CN101654693A - Method for preparing rapeseed peptides by microorganism fermentation - Google Patents
Method for preparing rapeseed peptides by microorganism fermentation Download PDFInfo
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Abstract
The invention relates to a method for preparing rapeseed peptides by microorganism fermentation, which sufficiently utilizes the functions of hydrolyzing proteins and degrading sulfuric glucoside by microorganism, thereby effectively improving the soluble nitrogen content and the bioactivity of the product. The method for the preparing rapeseed peptides by microorganism fermentation comprises thefollowing steps: (1) mixing rapeseed meal powder with nutrient solution to prepare liquid fermentation medium; (2) inoculating Bacillus licheniformis into the fermentation medium to carry out liquid fermentation; (3) carrying out solid-liquid separation on the liquid fermentation medium after fermentation to obtain the crude extract of the rapeseed peptides; and (4) purifying the crude extract toobtain the rapeseed peptides. The invention has simple preparation process and low cost, selects the Bacillus licheniformis for liquid fermentation to generate a plurality of metabolites and enzymes and sufficiently utilizes the functions of hydrolyzing proteins and degrading sulfuric glucoside by the Bacillus licheniformis, thereby effectively degrading the toxic substances of the rapeseed meal,hydrolyzing proteins, and improving the soluble nitrogen content, the bioactivity and the yield of the rapeseed peptides.
Description
Technical field
The present invention relates to a kind of method of preparing rapeseed peptides by microorganism fermentation.
Background technology
China is the country of Semen Brassicae campestris output maximum in the world, nearly 1,200 ten thousand tons of annual production, and rapeseed meal is the main by product of rapeseed oil processing, accounts for 65% of vegetable seed total amount, wherein contains albumen 35%～42%.Rape seed protein amino acid is formed rationally, and the content of Methionin, Gelucystine and methionine(Met) is higher, and the amino acid compositional model of recommending with WHO/FAO is close, and nutritive value is parity with or superiority over animal proteinum, is a kind of good vegetable-protein.But because there are some antinutritional factor in rapeseed meal,, be difficult in the course of processing remove, make rapeseed meal can only be applied to feed or fertilizer industry, utilize usefulness low, serious waste of resources as glucosinolate, phytic acid, polyphenols etc.Therefore, development and use vegetable seed protein resource has become trend of the times, because after rape seed protein is hydrolyzed into polypeptide, have better biological activity and processing characteristics, therefore strengthen research and development, and make it industrialization, effectively utilizing the plant protein resource of China to rapeseed peptide (rsp), improve China people's trophic level, have important social meaning and good economic benefit.
At present China is at the early-stage to researching and producing of rapeseed peptide (rsp), and its method is mainly enzyme process, promptly is to be raw material with the vegetable seed protein isolate, utilizes commercial protease preparation hydrolysis rape seed protein to prepare rapeseed peptide (rsp).This method is a raw material with the vegetable seed protein isolate, it is big and cost an arm and a leg to produce the used enzyme preparation consumption, cause production cost higher, and because antinutritional factor can not remove fully in rapeseed meal or the vegetable seed protein isolate, influenced protease activities, hydrolysis degree is not high, is difficult to form large-scale industrial production.
CN200810243461.6, CN200810106919.3 disclose the method for biofermentation method to the rapeseed meal modification, though removed the glucosinolate objectionable impuritiess such as (hereinafter to be referred as the sulphur glucosides) in most of rape seed protein, obtain rapeseed peptide (rsp) or contain the product of rapeseed peptide (rsp), but the soluble nitrogen content of product and biological activity still remain further to be improved, and still have a certain amount of glucosinolate to residue in the product.
Summary of the invention
The invention provides a kind of method of preparing rapeseed peptides by microorganism fermentation, give full play to protein hydrolysate effect and the effect of degraded sulphur glucoside of microorganism, can effectively improve the soluble nitrogen content and the biological activity of product.
The method of described preparing rapeseed peptides by microorganism fermentation may further comprise the steps:
(1) rapeseed meal powder and nutritive medium are mixed with the liquid state fermentation substratum;
(2) Bacillus licheniformis is inserted the liquid state fermentation substratum, carried out liquid state fermentation;
(3) the liquid substratum after the fermentation obtains the crude extract of rapeseed peptide (rsp) through solid-liquid separation;
(4) crude extract obtains rapeseed peptide (rsp) through purification.
In the step (1), rapeseed meal powder and nutritive medium are by solid-to-liquid ratio 1: 5～1: 40g/mL preparation liquid state fermentation substratum, and wherein the pH of nutritive medium is 8.0～10.0, comprises glucose 0.1%～2% in the nutritive medium, MnSO
40.1%～2.0%, above-mentioned per-cent is quality volume percent (the quality volume percent refers to contain in the 100ml solution how many gram, i.e. 1%=1g/100ml of certain material).Described solid-to-liquid ratio is preferably 1: 15～and 1: 20g/mL, the pH of nutritive medium are 9.0～10.The particle diameter of rapeseed meal powder is not more than 200 μ m.
