CN101492663B - Fermentation preparation and extraction method for bacillus subtilis debitterized aminopeptidase - Google Patents

Abstract

The invention discloses a method for preparing and extracting Bacillus subtilis debittering aminopeptidase by fermentation, which belongs to the technical field of enzyme preparation and food additive. The invention uses the fermentation cylinder production condition of Bacillus subtilis Zj016 to collect fermentation liquor and obtain the product of debittering aminopeptidase by carrying out clarifying, ultrafiltration concentration and salting out on the fermentation liquor. The aminopeptidase produced by the selected Bacillus subtilis Zj016 is exopeptidase which can dissociate amino acid from a polypeptide chain amino terminal. Researched by the laboratory, the aminopeptidase cooperates with alkali protease to hydrolyze soybean protein isolate; the prepared soybean peptide does not have bitterness, and the contents of oligopeptide like dipeptide and tripeptide are higher. Besides, researches on the enzyme lines produced by the Bacillus subtilis show that the Bacillus subtilis only produces circumscribed prolease and does not have the activity of endo protease; moreover, the circumscribed prolease has a stronger hydrolysis capacity to the terminal amino acid with strong hydrophobicity. The research further explains that the invention can reduce or eliminate the bitterness generated during hydrolysis of the soybean protein.

Description

A kind of fermentative preparation of bacillus subtilis debitterized aminopeptidase and the method for extraction

Technical field

The method of a kind of bacillus subtilis debitterized aminopeptidase mass-producing fermentative preparation and extraction; specifically utilize fermentation of bacillus subtilis, collect fermented liquid, by clarification, ultrafiltration and concentration with saltout; obtain the debitterized aminopeptidase goods, belong to zymin, technical field of food additives.

Background technology

Proteolysis produces peptide matters and a small amount of amino acid, its solvability, thermostability and nutritive property be improved significantly, be important material in the modern food course of processing.But often produce the bitter peptides that is bitter taste in the hydrolyzed solution, this bitter peptides generally is the polypeptide chain that the ammonia end has single or multiple hydrophobic amino acids, also may be carboxylic end band hydrophobic amino acid sometimes, and bitter taste has limited the application of protein hydrolystate.Therefore, reduce, stop and remove the bitter taste of protein hydrolystate, become the important topic of current research.

Debittering method rationalization and enzyme process debitterize, physics and chemistry debitterize comprise separation, extract, adsorb, cover, protein transformation and acid and alkali hydrolysis etc.But these method debitterizes are not thorough, can destroy some amino acid, and especially necessary amino acid reduces proteinic nutritive value.Compare the various key properties advantages such as (as solvability, low antigenicities) that enzyme process has hydrolysis efficiency height, mild condition, the nutritive ingredient of protein polypeptide lost, hydrolytic process is easy to control, do not change protein peptide.Utilize the bitter taste of enzyme control peptide, aminopeptidase method is arranged, carboxypeptidase method, alkalescence/neutral protein enzyme process, aminopeptidase method is more more extensive than back two kinds of method sphere of action comparatively speaking, and de-bittering effect is better.The existing report of domestic research about the production by biological carboxypeptidase, then less about the report of production by biological aminopeptidase.In addition, aminopeptidase also can be widely used in meat product processing, Dairy industry etc., reduces bitter taste and increases total free aminoacids, improves food value.Aminopeptidase also can be used as the molecular tool of protein sequence mensuration and the solidifying agent of recombinant protein or fusion product.

Enzymolysis protein matter is produced polypeptide has become current popular research, can obtain the active polypeptide of difference in functionality by different enzyme butt formulas, about the existing a lot of relevant reports of this respect.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 bitter taste that the soybean polypeptide product has in various degree can directly influence flavours in food products, has " unhappiness " when eating to people, has 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.

Summary of the invention

The purpose of this invention is to provide a kind of fermentative preparation of bacillus subtilis debitterized aminopeptidase and the method for extraction, adopt a strain can produce microorganism with de-bittering effect aminopeptidase, by fermentation culture, centrifugal, clarification, ultrafiltration and concentration and technology such as saltout, finally obtain aminopeptidase.

