CN106434799A

CN106434799A - Protein nanoparticles and preparation method thereof

Info

Publication number: CN106434799A
Application number: CN201610864092.7A
Authority: CN
Inventors: 崔春; 相欢; 卢慧茵
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2016-09-28
Filing date: 2016-09-28
Publication date: 2017-02-22

Abstract

The invention discloses protein nanoparticles and a preparation method thereof. The preparation method comprises the following steps: mixing protein with water 20-200 times the weight of the protein; adding glutaminase accounting for 0.01-1% of the protein weight; adjusting the pH; preserving heat for 0.5-24h at 20-55 DEG C; preserving heat for 5-30min at 70-95 DEG C and deactivating enzyme; centrifuging; and fetching the supernate to obtain the protein nanoparticles. Without adding any food additive, the method for preparing protein nanoparticles disclosed by the invention has the advantages of simple preparation technology, mild reaction conditions, low equipment requirements and the like.

Description

A kind of protein nanoparticles and preparation method thereof

Technical field

The present invention relates to the field of deep of protein is and in particular to a kind of protein nanoparticles and preparation method thereof.

Background technology

Domestic protein nanoparticles are done with more research.Lin Zhangchun in 2010 etc. is resisted molten using supercritical carbon dioxide Agent method be obtained from the acetone-dimethyl sulfoxide mixed solution of zein corn protein nano-particles (Lin Zhangchun, Sun Lijun, Zhao Yaping. supercritical carbon dioxide anti-solvent method prepares corn protein nano-particles [J]. food industry science and technology, 2010 (9): 216-219.).2013 Nian Liuyan armies using reverse microemulsion process prepare the gliadin nano-particle of particle diameter 100nm (Liu Yanjun, Chen Jing, Hu Shaodong, etc. reverse microemulsion process prepares the preliminary study [J] of gliadin nano-particle. biomedical engineering with face Bed, 2013 (5):411-415.).University of Fuzhou in 2014 discloses a kind of Radix Puerariae albumen and its nanometer grain preparation method (specially Sharp application number 201410541087.3).Key step：After Radix Puerariae powder is mixed with water, through Tris-HCl buffer extractions, second Alcohol class settling, ion exchange mechansim, obtain Radix Puerariae albumen, and sequence is DFVYDMCGNVLN.The water of this Radix Puerariae albumen Solution is 1mg/mL in protein concentration, under the conditions of pH6.05, after 100 DEG C of heating 60min, prepares Radix Puerariae protein nano Granule, Radix Puerariae protein nano granule all shows relatively low cytotoxicity in vitro.Radix Puerariae protein nano granule dried frozen aquatic productses flow Property good it is easy to stable preserve.Applicant declares that the nano-particle of its preparation is not related to any cross-linking agent, and its finished product is widely present In the decoction of all kinds of use Radix Puerariaes, it is that many people takes for many years, safe.The same year, Chinese Academy of Sciences's Suzhou nanometer skill Art and nano bionic institute disclose a kind of magnetic nanoparticle of the single functionalization based on ferritin (201410066418.2), it includes single functionalization ferritin shell and magnetic nanoparticle kernel, this single functionalization ferritin Shell is the asymmetric globulin with tetrahedral structure, and this asymmetric globulin is mainly by 1 saltant type Dps subunit and 11 Wild type Dps subunit forms, and this Dps is the DBP of hungry induction.Chinese Academy of Agricultural Sciences's processing of farm products in 2015 Institute discloses a kind of preparation method (201510252856.2) of Semen arachidis hypogaeae protein nano-particle, and the method comprising the steps of：Join Semen arachidis hypogaeae protein solution processed, alkali process, heat treatment, cooling, add metal ion in the modified peanut protein solution obtaining, fully Obtain Semen arachidis hypogaeae protein nanoparticles solution after reaction, the Semen arachidis hypogaeae protein obtaining nanoparticles solution carried out desalting processing, be dried, Obtain final product solid Semen arachidis hypogaeae protein nano-particle.The preparation method technological operation of this invention Semen arachidis hypogaeae protein nano-particle is simple, need not be special Different equipment, and it is not directed to organic reagent in preparation process, suitable industrialized production.The inventive method prepares Semen arachidis hypogaeae protein and receives Rice grain is spherical in shape and roundness is good, adhesion, and particle size range is 10-700nm.Liu Yong creates with soybean protein (Soy within 2015 Protein isolate, SPI) for raw material, a kind of nano-particle with multi-functional property is prepared for by gel crush method, And have studied this nano-particle stability under various circumstances and interfacial property.

