CN101139580A - Method for preparing debittering aminopeptidase by fermentation of bacillus subtilis - Google Patents
Method for preparing debittering aminopeptidase by fermentation of bacillus subtilis Download PDFInfo
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- CN101139580A CN101139580A CNA2007100258649A CN200710025864A CN101139580A CN 101139580 A CN101139580 A CN 101139580A CN A2007100258649 A CNA2007100258649 A CN A2007100258649A CN 200710025864 A CN200710025864 A CN 200710025864A CN 101139580 A CN101139580 A CN 101139580A
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Abstract
The invention provides a method for preparing debittered aminopeptidase by using bacillus subtilis and the application of the debittering aminopeptidase, which pertains to the technical field of enzyme preparations and food additives. The invention relates to the fermentation and production by using bacillus subtilis Zj016, and aminopeptidase product is obtained by staged alcohol settling the fermented fluid. The invention relates also to the application of the aminopeptidase, which coordinates some alkaline protease to hydrolyze protein for 4h, the hydrolyzed degree (DH) can be up to 25.03%, and the hydrolyzing fluid is free from any bitter, can solve well the problem of bitter taste in producing bean peptide by using other enzyme.
Description
Technical field
A kind of method and application for preparing debitterized aminopeptidase with fermentation of bacillus subtilis, specifically utilize fermentation of bacillus subtilis production to collect fermented liquid, by ethanol precipitation, obtain the debitterized aminopeptidase goods, belong to zymin, technical field of food additives.
Background technology
It is an important directions of soybean deep processing that soybean protein hydrolysis is produced soybean polypeptide, because soybean polypeptide not only has good nutritive property, also has physiological and pharmacological effect widely and than soybean protein rich functions characteristic more, so soybean polypeptide is a kind of very promising functional food ingredient.Progress and development of life science along with biotechnology, the physiological function of physiologically active peptide progressively be it is found that, and it is clear and definite gradually to be subjected to the structure and the physiological function of increasing attention, especially some bioactive peptides, promote the research of bioactive peptide, also promoted the research and development of soybean peptides.
The U.S. is after early 1970s has worked out the soybean polypeptide product, and u s company has built up the human consumption soybean peptide factory that produces 5000 tons per year.Japan carries out in this respect research the eighties in 20th century, and dairy industry companies such as the oily company of Japanese only system, Seichin and Sen Yong all successfully are used for soybean peptides the food biotechnology industry.China has also begun the research of soybean peptides in the middle and later periods eighties 20th century, and has obtained some gratifying achievements.But, soybean polypeptide product market at home still belongs to the elementary development phase, its production technique is still needed further perfect, the subject matter that will solve is the regulation and control of enzymolysis process, the content of total free aminoacids, the separation of crossing small-molecular peptides and the improvement of soybean active peptide local flavor at present, avoids producing problems such as bitter peptides.
The bitter taste that the soybean polypeptide product has in various degree can directly influence flavours in food products, and people have " unhappiness " when edible, have also limited its applying in foodstuffs industry to a certain extent.Studies confirm that polypeptide presents bitter taste when hydrophobic amino acid residues is in the outside of polypeptide chain, and therefore low many of one hydrophobic amino acid bitter taste adopt excision enzyme that hydrophobic amino acid is cut from the polypeptide chain end and can reduce bitter taste effectively.Based on this theory, this laboratory screening goes out a bacillus subtilis, the debitterized aminopeptidase of producing is an excision enzyme, amino acid can dissociate from the aminoterminal of polypeptide chain, through this aminopeptidase of this laboratory study and Sumizyme MP synergetic hydrolysis soybean protein isolate, the soybean polypeptide of preparation does not have bitter taste, and oligopeptides content such as dipeptides tripeptides is higher.
Summary of the invention
The objective of the invention is to screen a strain and can produce microorganism with de-bittering effect aminopeptidase, by technologies such as fermentation culture, frozen centrifugation, ethanol precipitations, the purer aminopeptidase of final acquisition, the yield of whole process flow is 55%, the ratio of enzyme is lived and is 1700U/mL, 50 ℃ of optimum temperutures, 45～70 ℃ of temperature-stable scopes, optimal pH 8.0, stable pH range 7.0～9.5.With this enzyme and Sumizyme MP synergetic hydrolysis soybean protein isolate, hydrolyzed solution presents bitter taste hardly, and de-bittering effect is remarkable.