Technical scheme of the present invention: it is in the substratum of carbon, nitrogenous source that subtilis is inserted with Semen Maydis powder, okara powder, regulates the pH of fermention medium, and fermentation culture is collected fermented liquid, by clarification, ultrafiltration and concentration with saltout, obtains thick enzyme, and technology is:

The A fermentation of bacillus subtilis, collect fermented liquid:

(1) starting strain: adopting subtilis Zj016 is starting strain; This bacterial strain is seen [Wei Yajuan etc., ' applied research of bacillus subtilis debitterized aminopeptidase in the hydrolyzed soy protein isolate ', foodstuffs industry science and technology Vol 29, No.04,2008.p149-151];

(2) seed culture medium: peptone 10g/L, extractum carnis 5g/L, NaCl 5g/L, pH7.2;

(3) seed culture: be divided into first order seed cultivation and secondary seed and cultivate, the firsts and seconds seed culture medium is identical;

First order seed is cultivated: get in the 50mL seed culture medium in the good bacterial classification access 250mL culturing bottle of 0.2mL preservation, rotating speed 200rpm, cultivates 8h by 37 ℃;

Secondary seed is cultivated: get primary seed solution and be inoculated in the seed culture medium, rotating speed 200rpm, cultivates 6h by 37 ℃;

(4) fermention medium is formed: Semen Maydis powder 25～35g/L, okara powder 6～12g/L, K ₂HPO ₄2.7～3.8g/L, MgSO ₄7H ₂O 0.5～0.7g/L, the CoCl of concentration 0.1mol/L ₂The every L substratum of 1～3mL/ is regulated pH to 7.5;

(5) pre-treatment of raw material:

Liquefaction processing: press raw material and a certain amount of water mixing of the composition of fermention medium with fermention medium, temperature is elevated to 85 ℃, transfer pH to 5.6, diastatic consumption 40U/g culture medium raw material ceaselessly stirs hydrolysis 2h;

Saccharification is handled: will be cooled to 65 ℃ through the culture medium raw material after the liquefaction processing, and transfer pH to 4.5, the consumption 500U/g culture medium raw material of saccharifying enzyme ceaselessly stirs hydrolysis 1.5h;

(6) ferment tank condition: 7L fermentor tank liquid amount 4L, the inoculum size by volume is 4%, initial pH7.0-8.0, culture temperature 35-40 ℃, rotating speed 400～1000rpm, air flow are 2: 1v/vmin, dissolved oxygen DO coordinates mutually with rotating speed, and control DO 〉=60% is cultivated 15h;

Feed supplement operation in the fermenting process: the solution of feed supplement is 200g glucose/L, the feed supplement volume is 200mL, the mode of feed supplement is selected disposable feed supplement or twice feed supplement for use, determine that concentration of reduced sugar is not less than 2g/L in the maintenance fermented liquid opportunity of feed supplement according to the change in concentration of the reducing sugar in the fermenting process;

(7) collect fermented liquid: behind 4 ℃ of fermented liquids, the centrifugal 10min of 10000rpm, abandon precipitation, collect supernatant liquor;

The extraction of B aminopeptidase:

(8) adding massfractions down at 4 ℃ in the supernatant liquor of collecting is 15% (NH ₄) ₂SO ₄, 4 ℃, the centrifugal 10min of 10000rpm abandon precipitation, collect clear liquor;

(9) clear liquor of collecting is selected for use to hold back relative molecular mass be that the ultra-filtration membrane of 30kDa carries out ultrafiltration and concentration, concentrated 3-5 doubly;

(10) adding massfraction again in concentrated solution is 25% (NH ₄) ₂SO ₄Saltout, 4 ℃, the centrifugal 10min of 10000rpm abandon supernatant, get precipitation;

(11) will precipitate lyophilize, promptly obtain the solid aminopeptidase.

The aminopeptidase of preparation is as stated above pressed its hydrolysis situation to several substrates of certain methods analyst, find its no endo type protease activity (acidity, neutrality or Sumizyme MP), only having circumscribed amino-peptidase activity, is that the stronger amino acid whose effect of hydrophobicity is stronger to end.

Beneficial effect of the present invention: the microorganism subtilis Zj016 that the present invention adopts a strain to produce to have the de-bittering effect aminopeptidase, by fermentation culture, centrifugal, clarification, ultrafiltration and concentration and technology such as saltout, the final aminopeptidase that obtains, the yield of whole process flow is 87.7%, the ratio of enzyme is lived and is 1102055U/mg, 50 ℃ of optimum temperutures, 45～70 ℃ of temperature-stable scopes, optimal pH 8.0, stable pH range 7.0～9.5.

The aminopeptidase that makes is an exopeptidase, the amino acid that can dissociate from the aminoterminal of polypeptide chain, and 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.In addition, by discovering to enzyme that this subtilis produces system, it only produces circumscribed-type proteolytic enzyme, the activity that does not possess endo-protease, and this circumscribed proteolytic enzyme is stronger to the strong amino acid whose hydrolysis ability of terminal hydrophobicity, and this research illustrates further it and can reduce or eliminate the bitter taste that produces in the soybean protein hydrolysis.