In the step (2), Bacillus licheniformis is inserted the liquid state fermentation substratum, making cell concentration is 10
5～10
7Individual/mL, 25～40 ℃ of leavening temperatures, fermentation time 1～5 day.Preferred scheme is, Bacillus licheniformis is activated respectively, to obtain cell concentration be 10 to enlarged culturing
7～10
8The seed liquor of individual/mL inserts the liquid state fermentation substratum.The inoculum size of seed liquor is preferably 2%～8%, and 3%-5% more preferably is in the per-cent of seed liquor with respect to the liquid state fermentation culture volume.
Described Bacillus licheniformis activation, enlarged culturing all adopt ordinary method, and the method that generally obtains seed liquor is as follows: Bacillus licheniformis is inoculated on the slant medium cultivates, the picking Bacillus licheniformis is inoculated in and carries out enlarged culturing in the seed culture medium then, wherein the slant culture based formulas is: extractum carnis 3g, peptone 10g, NaCl 5g, agar 20g, water 1000mL, pH 7.2-7.4, the seed culture based formulas is: extractum carnis 3g, peptone 10g, NaCl 5g, water 1000mL, pH 7.0.
Step (3) can known method be carried out solid-liquid separation, removes residue as adopting vacuum filter or pressure filter, also can adopt centrifuging, and relative centrifugal force is 2500g～10000g.
As preferred version of the present invention, in the step (4), described method of purification is: decolouring, desalination and drying.Preferably scheme is, crude extract is adopted activated carbon decolorizing, and concentrating under reduced pressure, warm air drying, lyophilize or spraying drying mode drying are adopted in ion exchange resin or reverse osmosis desalination, obtain the rapeseed peptide (rsp) powder.
As another kind of preferred version of the present invention, in the step (4), described method of purification comprises ion-exchange, hydrophobic chromatography and gel-filtration, collects to have the active component of antioxidation biology, obtains the single-minded rapeseed peptide (rsp) of oxidation-resistance through lyophilize.Can certainly adopt remaining separation purification method, carry out ultrafiltration, collect the filtrate of PSPP,,, collect the single-minded component of biologically active, after lyophilize, obtain rapeseed peptide (rsp) through gel-filtration through the desalting column desalination as ultra-filtration membrane.When the ultra-filtration membrane molecular weight cut-off hour, then can be directly through gel-filtration, obtain rapeseed peptide (rsp), ultra-filtration membrane is that molecular weight cut-off is the ultra-filtration membrane of 3kDa as described, the component of molecular weight≤3kDa is through gel-filtration, and the chromatographic condition condition is: filler is the reversed phase chromatography gel, flow velocity 1mL/min, 25 ℃ of temperature, moving phase are the phosphate buffered saline buffer of pH value 7.0.
The present invention compared with prior art; be the single-minded rapeseed peptide (rsp) of raw material microorganism liquid state fermentation production biological activity directly with the rapeseed meal; raw material sources are extensive, and are cheap, do not need it is carried out the processing of acid, alkali; more do not need to isolate rape seed protein; production technique is simple, cost is low, and turns waste into wealth, and helps environment protection; replace pure zymin hydrolysis rape seed protein with microbial fermentation and prepare polypeptide, can save a large amount of expensive biological enzyme preparations.Select Bacillus licheniformis for use, carry out liquid state fermentation, produce multiple meta-bolites and enzyme, give full play to its protein hydrolysate effect and the effect of degraded sulphur glucoside, the toxic substance of the rapeseed meal kind of effectively degrading, protein hydrolysate reaches the food sanitation standard of humans and animals, improve soluble nitrogen content, biological activity and the rapeseed peptide (rsp) yield of product, also contain multiple functional factor the human body beneficial.In a word, the present invention is environmentally friendly, and the reaction conditions gentleness can be given full play to protein hydrolysate effect and the effect of degraded sulphur glucoside of microorganism, is the method that a kind of low cost, suitable industrial mass prepare rapeseed peptide (rsp).
Embodiment
Embodiment 1
(1) preparation liquid state fermentation substratum
Nutrient composition: glucose 2.0g, MnSO
41.0g tap water 1000mL, pH are 9.5.It is about 100 μ m that double lower rapeseed dreg is crushed to particle diameter, mixes with the ratio and the rapeseed meal of nutritive medium in solid-to-liquid ratio 1: 15g/mL, obtains the liquid state fermentation substratum and place in the fermentor tank after 121 ℃ are sterilized 30min down.
(2) liquid state fermentation
1. the preparation of seed liquor:
The slant culture based formulas: extractum carnis 3g, peptone 10g, NaCl 5, agar 20g, tap water 1000mL, pH7.2-7.4.