Technical scheme of the present invention: it is in the substratum of nitrogen, carbon source that subtilis is inserted with okara powder, W-Gum, regulates fermention medium pH with sodium hydroxide, and fermentation culture is collected fermented liquid, uses ethanol precipitation, obtains thick enzyme, and technology is:
The A fermentation of bacillus subtilis, collect fermented liquid:
(1) starting strain: adopting subtilis Zj016 is starting strain; [food and fermentation industries] 2006 32 volumes are open the 3rd the sixth of the twelve Earthly Branches phase.
(2) fermention medium is formed: W-Gum 33～42g/L, okara powder 27～35g/L, KH
2PO
412H
2O 0.16～0.32g/L, K
2HPO
42.68～5.0g/L, NaCl 1g/L, MgSO
47H
2O0.1～0.3g/L, CoCl
21～3ml (0.1mol/L) transfers pH7.5 with sodium hydroxide;
(3) fermentation condition: inoculum size 8%, initial pH7.5～8.0,35～40 ℃, 200～400rpm cultivates 15～16h;
(4) collect fermented liquid: 4 ℃ of fermented liquids with behind the centrifugal 10min of 10000rpm, are abandoned precipitation, collect supernatant liquor;
The B ethanol precipitation:
(1) add-20 ℃ of ethanol in the supernatant liquor of collecting, making the ethanol final concentration is 20% to carry out alcohol precipitation first time, and 4 ℃ with behind the centrifugal 10min of 10000rpm, abandons precipitation, the collection supernatant liquor;
(2) add-20 ℃ of ethanol in the supernatant liquor behind the alcohol precipitation for the first time, making the ethanol final concentration is 40% to carry out alcohol precipitation, and 4 ℃ with behind the centrifugal 10min of 10000rpm, abandons supernatant, gets precipitation;
(3) the Tris-HCL damping fluid of a small amount of pH8.5 of precipitation adding (contains 0.1mmol/LCoCl
2) dissolving, 4 ℃ with behind the centrifugal 10min of 10000rpm, abandons precipitation, collects supernatant liquor, is aminopeptidase solution.
The application of C debitterized aminopeptidase
90 ℃ of water-bath 10min of soybean protein isolate aqueous solution pre-treatment with 60g/L, be cooled to room temperature, transfer to pH 8.5, place 50 ℃ of water-baths with 1mol/L sodium hydroxide, treat that the suspension temperature rises to 50 ℃, the amount ratio of pressing aminopeptidase, basic protein enzyme-to-substrate soybean protein isolate all is 10000U/g, adds two kinds of enzymes simultaneously, slowly stirs, use the 1mol/LNaOH titration, keep pH 8.5 ± 0.1, hydrolysis 4h, percent hydrolysis reaches 25%.After finishing, hydrolysis reaction make hydrolyzed solution rise to 90 ℃ of heating 20min, passivation proteolytic enzyme rapidly.The centrifugal 20min of hydrolyzed solution 4000rpm gets the supernatant liquor lyophilize, is soybean polypeptide.
(1) the bitter taste analysis of hydrolysate
Bitter taste definition: take by weighing the 2g Leaf of Chinese Holly, add 500mL distilled water and boil 30min, filter constant volume 500mL.Dilute 20,40,60,80,100 times respectively, the definition bitterness value for by force, strong, in, weak, nothing.
The assessment method of bitter taste: subjective appreciation group is made up of 8 people, after the evaluation personnel gargle with distilled water, get hydrolyzed solution 3～5mL to be measured and place mouth, spue behind the 10s, get the reference liquid that bitterness is close with it after gargling and taste, as confirm that two bitter tastes are close, can be defined as hydrolyzed solution to be measured the bitterness value of this reference liquid, taste otherwise get other reference liquid, until definite its bitterness value.
Bitter taste evaluation result such as following table:
| Bitterness value | By force | Stronger | In | A little less than | Do not have |
| Two enzyme synergetic hydrolysis liquid caustic soda proteolytic enzyme single enzymolysis liquid | +++ | +++++ | ++ | ++++++ |
Evaluation result is analyzed: can find out that from last table this aminopeptidase greatly reduces the bitter taste of soybean peptides, tangible de-bittering effect is arranged, its hydrolyzed solution almost can not tasted bitter taste.
