cell suspensions and different concns in 96 each hole of hole flat underside, put 37 ℃, 5%CO
2Incubator; Take out culture plate after cultivating 22 h, abandon supernatant, the toluylene red 50uL that adds 0.073 % in every hole continues to cultivate 30min; Discard toluylene red; Wash 2 times with the preparatory PBS of temperature, add again the lysis agent (acetic acid: the 100 μ L of absolute ethyl alcohol=1:1), behind the 2h with ELIASA at the 540nm place mensuration absorbancy.The result is with phagocytic index (phagocytic index, PI) expression: PI=experimental port OD
540Value/control wells OD
540Average.
Beneficial effect of the present invention: the present invention adopts enzymolysis and membrane separation technique, obtains in hydrolysis reaching purposes such as clarification, enrichment immunocompetence soybean peptides and desalination on the basis of soybean peptides, has improved the immunocompetence quality of soybean peptides product greatly.
Embodiment
Below in conjunction with specific examples the present invention is further set forth.
Embodiment 1
Low-temperature defatted bean powder pickling 4 times.Add mass concentration 2g/LNa during pickling
2SO
3Solution is regulated pH4.5, adds pickle solution by 1:10 (w/v), 55 ℃ of temperature, and washing time 10min, the centrifugal supernatant of removing after the pickling obtains the heavy albumen of acid.Through the Sunlover 10 of overpickling, add water by 1:10 (w/v), regulate pH to 8.0 and (transfer in the pH8.0 process; Protein viscosity increases gradually, stir), 55 ℃ of attemperation; Add Alcalase proteolytic enzyme (E/S=1.0%), keep the pH value constant in the reaction process.When degree of hydrolysis DH=20%, transfer pH to 4.0 enzyme that goes out, centrifugal 15min under the 3000r/min, gained supernatant are thick soybean peptides liquid.Adopt the micro-filtration technology that thick peptide liquid is clarified removal of impurities, adopting the aperture is the microfiltration membrane (PS membrane) of 0.45 μ m.In treating processes, microfiltration membrane is a cycle of operation with 180min, shuts down and carries out the cleaning and the regeneration of membrane module.The clear peptide liquid that collection obtains.At room temperature the soybean peptides liquid behind the micro-filtration is carried out the total reflux ultrafiltration, adopting molecular weight cut-off (MWCO) is that 8000 composite package (cellulose acetate+polysulfones) is as ultra-filtration membrane.The feed liquid starting point concentration is 8.0%, working pressure 0.55MPa, 40 ℃ of feed temperatures, material liquid pH value 4.0, cycle running time 180min.Collect filtrate, thereby realize enrichment active soybean peptides liquid.At room temperature the soybean peptides liquid after the ultrafiltration is carried out nanofiltration then and handle, using molecular weight cut-off (MWCO) is that 300 composite package is as nf membrane.Operation pH is 4.0, and pressure 0.4MPa collects filtrate.Obtain powdered immunocompetence soybean peptides through spraying drying at last.
Embodiment 2
Low-temperature defatted bean powder pickling 2 times.Add mass concentration 2g/LNa during pickling
2SO
3Solution is regulated pH4.0, adds pickle solution by 1:20 (w/v), 50 ℃ of temperature, and washing time 15min, the centrifugal supernatant of removing after the pickling obtains the heavy albumen of acid.Through the Sunlover 10 of overpickling, add water by 1:15 (w/v), regulate pH to 8.0 and (transfer in the pH8.0 process; Protein viscosity increases gradually, stir), 60 ℃ of attemperation; Add Alcalase proteolytic enzyme (E/S=3.0%), keep the pH value constant in the reaction process.When degree of hydrolysis DH=20%, regulate pH to 4.0 enzyme that goes out, centrifugal 15min under the 3000r/min, gained supernatant are thick soybean peptides liquid.Adopt the micro-filtration technology that thick peptide liquid is clarified removal of impurities.Adopt the microfiltration membrane (polysulfones) of 0.10 μ m.In treating processes, microfiltration membrane is a cycle of operation with 180min, shuts down and carries out the cleaning and the regeneration of membrane module.Collect the clear peptide liquid that obtains at last.At room temperature soybean peptides solution is carried out the total reflux ultrafiltration.The ultra-filtration membrane molecular weight cut-off (MWCO) that adopts is 5000 PES films (polyethersulfone).The feed liquid starting point concentration is 7.0%, and working pressure 0.55MPa, 40 ℃ of feed temperatures, material liquid pH value 4.0, cycle running time 180min collect filtrate, thereby realizes the enrichment to active soybean peptides liquid.At room temperature soybean peptides solution being carried out nanofiltration then handles.The nf membrane molecular weight cut-off (MWCO) that adopts is 150 composite packages.Operation pH is 4.0, and pressure 0.6MPa collects filtrate.Obtain powdered immunocompetence soybean peptides through spraying drying at last.