The seed culture based formulas: extractum carnis 3g, peptone 10, NaCl 5g, tap water 1000mL, pH 7.0.
From the spawn culture inclined-plane after the activation, picking two ring Bacillus licheniformis are inoculated in the seed culture medium, and 8 layers of gauze seal, and 35 ℃, 120r/min, shaking table is cultivated 24h, obtains cell concentration and reaches 10
8The seed liquor of individual/mL.
2. get the seed liquor access and be equipped with in the fermentor tank of fermention medium, making cell concentration is 10
6Individual/mL, readjusting the distribution the ferment temperature is 31 ℃, and rotating speed 180r/min shakes cultivation, regularly ventilates, and ferments 1～2 day.
(3) solid-liquid separation obtains the crude extract of rapeseed peptide (rsp)
The taking-up fermention medium separates with 2500g relative centrifugal force low-speed centrifugal earlier and removes dregs of rice slag, uses the impurity elimination of 10000g relative centrifugal force high speed centrifugation again, gets the rapeseed peptide (rsp) crude extract, is 30～32% with recording the rapeseed peptide (rsp) yield after this rapeseed peptide (rsp) crude extract spraying drying.
(4) purify
Above-mentioned crude extract is that the ultra-filtration membrane of 10kDa carries out ultrafiltration by molecular weight cut-off respectively, with molecular weight less than the component of 10kDa through ion exchange column (HiTrap
TMIon exchange column Q XL; Flow velocity: 1mL/min; Tris-HCL pH of buffer 8.0, temperature: 25 ℃), hydrophobic chromatography (HiTrap
TMHydrophobic chromatography post PhenyL FF; Flow velocity: 1mL/min; Temperature: 25 ℃) and gel-filtration (Superdex Peptide HR 10/30; Flow velocity: 0.2mL/min; Temperature: 25 ℃), collection has the active component of antioxidation biology, obtains the single-minded rapeseed peptide (rsp) of oxidation-resistance after lyophilize.
The antioxidation biology activity index of gained rapeseed peptide (rsp) is as follows:
The rapeseed peptide (rsp) reducing power: during product concentration 1mg/mL, A
700nmBe 0.951;
Free radical scavenging activity: during product concentration 1mg/mL, the DPPH free radical scavenging activity is 85%;
Metal chelating is made a concerted effort: during product concentration 5mg/mL, it is 45% that metal chelating is made a concerted effort;
The lipid oxidation inhibiting rate: during product concentration 1mg/mL, the lipid oxidation inhibiting rate is 50%.
Embodiment 2
Difference from Example 1 is, carries out (4) as follows and purifies:
With above-mentioned crude extract is that the ultra-filtration membrane of 3kDa carries out ultrafiltration by molecular weight cut-off respectively, and the component of molecular weight≤3kDa is through gel-filtration (post: SOURCE
TM5RPC, flow velocity 1mL/min, 25 ℃ of temperature; Phosphate buffered saline buffer pH7.0), collection has hypotensive bioactive component, obtains the single-minded rapeseed peptide (rsp) of biological activity after lyophilize.The single-minded hypotensive index of biological activity of rapeseed peptide (rsp) of this biological activity is: the concentration (IC of the active required inhibitor of ACE (angiotonin transferring enzyme) of inhibition 50%
50) be 0.15mg/mL.
Embodiment 3
Difference from Example 1 is, carries out (4) as follows and purifies:
Regulate rapeseed peptide (rsp) crude extract pH to 6.5～7.5, add the gac of 10g/L, in 50 ℃ of decolourings, debitterize 1h, high speed centrifugation (relative centrifugal force is 12000g) impurity elimination then is through desalting column (HiPrep
TM26/10, flow velocity 30mL/min) after the desalination, spray-dried again acquisition rapeseed peptide (rsp), its protein content (butt, N * 6.25) 82.5%, soluble nitrogen content 90%, molecular weight is at the peptide content 60% of 500Da～5000Da, and the sulphur glucoside does not detect.Gained rapeseed peptide (rsp) soluble nitrogen content of the present invention and molecular weight are all very high at the peptide content of 1000Da～5000Da, so the solvability of product is better, helps the performance of intestinal absorption and bioactive functions, and the sulphur glucoside does not detect, and Product Safety is better.
Claims (11)
1. the method for a preparing rapeseed peptides by microorganism fermentation is characterized in that may further comprise the steps:
(1) rapeseed meal powder and nutritive medium are mixed with the liquid state fermentation substratum;
(2) Bacillus licheniformis is inserted the liquid state fermentation substratum, carried out liquid state fermentation;
(3) the liquid substratum after the fermentation obtains the crude extract of rapeseed peptide (rsp) through solid-liquid separation;
(4) crude extract obtains rapeseed peptide (rsp) through purification.