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 4% again, fermention medium is formed as described in the specification sheets, earlier through liquefaction and saccharification processing.Initial pH7.0～8.0,35～40 ℃, 400～1000rpm, air flow 2: 1, minimum DO is controlled at 60%, and DO and rotating speed coupling mutually cultivate 15h.At 4 ℃, the centrifugal 10min of 10000rpm collects the 3.28L fermented liquid with fermented liquid, and the adding massfraction is 15% (NH under 4 ℃ ₄) ₂SO ₄, 4 ℃, the centrifugal 10min of 10000rpm collects clear liquor, selects for use the ultra-filtration membrane of 30kDa to concentrate suitable multiple, adds massfraction again and be 25% (NH in concentrated solution ₄) ₂SO ₄Saltout, 4 ℃, the centrifugal 10min collecting precipitation of 10000rpm will precipitate lyophilize, promptly obtain the solid aminopeptidase.

Embodiment 2

Aminopeptidase prepared among the embodiment 1 is carried out enzyme system and substrate specificity Journal of Sex Research, measure its acidity, neutrality and alkaline protease activity respectively, the result does not have relevant endo-type protein-active.Discover that further it has circumscribed amino-peptidase activity, it is that the activity of L-leucine p-Nitroaniline (Leu-pNA) is the strongest for substrate, can reach about 3900U/mL, for substrate is that L-L-glutamic acid p-Nitroaniline (Glu-pNA) activity only is 391U/mL, and be L-glycine-no hydrolysis ability of proline(Pro) p-Nitroaniline (Gly-Pro-pNA) for substrate, show that it is that the stronger amino acid whose effect of hydrophobicity is stronger to end.

Claims (1)

1. the method for the fermentative preparation of a subtilis aminopeptidase and extraction is characterized in that technology is: the A fermentation of bacillus subtilis, collect fermented liquid:

(1) starting strain: adopting subtilis Zj016 is starting strain;

(2) seed culture medium: peptone 10g/L, extractum carnis 5g/L, NaCl 5g/L, pH7.2;

(3) seed culture: be divided into first order seed cultivation and secondary seed and cultivate, the firsts and seconds seed culture medium is identical;

First order seed is cultivated: get in the 50mL seed culture medium in the good bacterial classification access 250mL culturing bottle of 0.2mL preservation, rotating speed 200rpm, cultivates 8h by 37 ℃;

Secondary seed is cultivated: get primary seed solution and be inoculated in the seed culture medium, rotating speed 200rpm, cultivates 6h by 37 ℃;

(4) fermention medium is formed: Semen Maydis powder 25～35g/L, okara powder 6～12g/L, K ₂HPO ₄2.7～3.8g/L, MgSO ₄7H ₂O 0.5～0.7g/L, the CoCl of concentration 0.1mol/L ₂The every L substratum of 1～3mL/ is regulated pH to 7.5;

(5) pre-treatment of raw material:

Liquefaction processing: press raw material and a certain amount of water mixing of the composition of fermention medium with fermention medium, temperature is elevated to 85 ℃, transfer pH to 5.6, diastatic consumption 40U/g culture medium raw material ceaselessly stirs hydrolysis 2h;

Saccharification is handled: will be cooled to 65 ℃ through the culture medium raw material after the liquefaction processing, and transfer pH to 4.5, the consumption 500U/g culture medium raw material of saccharifying enzyme ceaselessly stirs hydrolysis 1.5h;

(6) ferment tank condition: 7L fermentor tank liquid amount 4L, the inoculum size by volume is 4%, initial pH 7.0-8.0, culture temperature 35-40 ℃, rotating speed 400～1000rpm, air flow are 2: 1v/vmin, dissolved oxygen DO coordinates mutually with rotating speed, and control DO 〉=60% is cultivated 15h;

Feed supplement operation in the fermenting process: the solution of feed supplement is 200g glucose/L, the feed supplement volume is 200mL, the mode of feed supplement is selected disposable feed supplement or twice feed supplement for use, determine that concentration of reduced sugar is not less than 2g/L in the maintenance fermented liquid opportunity of feed supplement according to the change in concentration of the reducing sugar in the fermenting process;

(7) collect fermented liquid: behind 4 ℃ of fermented liquids, the centrifugal 10min of 10000rpm, abandon precipitation, collect supernatant liquor;

The extraction of B aminopeptidase:

(8) adding massfractions down at 4 ℃ in the supernatant liquor of collecting is 15% (NH ₄) ₂SO ₄, 4 ℃, the centrifugal 10min of 10000rpm abandon precipitation, collect clear liquor;

(9) clear liquor of collecting is selected for use to hold back relative molecular mass be that the ultra-filtration membrane of 30kDa carries out ultrafiltration and concentration, concentrated 3-5 doubly;

(10) adding massfraction again in concentrated solution is 25% (NH ₄) ₂SO ₄Saltout, 4 ℃, the centrifugal 10min of 10000rpm abandon supernatant, get precipitation;

(11) will precipitate lyophilize, promptly obtain the solid aminopeptidase.

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