A method for extracting a cereal thermostable lipase

Abstract

The application discloses a kind of extraction methods of cereal heat-resistant lipase, and it relates to the field of biological extraction technology.The extraction method comprises the following steps: (1) the cereal bran is carried out high-temperature stabilization treatment, and the stabilized bran is obtained;(2) the solution containing surfactant is used to extract the stabilized bran, and the cereal heat-resistant lipase is obtained.The advantages of the extraction method are: (1) by adding surfactant in the buffer solution, heat-resistant lipase can be extracted from the stabilized bran;(2) the extracted lipase has good heat resistance, and the extraction method is simple.The application explores the enzymatic characteristics of cereal heat-resistant lipase, thereby enriching the types of cereal endogenous lipase, and providing theoretical basis and guidance for the development of efficient and stable processing technology of cereal bran.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological extraction, in particular to a method for extracting heat-resistant lipase from cereal bran. BACKGROUND

[0002] Rice and wheat are the main food crops in China, and a large amount of by-products bran is produced during their processing. Bran contains rich high-quality protein, oil, dietary fiber, vitamins and minerals, and has great development value. However, at present, most of the bran by-products are used for feed, and the level of high-value utilization is low, which seriously affects the economic benefits of grain processing enterprises.

[0003] The bottleneck restricting the deep processing and utilization of bran is that it is prone to oxidation and rancidity. Bran contains rich oil, accounting for 15-20% (w / w), which undergoes strong hydrolysis and oxidation under the action of endogenous enzymes, causing rapid rancidity and deterioration of bran, producing toxic and harmful components, and affecting its high-value utilization. A series of biological, physical and thermal processing methods are used to inactivate bran lipase and improve its storage stability. Among them, the method of heat treatment to inactivate endogenous enzymes of bran is relatively effective, but even so, the stable bran still retains about 10-20% of lipase activity, affecting the long-term storage stability of bran.

[0004] Taking rice bran as an example, there are currently 7 kinds of lipase reported from rice bran, 6 of which belong to normal temperature lipase, and their optimal reaction temperature and thermal stability are between 30-50℃. Only (Phospho) Lipase reported by Bhardwaj et al. has an optimal reaction temperature of 80℃, good thermal stability at 60℃, and significantly reduced thermal stability above 60℃ (Table 1). These results show that the reported rice bran lipases are not outstanding in heat resistance, and high-pressure steam treatment at 121℃ for 20 min should completely inactivate the lipase in rice bran, but in fact, the rice bran treated by this method still retains 10-15% of lipase activity. The reason may be related to the fact that heat-resistant lipase has not been found in rice bran.

[0005] Table 1 Overview of different rice bran lipase properties

[0006]

[0007]

[0008] Limited to this, the present application aims to extract heat-resistant lipase from cereal bran, thereby providing a theoretical basis for developing efficient cereal bran stabilization methods. SUMMARY

[0009] The application aims to provide a method for extracting heat-resistant lipase from cereal bran, so as to solve the problems in the prior art, and enrich the types of endogenous lipase in cereal, and provide a theoretical basis for the development of high-efficiency and stable processing technology of cereal bran.

[0010] To achieve the above-mentioned purpose, the application provides the following solutions.

[0011] The application provides a method for extracting heat-resistant lipase from cereal, comprising the following steps.

[0012] (1) subjecting the cereal bran to high-temperature stabilization treatment to obtain stabilized bran;

[0013] (2) extracting the stabilized bran by using a solution containing a surfactant to obtain the heat-resistant lipase from cereal.

[0014] Further, in step (1), the high-temperature stabilization treatment is performed at a temperature of 100-130 DEG C for 10-30 min.

[0015] Further, in step (2), the solution is one of phosphate, citric acid, Tris (tris-hydroxymethyl aminomethane), HEPES and the like.

[0016] Further, in step (2), the surfactant is at least one of an anionic surfactant, a cationic surfactant and a non-ionic surfactant.

[0017] Further, the anionic surfactant is sodium dodecyl sulfate.

[0018] Further, the cationic surfactant is trimethyl 1-hexadecyl ammonium bromide.

[0019] Further, the non-ionic surfactant is Triton X-100.

[0020] Further, in step (2), the mass-volume ratio of the stabilized bran to the solution containing the surfactant is 1: (5-10) mL.

[0021] Further, the mass fraction of the surfactant in the solution is 5-50 mM.

[0022] The application also provides heat-resistant lipase from cereal extracted by the above-mentioned extraction method.

[0023] The application discloses the following technical effects:

[0024] The application provides a method for extracting heat-resistant lipase from cereals, which has the advantages of (1) heat-resistant lipase can be extracted from stabilized bran by adding a surfactant in a buffer solution; and (2) the extracted lipase has good heat resistance and the extraction method is simple. The application enriches the types of endogenous lipase in cereals and provides a theoretical basis for the development of efficient and stable processing technology of cereal bran. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0026] Figure 1 Optimum reaction temperature test results of the lipases of Example 3 and Comparative Example 1. DETAILED DESCRIPTION

[0027] The various exemplary embodiments of the present application will now be described in detail below, which should not be considered as limiting the present application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.

[0028] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value in the range, and any other stated value or intermediate value in the range, is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are concerned. In the event of any conflict between the content of this specification and the documents incorporated by reference, the content of this specification will control.

[0030] Many modifications and variations of the specific embodiments of the application can be practiced in accordance with the teachings of the description of the application, which are within the scope of the present application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples of the application are exemplary only.

[0031] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", and the like are open-ended terms that are intended to mean including, but not limited to.