Embodiment 3
Low-temperature defatted bean powder pickling 3 times.Add mass concentration 2g/LNa during pickling
2SO
3Solution is regulated pH5.0, adds pickle solution by 1:20 (w/v), 60 ℃ of temperature, and washing time 15min, the centrifugal supernatant of removing after the pickling obtains the heavy albumen of acid.Through the Sunlover 10 of overpickling, add water by 1:20 (w/v), regulate pH to 8.0 and (transfer in the pH8.0 process; Protein viscosity increases gradually, stir), add Alcalase proteolytic enzyme (E/S=2.0%); 60 ℃ of attemperation keep the pH value constant in the reaction process.When degree of hydrolysis DH=20%, transfer pH to 4.0 enzyme that goes out, centrifugal 15min under the 3000r/min, gained supernatant are thick soybean peptides liquid.Adopt the micro-filtration technology that thick peptide liquid is clarified removal of impurities.The microfiltration membrane membrane pore size that adopts is 0.45 μ m (polysulfones).In treating processes, microfiltration membrane is a cycle of operation with 180min, shuts down and carries out the cleaning and the regeneration of membrane module.Collect the clear peptide liquid that obtains at last.At room temperature soybean peptides solution is carried out the total reflux ultrafiltration.Adopting molecular weight cut-off (MWCO) is that 5000 composite package (polyethersulfone) is as ultra-filtration membrane.The feed liquid starting point concentration is 6.0%, and pH is 4.0, pressure 0.8MPa, 40 ℃ of feed temperatures, running time 180min.Collect filtrate, thereby realize enrichment active soybean peptides liquid.At room temperature soybean peptides solution being carried out nanofiltration then handles.The nf membrane molecular weight cut-off (MWCO) that adopts is 150 composite packages.Operation pH is 4.0, and pressure 0.5 MPa collects filtrate.Obtain powdered immunocompetence soybean peptides through spraying drying at last.
The peptide content and the immunocompetence of the immunocompetence soybean peptides product that obtains among table 1 embodiment
* there were significant differences to represent sample and control group.
Claims (1)
1. enzymolysis and membrane sepn prepare the method for immunocompetence soybean peptides, it is characterized in that said method steps is:
(1) defatted soybean protein powder is carried out pickling and obtain the pickling Sunlover 10: being raw material with the defatted soybean protein powder, is that 1:10 ~ 1:20 adds pickle solution by g/mL, and pickle solution adopts the Na of mass concentration 2g/L
2SO
3The aqueous solution is regulated pH4.0 ~ 5.0,50 ~ 60 ℃ of temperature, and washing time 10 ~ 20min, pickling 2 ~ 4 times, the centrifugal supernatant of removing obtains the pickling Sunlover 10 after the pickling;
(2) use Alcalase proteolytic enzyme to carry out enzymic hydrolysis under certain condition and obtain thick soybean peptides liquid: with the pickling Sunlover 10 of step (1) gained, be that 1:10 ~ 1:20 adds water, regulate pH to 8.0 by g/mL; Transfer in the pH8.0 process, protein viscosity increases gradually, stir; Attemperation is 55 ~ 65 ℃, adds Alcalase proteolytic enzyme, E/S=1.0% ~ 3.0%; Keep pH value constant in the reaction process, when degree of hydrolysis DH=20%, adjusting pH to 4.0 enzyme that goes out; The centrifugal 15min of 3000r/min, gained supernatant are thick soybean peptides liquid;
(3) adopt micro-filtration, ultrafiltration and nanofiltration to the prepared thick soybean peptides liquid that obtains clarify, enrichment, desalting treatment;