In above-mentioned paper and patent, the not disclosed research report preparing protein nano granule using transglutaminase.

Content of the invention

It is an object of the invention to overcoming deficiencies of the prior art, provide a kind of protein nanoparticles and its Preparation method.

The purpose of the present invention is achieved through the following technical solutions.

A kind of preparation method of protein nanoparticles, comprises the steps：After protein is mixed with water, add paddy ammonia Amidase, adjusts pH, is incubated enzyme denaturing, centrifugation, takes supernatant, obtain protein nanoparticles.

Further, described protein includes soybean protein, Semen arachidis hypogaeae protein, wheat gluten protein, avenin, Semen Maydiss egg In vain, one of rice protein, lactalbumin, casein and lactoprotein.

Further, the weight of described protein and water is than for 1：20-200.

Further, the addition of described transglutaminase is the 0.01-1%, preferably 0.01- of protein wt 0.2%.

Further, adjusting pH value is 7.1-10.0.

Further, described insulation enzyme denaturing is after 20-55 DEG C of insulation 0.5-24h, then 70-95 DEG C of insulation 5-30min.

Further, described centrifugation is 8000g-10000g centrifugation 10-20min.

Further, described transglutaminase is from bacillus amyloliquefaciens, and described bacillus amyloliquefaciens preserve In China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC8425.

Further, described transglutaminase is from Japanese Tian Ye company transglutaminase.

Described in any of the above-described, preparation method is obtained protein nanoparticles.

Compared with prior art, the invention has the advantages that：

(1) present invention first public utilization transglutaminase catalytic proteins polymerization, forms protein nanoparticles, obtains Protein nanoparticles granularity in below 200nm.

(2) present invention is compared with supercritical carbon dioxide anti-solvent method, reverse microemulsion process, has that equipment is few, reaction bar Part is gentle, without any cross-linking agent the advantages of, there is preferable industrial prospect.

Specific embodiment

The present invention is further elaborated with reference to embodiments, but the invention is not restricted to following examples.

The transglutaminase that the present invention derives from bacillus amyloliquefaciens obtains by the following method：

Take out bacillus amyloliquefaciens SWJS22 (Bacillus amyloliquefaciensSWJS22, the CGMCC of preservation No.8425) test tube slant, in aseptic operating platform, scrapes a little bacterium colony, rules, in 37 DEG C of perseverances on neutral casein plate Cultivate 24h in warm incubator, then picking single bacterium colony is transferred to another flat board and is cultivated, and is so repeated 3 times, makes bacterial strain completely multiple Strong；The SWJS22 bacterial strain access liquid amount of activation three generations or more on neutral casein plate is 10% to picking one ring Inside LB culture medium (by tryptone 10%, yeast extract 0.2%, sodium chloride 10%, water 78% forms LB culture medium), 150rpm, cultivates 12h at 37 DEG C, obtains seed liquor.

In wheat bran：Water=5:The addition 0.5% (w/w) of 3 (w/w), sucrose ester S-1170, bacillus amyloliquefaciens The inoculum concentration 2.0% (v/w) of SWJS22 seed liquor, under conditions of 37 DEG C, ferment 48h, obtains transglutaminase, transglutaminase enzyme Vigor is 2.72GTU/g.

Embodiment 1

After 10g soybean protein is mixed with 990g water, add protein wt 0.01%, 0.02%, 0.1%, 0.2% respectively With 1% transglutaminase (Japanese Tian Ye company transglutaminase), adjust pH to 8.0, after 37 DEG C of insulation 6h, 90 DEG C of insulations 15min enzyme denaturing, 9000g is centrifuged 15min, takes supernatant, obtains protein nanoparticles；The protein concentration of reactant liquor is diluted to After 0.1wt%, its mean diameter, PDI and zeta current potential are measured using nano particle size instrument, the results are shown in Table 1.