(2) degree of hydrolysis of hydrolysate and free aminoacid content analysis
Two enzyme synergetic hydrolysis 4h degree of hydrolysis reach 25.0%, and free aminoacid content is 1610mg/L; The degree of hydrolysis of Sumizyme MP single enzymolysis is 22.9%, and free aminoacid content is 1250mg/L, illustrates that aminopeptidase is a kind of excision enzyme, can dissociate total free aminoacids from polypeptide chain ammonia end.
(3) the molecular weight distribution characteristics of hydrolysate
| Single enzymolysis | Peak top molecular weight proportion (%) bitterness value hydrophobic value (S 0) | 5248.1 it is strong by 783.56 in 21.62 | 2011.3 the last 51.35 833.3 | 1345.6 it is strong by 646.62 in 16.22 | 293.4 10.81 do not have 100.36 |
| Two enzyme synergetic hydrolysis | Peak top molecular weight proportion (%) bitterness value hydrophobic value (S 0) | 2570.7 33.34 do not have 324.11 | 1,885 22.22 sour and astringent 98.36 | 1013.4 24.07 do not have 493.15 | 261.7 20.37 do not have 100.11 |
A: double-enzyme hydrolysis liquid lyophilized powder
B: Sumizyme MP single enzymolysis liquid lyophilized powder
(4) the hydrophobic value S of hydrolysate
0Analysis
| Sample | A | B | C | D | E |
| Hydrophobic value (S 0) | 163.11 | 2107.7 | 1345.6 | 5163.9 | 635.25 |
A: soybean protein isolate
B: double-enzyme hydrolysis liquid lyophilized powder
C: wait electricity (pH4.3) precipitation supernatant liquor lyophilized powder behind the double-enzyme hydrolysis
D: Sumizyme MP single enzymolysis liquid lyophilized powder
E: wait electricity (pH4.3) precipitation supernatant liquor lyophilized powder behind the Sumizyme MP single enzymolysis
Measure the hydrophobic value S of hydrolyzed solution lyophilized powder by the ANS method
0, the S of the hydrolyzed solution of two enzyme synergetic hydrolysis
0Be 2107.7, and the S of the hydrolyzed solution of Sumizyme MP single enzymolysis
0Be 5163.9, aminopeptidase makes the hydrophobic value of hydrolyzed solution reduce by 60%, and both are waited electricity (pH4.3) precipitation process, it is less that the former hydrophobic value reduces amplitude, and the latter's hydrophobic value reduction amplitude is about an order of magnitude, so may have the polypeptide that contains a large amount of hydrophobic amino acids in the explanation Sumizyme MP single enzymolysis liquid, it is reported that the bitter taste of soybean polypeptide generally is that the polypeptide chain that ammonia end or carboxylic end have single or multiple hydrophobic amino acids causes, so this aminopeptidase of proof has the effect of removing the polypeptide solution bitter taste by the hydrophobic amino acid of the bitter peptides end that dissociates really;
Above result shows, this aminopeptidase is as a kind of excision enzyme, greatly reduces the bitter taste of soybean peptides by the hydrophobic amino acid of excision soybean polypeptide ammonia end, can solve other enzyme of domestic usefulness and produce the difficult problem that soybean polypeptide easily produces bitter taste; Confirmed that also bitter taste and amino acid whose hydrophobicity are irrelevant, and relevant with the hydrophobicity of polypeptide chain.
Beneficial effect of the present invention: the fermentative production debitterized aminopeptidase can enlarge, and extracting method enlarges production easily, and this enzyme and Sumizyme MP synergetic hydrolysis soybean protein isolate produce soybean peptides, and de-bittering effect is remarkable, and the gained soybean peptides aqueous solution presents bitter taste hardly.
Description of drawings
Fig. 1 bacillus subtilis debitterized aminopeptidase preparation technology synoptic diagram.