Adopting micro-filtration that the prepared thick soybean peptides liquid that obtains is clarified removal of impurities handles: at room temperature soybean peptides liquid is carried out the operation of total reflux micro-filtration, the microfiltration membrane of employing is the PS membrane of aperture 0.10 ~ 0.45 μ m; In treating processes, micro-filtration is a cycle of operation with 180min, collects the thick soybean peptides liquid that obtains clear; The cycle of operation finishes back shutdown the carrying out cleaning and the regeneration of membrane module;
Thick soybean peptides liquid after adopting ultrafiltration to micro-filtration carries out enrichment to be handled: at room temperature the thick soybean peptides liquid behind the micro-filtration is carried out the total reflux ultrafiltration; The molecular weight cut-off MWCO of the ultra-filtration membrane that adopts is 5000 ~ 8000; The soybean peptides starting point concentration of feed liquid is 6.0% ~ 8.0%, and pH is 4.0, and pressure is between 0.35～0.8MPa; Collect filtrate, realize enrichment active soybean peptides liquid;
Thick soybean peptides liquid after adopting nanofiltration to ultrafiltration carries out desalting treatment: at room temperature the thick soybean peptides liquid after the ultrafiltration is carried out total reflux nanofiltration operation; The molecular weight cut-off MWCO of the nf membrane that adopts is 150 ~ 300; Operation pH is 4.0, and pressure is collected filtrate between 0.4 ~ 0.7MPa;
(4) filtrate of collecting after the nanofiltration is obtained powdered immunocompetence soybean peptides through spraying drying;
(5) neutral red test or lymphopoiesis method mensuration soybean peptides activity are engulfed in employing, show that the soybean peptides for preparing gained has good immunocompetence.
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| CN2010105237477A CN101974589B (en) | 2010-10-29 | 2010-10-29 | Method for preparing immunocompetent soybean peptide by enzymolysis and membrane separation |
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| CN102178028B (en) * | 2011-06-17 | 2013-03-20 | 武汉百安生物科技有限责任公司 | Method for producing soybean peptide |
| CN103014112B (en) * | 2012-12-20 | 2015-05-06 | 广州合诚实业有限公司 | Soybean peptide and industrial production method and application of soybean peptide |
| CN103146788B (en) * | 2013-02-28 | 2014-11-19 | 珠海博康药业有限公司 | Enzymic-method based preparation method of soybean peptide |
| CN103266157B (en) * | 2013-05-23 | 2014-09-24 | 江南大学 | Preparation method of plant protein derivative with physiological activity and nutrition supplementing function |
| CN103435680A (en) * | 2013-07-31 | 2013-12-11 | 招远金汇膜科技有限公司 | Method for extracting protein peptide from beans |
| CN103695513B (en) * | 2013-10-14 | 2016-04-06 | 诺利如一（安阳）生物科技有限公司 | A kind of method improving yield of soybean peptide with low molecular weight |
| CN103555799B (en) * | 2013-10-26 | 2015-09-23 | 吉林大学 | A kind of immunocompetence peptide freeze-dried powder and preparation method thereof |
| CN105166321A (en) * | 2015-09-18 | 2015-12-23 | 山东农业大学 | High immune activity soybean peptide preparation method obtained through combination of microcalorimetric method and immune activity evaluation experiment |
| EP3439490A1 (en) | 2016-04-08 | 2019-02-13 | Fraunhofer Gesellschaft zur Förderung der Angewand | Techno-functional plant protein fraction from leguminous or oil seeds |
| CN107674900B (en) * | 2017-06-12 | 2021-05-14 | 诺利如一（安阳）生物科技有限公司 | Process method for concentrating soybean peptide by adopting tandem membrane |
| CN114903176B (en) * | 2022-05-09 | 2023-11-21 | 博厚健康科技股份有限公司 | Low-sensitization polypeptide nutrition powder and preparation method thereof |
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