Method for improving preparation efficiency of starch ester compound

Abstract

The invention belongs to the technical field of food engineering, discloses a method for improving the preparation efficiency of a starch ester compound, and particularly relates to application of GSDL lipase OsGLIP11 in preparation of the starch ester compound. The invention discloses an amino acid sequence of OsGLIP11, which is shown in an ST.26 standard sequence nucleotide or amino acid sequence table. The starch is rice starch, and the lipid is 1, 2-dioleoyl-sn-glycerol-3-phosphorylethanolamine (DOPE) and 1, 2-distearoyl-sn-propyltriyl-3-phosphatidyl ethanolamine (DSPE), and the lipid is one or more than one or more than one or more than one or more than one or more than one or more than one or more than one or more than one or more than one or more than one or more than one or more than one or more than one or more than one or By applying more active GDSL lipase OsGLIP11, the content of a starch ester compound formed by rice starch, DOPE and DSPE can be remarkably increased. According to the method disclosed by the invention, the GDSL lipase OsGLIP11 is added, so that the starch interacts with the exogenous lipid to generate the starch lipid compound more easily. The method can provide a theoretical reference for increasing the content of the resistant starch in the rice and further improving the digestion characteristic of the rice, and the high-resistant starch rice suitable for diabetics to eat is created.

Description

Technical Field

[0001] The invention belongs to the technical field of food engineering, discloses a method for improving the preparation efficiency of starch-lipid complexes, and particularly relates to the application of GSDL lipase OsGLIP11 in the preparation of starch-lipid complexes. Background Art

[0002] Starch lipid complexes are widely present in processed starch foods, especially in heat-processed products. The formation of starch lipid complexes can promote the transformation of starch quality processing characteristics and expand the application market. Starch lipid complexes have stronger resistance to enzymatic hydrolysis than ordinary starch, so they are called RS5 resistant starch. It has certain effects in reducing digestibility, inhibiting postprandial blood sugar, and preventing colon cancer and coronary heart disease. The complex of starch and lipid is restricted by many factors, among which the selection of starch and lipid has the most direct impact on the complex of the two. The preparation of starch lipid complexes is mainly through heating method, freezing method, ultrahigh pressure method, steam jet method, extrusion method, enzyme method, solvent method, cooking method, rapid viscometer method (RVA method), differential scanning calorimeter method (DSC method), high pressure treatment method, etc. However, selecting a simple, efficient and environmentally friendly preparation method has also become the focus of many researchers. The enzyme method meets the requirements of simplicity and environmental protection, but the preparation efficiency of the enzyme method is generally low.

[0003] Studies have found that the lipids in rice that can form starch lipids with starch are mainly lysophospholipids, such as lysophosphatidylcholine and lysophosphatidylethanolamine. Lysophospholipids mainly form starch lipids with amylose. Lysophospholipids are the products of phospholipase A2 (PLA2) hydrolyzing phospholipids such as phosphatidylcholine and phosphatidylethanolamine. In the early stage of the present invention, it was found that GDSL lipase OsGLIP11 has the enzyme activity of phospholipase A2 (PLA2). So far, there has been no report on the use of OsGLIP11 protein to prepare starch lipid complexes. Summary of the invention

[0004] In view of this, the present invention aims at the problems existing in the current research on starch lipid complexes, and discloses a new enzyme that can be used to prepare starch lipid complexes. The enzyme is GDSL lipase, and its amino acid sequence is shown in SEQ ID NO.1 in the ST.26 standard sequence nucleotide or amino acid sequence table. The present invention also discloses an application method of GDSL lipase OsGLIP11 in preparing starch lipid complexes, comprising the following steps: 1) extracting OsGLIP11 protein from rice. First, the coding region sequence of the OsGLIP11 gene is constructed in the overexpression vector pCAMBIA1305.1 (UBQ10-OsGLIP11-Flag), and then the conventional japonica rice variety Nipponbare is selected as the background transformation to obtain an overexpression homozygous strain, and finally the OsGLIP11 protein is extracted and purified by a Flag tag protein immunoprecipitation kit (magnetic bead method); 2) preparing starch lipid complexes. 3.0 g of Nipponbare rice flour and 25.0 g of ddH2O, 45.0 mg of 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and 45.0 mg of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) and inactivated OsGLIP11-FLAG as the control group were accurately weighed and added into a RVA special aluminum pot and stirred evenly, and then placed in a 37 ℃ constant temperature incubator for 12 h. The experimental group was replaced with active OsGLIP11 except that the inactivated OsGLIP11 was replaced with the active OsGLIP11. The rice paste was formed according to the process of Tech Master RVA rapid viscosity analyzer (Perten, Swiss), and its RVA spectrum was measured at the same time. The ratio of DOPC, DSPE to rice flour was 1.5:100, and the amount of OsGLIP11 protein added was 1.0 μg and 2.0 μg; 3) Determination of the composite index. Weigh 2.0 g iodine and 20.0 g potassium iodide, dissolve in ddH2O, and dilute to 100.0 mL, which is the iodine stock solution. Weigh 5.0 g starch paste, place in a centrifuge tube, add 25.0 mL ddH2O, mix and shake for 2 minutes, then centrifuge at 4000 rpm / min for 10 min, pipette 2.0 mL supernatant, add 8.0 mL iodine stock solution, mix and shake, and measure the absorbance at 690 nm with an ELISA reader. The calculation formula is as follows: CI = (100 × (A1-A2) / A1), where A1 is the absorbance of the starch solution with inactivated OsGLIP11 protein added, and A2 is the absorbance of the starch solution with active OsGLIP11 protein added; 4) Observe the complex scanning electron microscope. The processed rice paste was dried in a vacuum freeze dryer for 12 h, the dried sample was ground into powder, a proper amount of powder was taken with a toothpick and spread evenly on the black double-sided tape, and the particle morphology was observed under a scanning electron microscope after gold spraying.