2. the method for preparing rapeseed peptides by microorganism fermentation as claimed in claim 1, it is characterized in that in the step (1), rapeseed meal powder and nutritive medium are by solid-to-liquid ratio 1: 5～1: 40g/mL preparation liquid state fermentation substratum, wherein the pH of nutritive medium is 8.0～10.0, comprise glucose 0.1%～2% in the nutritive medium, MnSO40.1%～2.0%, above-mentioned per-cent are the quality volume percent.
3. the method for preparing rapeseed peptides by microorganism fermentation as claimed in claim 2, it is characterized in that in the step (1), described solid-to-liquid ratio is 1: 15～1: 20g/mL, the pH of nutritive medium are 9.0～10.
4. the method for preparing rapeseed peptides by microorganism fermentation as claimed in claim 1 is characterized in that in the step (1), the particle diameter of rapeseed meal powder is not more than 200 μ m.
5. as the method for each described preparing rapeseed peptides by microorganism fermentation among the claim 1-4, it is characterized in that in the step (2) Bacillus licheniformis being inserted the liquid state fermentation substratum, making cell concentration is 105～107/mL, 25～40 ℃ of leavening temperatures, fermentation time 1～5 day.
6. the method for preparing rapeseed peptides by microorganism fermentation as claimed in claim 5 is characterized in that Bacillus licheniformis is activated respectively in the step (2), enlarged culturing obtains the seed liquor that cell concentration is 107～108/mL, inserts the liquid state fermentation substratum.
7. the method for preparing rapeseed peptides by microorganism fermentation as claimed in claim 6 is characterized in that in the step (2), the inoculum size of seed liquor is 2%～8%.
8. as the method for each described preparing rapeseed peptides by microorganism fermentation among the claim 1-4, it is characterized in that described method of purification is in the step (4): decolouring, desalination and drying.
9. as the method for each described preparing rapeseed peptides by microorganism fermentation among the claim 1-4, it is characterized in that in the step (4), described method of purification comprises ion-exchange, hydrophobic chromatography and gel-filtration, collection has the active component of antioxidation biology, obtains the single-minded rapeseed peptide (rsp) of oxidation-resistance through lyophilize.
10. as the method for each described preparing rapeseed peptides by microorganism fermentation among the claim 1-4, it is characterized in that in the step (4) that described method of purification comprises by ultra-filtration membrane carries out ultrafiltration, through gel-filtration, the active single-minded component of collection of biological.
11. the method for preparing rapeseed peptides by microorganism fermentation as claimed in claim 10, it is characterized in that in the step (4), described ultra-filtration membrane is that molecular weight cut-off is the ultra-filtration membrane of 3kDa, the component of molecular weight≤3kDa is through gel-filtration, the chromatographic condition condition is: filler is the reversed phase chromatography gel, flow velocity 1mL/min, 25 ℃ of temperature, moving phase are the phosphate buffered saline buffer of pH value 7.0.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710416A (en) * | 2013-12-20 | 2014-04-09 | 安徽省农业科学院农产品加工研究所 | Preparation method and applications of rapeseed cake-sourced metal chelating peptide and peptide metal chelate |
| CN105543317A (en) * | 2016-02-17 | 2016-05-04 | 成都美溢德生物技术有限公司 | Method for liquid synchronous fermentation and enzymolysis of plant protein and prepared protein hydrolysate |
| CN111534560A (en) * | 2020-06-17 | 2020-08-14 | 青岛科瑞培养基有限公司 | Method for preparing active polypeptide by fermenting oil pressing meals |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101464981A (en) * | 2007-12-18 | 2009-06-24 | 黄金富 | Bank card account security system and method through mobile phone orientation authentication card owner identification |
| CN101492708B (en) * | 2008-12-25 | 2011-06-29 | 南京财经大学 | Method for preparing vegetable seed peptide with single bioactivity by mix bacterium solid state fermentation |
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2009
- 2009-08-21 CN CN 200910305946 patent/CN101654693B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710416A (en) * | 2013-12-20 | 2014-04-09 | 安徽省农业科学院农产品加工研究所 | Preparation method and applications of rapeseed cake-sourced metal chelating peptide and peptide metal chelate |
| CN103710416B (en) * | 2013-12-20 | 2016-08-17 | 安徽省农业科学院农产品加工研究所 | Rapeseed cake source metal chelating peptide, the Preparation method and use of peptide metallo-chelate |
| CN105543317A (en) * | 2016-02-17 | 2016-05-04 | 成都美溢德生物技术有限公司 | Method for liquid synchronous fermentation and enzymolysis of plant protein and prepared protein hydrolysate |
| CN111534560A (en) * | 2020-06-17 | 2020-08-14 | 青岛科瑞培养基有限公司 | Method for preparing active polypeptide by fermenting oil pressing meals |
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