Table 1

From table 1, with the increase of transglutaminase addition, reactant liquor particle diameter is in rising trend, its zeta current potential Change between -28.2 to -36.5, but overall particle size is all in below 200nm.

Embodiment 2

After 100g lactoprotein is mixed with 2000g, 4000g, 10000g and 20000g water respectively, add protein wt 1% Transglutaminase (from bacillus amyloliquefaciens SWJS22), adjust pH to 7.1,20 DEG C insulation 24h after, 70 DEG C insulation 30min enzyme denaturing, 8000g is centrifuged 20min, takes supernatant, obtains protein nano granule；The protein concentration of reactant liquor is diluted to After 0.1wt%, its mean diameter, PDI and zeta current potential are measured using nano particle size instrument, the results are shown in Table 2.

Table 2

From table 2, with the increase of water addition in reactant liquor, reactant liquor particle diameter is integrally on a declining curve, its zeta Current potential changes between -26.5 to -40.2, but overall particle size is all in below 200nm.

Embodiment 3

After 10g Semen arachidis hypogaeae protein is mixed with 300g water, the transglutaminase adding protein wt 0.01% is (from Xie Dian Afnyloliquefaciens SWJS22), adjust pH to 7.1,8.0,9.0 and 10.0 respectively, after 55 DEG C of insulation 0.5h, 95 DEG C of insulation 5min Enzyme denaturing, 10000g is centrifuged 10min, takes supernatant, obtains protein nano granule；The protein concentration of reactant liquor is diluted to After 0.1wt%, its mean diameter, PDI and zeta current potential are measured using nano particle size instrument, the results are shown in Table 3.

Table 3

From table 3, with the increase of pH value in reactant liquor, reactant liquor particle diameter is in integrally downward trend after first rising, Its zeta current potential changes between -36.9 to -37.9, but overall particle size is all in below 200nm.

Claims

1. a kind of preparation method of protein nanoparticles is it is characterised in that comprise the steps：Protein is mixed with water Afterwards, add transglutaminase, adjust pH, be incubated enzyme denaturing, centrifugation, take supernatant, obtain protein nanoparticles.

2. a kind of preparation method of protein nanoparticles according to claim 1 is it is characterised in that described protein bag Include soybean protein, Semen arachidis hypogaeae protein, wheat gluten protein, avenin, zein, rice protein, lactalbumin, casein and One of lactoprotein.

3. a kind of protein nanoparticles according to claim 1 preparation method it is characterised in that described protein with The weight of water is than for 1：20-200.

4. a kind of preparation method of protein nanoparticles according to claim 1 is it is characterised in that described L-Glutamine The addition of enzyme is the 0.01-1% of protein wt.

5. a kind of preparation method of protein nanoparticles according to claim 1 is it is characterised in that described L-Glutamine The addition of enzyme is the 0.01-0.2% of protein wt.

6. a kind of preparation method of protein nanoparticles according to claim 1 is it is characterised in that regulation pH value is 7.1-10.0.

7. a kind of preparation method of protein nanoparticles according to claim 1 is it is characterised in that described insulation enzyme denaturing It is after 20-55 DEG C of insulation 0.5-24h, then 70-95 DEG C of insulation 5-30min；Described centrifugation is 8000g-1000g centrifugation 10- 20min.

8. a kind of preparation method of protein nanoparticles according to claim 1 is it is characterised in that described L-Glutamine Enzyme is from bacillus amyloliquefaciens；Described bacillus amyloliquefaciens are stored in China Committee for Culture Collection of Microorganisms Common micro-organisms center, preserving number is CGMCC8425.

9. a kind of preparation method of protein nanoparticles according to claim 1 is it is characterised in that described L-Glutamine Enzyme is from Japanese Tian Ye company transglutaminase.

10. the protein nanoparticles that preparation method described in any one of claim 1 ~ 9 is obtained.