[0005] The present invention discloses a method for improving the preparation efficiency of starch lipid complexes, and provides a new GDSL lipase OsGLIP11 that can be used to prepare starch lipid complexes. Adding the enzyme during the preparation of starch lipid complexes can significantly improve the preparation efficiency. The present invention can provide a theoretical reference for increasing the content of resistant starch in rice and thus improving its digestive properties, and create high-resistant starch rice suitable for diabetic patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 The present invention is used to construct a vector pCAMBIA1305.1 for overexpressing the OsGLIP11 gene, wherein UBQ10 is used as a strong promoter to drive the overexpression of OsGLIP11 in the conventional japonica rice variety Nipponbare, and the Flag tag protein is used to extract and purify the OsGLIP11 protein in Nipponbare.

[0007] Figure 2 .Determination of the complex index and scanning electron microscopy observation of starch lipid complex (CI). Figure A shows the CI values ​​when 1.0 μg and 2.0 μg DOPE are added. The CI value when 2.0 μg of active DOPE is added is significantly higher than that when 1.0 μg is added. At the same time, Figure C also shows that the RVA spectrum when 2.0 μg DOPE is added is significantly different from that when 1.0 μg is added; Figure B shows the CI values ​​when 1.0 μg and 2.0 μg DSPE is added. The CI value when 2.0 μg DSPE is added is significantly higher than that when 1.0 μg is added. At the same time, Figure C also shows that the RVA spectrum when 2.0 μg DSPE is added is significantly different from that when 1.0 μg is added; Figure E (i) and (iii) are scanning electron microscopy images when inactivated OsGLIP11 protein is added when preparing starch lipid complex (DOPE), and (ii) and (iv) are scanning electron microscopy images when 2.0 μg DSPE is added when preparing starch lipid complex (DOPE). Figures (v) and (vii) are scanning electron micrographs when inactivated OsGLIP11 protein is added to the preparation of starch-lipid complex (DSPE), and Figures (vi) and (viii) are scanning electron micrographs when 2.0 μg active OsGLIP11 protein is added to the preparation of starch-lipid complex (DSPE). The results show that the addition of active OsGLIP11 protein significantly increases the content of starch-DOPE and starch-DSPE complexes. * indicates P The yellow arrows in (v) and (vii) indicate that DSPE did not form a complex with starch, while the red arrows in (vi), (viii), (ii) and (iv) indicate the complex structure (small protrusion) formed by DOPE and DSPE with starch.

Claims

1. A method for improving the preparation efficiency of starch-lipid complexes, characterized in that: Application of OsGLIP11 protein can significantly increase the content of starch-lipid complex formed by rice starch and exogenous lipids.

2. A method for using the OsGLIP11 protein according to claim 1, characterized in that: The OsGLIP11 protein is a GDSL lipase, and its amino acid sequence is shown in the ST.26 standard sequence nucleotide or amino acid sequence table.

3. A method of using the exogenous lipids of claim 1 as 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE).

4. A method for improving the preparation efficiency of starch-lipid complexes using the method of claim 1, characterized in that: Adding OsGLIP11 protein can improve the efficiency of starch-lipid complex preparation.

5. The use according to claim 4, characterized in that: In overexpression OsGLIP11 OsGLIP11 protein was extracted from transgenic rice.

6. The use according to claim 4, characterized in that: Construct overexpression OsGLIP11 The forward sequence of the primer nucleotide used in the gene vector is 5′→3′: as shown in SEQ ID No. 2 in the ST.26 standard sequence nucleotide or amino acid sequence table; the reverse sequence of the primer nucleotide is 5′→3′: as shown in SEQ ID No. 3 in the ST.26 standard sequence nucleotide or amino acid sequence table.

7. The use according to claim 4, characterized in that: When preparing the starch-lipid complex, the mass ratio of exogenous lipid to rice flour was 1.5:

100.

8. The use according to claim 4, characterized in that: When preparing the starch-lipid complex, the amount of active OsGLIP11 protein added was 1.0 μg and 2.0 μg.

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