The specific examples mode
Embodiment 1
Subtilis Zj016 inclined-plane is inserted seed liquor, after the enlarged culturing, insert in the 4L fermention medium by inoculum size 8% again, fermention medium is formed as described in the specification sheets.Initial pH7.0～8.0,35～40 ℃, 400rpm, air flow cultivation in 1: 1 16h, at 4 ℃, the centrifugal 10min of 10000rpm collects the 3.4L fermented supernatant fluid fermented liquid, adds 0.85L-20 ℃ of dehydrated alcohol precipitation, 4 ℃, the centrifugal 10min of 10000rpm collects supernatant liquor, adds 1.42L-20 ℃ of dehydrated alcohol precipitation, 4 ℃, the centrifugal 10min collecting precipitation of 10000rpm, the Tris-HCL damping fluid that precipitation adds an amount of pH8.5 (contains 0.1mmol/LCoCl
2) dissolving, with 10000rpm, behind 4 ℃ of centrifugal 10min, abandon precipitation, collect supernatant liquor, be aminopeptidase solution.Be 60g/L, in pretreated soybean protein isolate, add two kinds of enzymes simultaneously toward concentration, aminopeptidase, Sumizyme MP are respectively 10000U/g with the concentration of substrate ratio, slowly stir, use the 1mol/LNaOH titration, keep pH 8.5 ± 0.1, every alkali lye consumption of 30min record, reaction 4h, calculating DH by the pH-stat method is 25%, and the soybean polypeptide hydrolyzed solution presents bitter taste hardly.
Claims (2)
1. the preparation method of bacillus subtilis debitterized aminopeptidase is characterized in that:
The A fermentation of bacillus subtilis, collect fermented liquid:
(1) starting strain: adopting subtilis Zj016 is starting strain;
(2) fermention medium is formed: W-Gum 33～42g/L, okara powder 27～35g/L, KH
2PO
412H
2O 0.16～0.32g/L, K
2HPO
42.68～5.0g/L, NaCl 1g/L, MgSO
47H
2O0.1～0.3g/L, the CoCl of 0.1mol/L
21～3ml transfers pH7.5 with sodium hydroxide;
(3) fermentation condition: inoculum size 8%, initial pH7.5～8,35～40 ℃, 200～400rpm cultivates 15～16h;
(4) collect fermented liquid: behind 4 ℃ of centrifugal 10min of 10000rpm of fermented liquid, abandon precipitation, collect supernatant liquor;
The B ethanol precipitation:
(1) add-20 ℃ of ethanol in the supernatant liquor of collecting, making the ethanol final concentration is 20% to carry out alcohol precipitation first time, and 4 ℃ with behind the centrifugal 10min of 10000rpm, abandons precipitation, the collection supernatant liquor;
(2) add-20 ℃ of ethanol in the supernatant liquor behind the alcohol precipitation for the first time, making the ethanol final concentration is 40% to carry out alcohol precipitation, and 4 ℃ with behind the centrifugal 10min of 10000rpm, abandons supernatant, gets precipitation;
(3) precipitation adds a small amount of pH8.5 and contains 0.1mmol/LCoCl
2Tris-HCl damping fluid dissolving, 4 ℃ with behind the centrifugal 10min of 10000rpm, abandons precipitation, collects supernatant liquor, is aminopeptidase solution.
2. use the application of the debitterized aminopeptidase of claim 1 method preparation, it is characterized in that:
(1) taking by weighing soybean protein isolate is dissolved in and is mixed with the suspension that concentration is 60g/L in the deionized water, 90 ℃ of water-bath 10min;
(2) be cooled to room temperature, with 1mol/L NaOH adjust pH to 8.5;
(3) place 50 ℃ of water-baths, treat that the suspension temperature rises to 50 ℃, the amount ratio of pressing aminopeptidase, basic protein enzyme-to-substrate soybean protein isolate all is 10000U/g, add two kinds of enzymes simultaneously, slowly stir, use the 1mol/LNaOH titration, keep pH 8.5 ± 0.1, every alkali lye consumption of 30min record, hydrolysis reaction 4h;
(4) make hydrolyzed solution rise to 90 ℃ of heating 20min, passivation proteolytic enzyme rapidly after hydrolysis reaction finishes;
(5) the centrifugal 20min of hydrolyzed solution 4000rpm gets the supernatant liquor lyophilize, is soybean polypeptide.
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| CN106119173B (en) * | 2016-08-22 | 2019-09-17 | 广东轻工职业技术学院 | A kind of shrimps low molecular peptide of no bitter taste and the preparation method and application thereof |
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