[0032] The lipase specific activity determination method in the following examples is as follows:

[0033] The lipase activity of the crude enzyme solution was determined by the p-nitrophenyl laurate method (Laokuldilok et al., Cereal Chemistry, 2014, 91(6): 560-565); A solution (0.1 g of p-nitrophenyl laurate (PNPL) was dissolved in 30 mL of isopropyl alcohol) and a B solution (0.4 g of triton X-100 and 0.1 g of gum arabic were dissolved in 90 mL of pH 7.0 phosphate buffer) were prepared, 0.4 mL of the A solution was mixed with 6.0 mL of the B solution, then 0.4 mL of the crude enzyme solution was added, and the mixture was reacted at 90°C for 40 min, then was placed in an ice bath for 2 min, and then was centrifuged at 8000 r / min at 4°C for 5 min. The absorbance of the supernatant was measured at 410 nm. A standard curve was prepared using p-nitrophenol, and one unit (U) of lipase activity was defined as the amount of p-nitrophenol generated per gram of sample per hour. Since the reaction temperature is relatively high (90°C), it is considered that the sample has lipase activity if it is determined to have lipase activity by this method.

[0034] The protein content in the crude enzyme solution was determined by the Kjeldahl method. The lipase specific activity was calculated based on the ratio of the total lipase activity of the crude enzyme solution to the protein content.

[0035] The following takes rice bran as an example to illustrate the extraction method of the cereal heat-resistant lipase of the application:

[0036] Example 1

[0037] The extraction method of the rice bran heat-resistant lipase is as follows:

[0038] (1) Stabilization of rice bran: The rice bran was placed in a rectangular container with holes and was placed in a sterilization kettle. The rice bran was stabilized at 100°C for 30 min. After stabilization, the rice bran was taken out of the sterilization kettle and was placed at room temperature for 2 h. The rice bran was stirred every 1 h for 2 times. After cooling to room temperature, the rice bran was dried and stored for use.

[0039] (2) Heat-resistant lipase extraction: The rice bran was weighed and placed in a sealed container with a lid. Buffer was added at a ratio of 10 mL buffer per 1 g of rice bran. The buffer was 50 mM phosphate buffer at pH 7.0 containing 50 mM anionic surfactant sodium dodecyl sulfate. After mixing the rice bran and buffer, the mixture was placed in a shaker for oscillation treatment at a shaking rate of 180 r / min for 60 min. Subsequently, the mixture was centrifuged at 8000 r / min for 30 min, and the crude enzyme solution (i.e., the supernatant containing the heat-resistant lipase) was collected and stored at room temperature.

[0040] The specific activity of the heat-resistant lipase of the crude enzyme solution prepared in this example was 126 U / mg.

[0041] Example 2

[0042] The extraction method of the heat-resistant lipase of rice bran was as follows:

[0043] (1) Stabilization of rice bran: The rice bran was placed in a rectangular container with holes and was placed in a sterilization kettle. The rice bran was stabilized at 120°C for 15 min. After stabilization, the rice bran was removed from the sterilization kettle and was placed at room temperature for 3 h, with stirring every 1 h for 2 times. After cooling to room temperature, the rice bran was dried and stored for use.

[0044] (2) Heat-resistant lipase extraction: The rice bran was weighed and placed in a sealed container with a lid. Buffer was added at a ratio of 5 mL buffer per 1 g of rice bran. The buffer was 50 mM citrate buffer at pH 6.0 containing 25 mM cationic surfactant trimethyl 1-hexadecyl ammonium bromide. After mixing the rice bran and buffer, the mixture was placed in a shaker for oscillation treatment at a shaking rate of 200 r / min for 40 min. Subsequently, the mixture was centrifuged at 7000 r / min for 20 min, and the crude enzyme solution (i.e., the supernatant containing the heat-resistant lipase) was collected and stored at room temperature.

[0045] The specific activity of the heat-resistant lipase of the crude enzyme solution prepared in this example was 82 U / mg.

[0046] Example 3

[0047] The extraction method of the heat-resistant lipase of rice bran was as follows:

[0048] (1) Stabilization of rice bran: The rice bran was placed in a rectangular container with holes and was placed in a sterilization kettle. The rice bran was stabilized at 130°C for 5 min. After stabilization, the rice bran was removed from the sterilization kettle and was placed at room temperature for 4 h, with stirring every 1 h for 3 times. After cooling to room temperature, the rice bran was dried and stored for use.

[0049] (2) Heat-resistant lipase extraction: Weigh rice bran and place it in a sealed container with a lid. Add buffer solution at a ratio of 10 mL buffer solution per 1 g of rice bran. The buffer solution is a 50 mM Tris buffer solution with pH 8.0 (containing 5 mM nonionic surfactant Triton X-100). After mixing the rice bran and buffer solution, place it in a shaker for 10 min at a shaking rate of 250 r / min. Then, centrifuge the mixture at 8000 r / min for 10 min, collect the crude enzyme solution (i.e., the supernatant containing heat-resistant lipase), and store it at room temperature.

[0050] The specific activity of rice bran heat-resistant lipase in the crude enzyme solution prepared in this embodiment is 152 U / mg.

[0051] Comparative Example 1

[0052] Same as Example 3, except that in step (2), the buffer solution does not contain Triton X-100. The specific activity of rice bran thermostable lipase in the crude enzyme solution prepared in this comparative example is 18 U / mg.

[0053] The optimal reaction temperature of the rice bran thermostable lipase (TRBL) crude enzyme solution extracted in Example 3 and the crude enzyme solution (PRBL) extracted in Comparative Example 1 were determined, and the results are as follows: Figure 1 As shown. By Figure 1 It is evident that there is a significant difference in the optimal reaction temperatures between the two crude enzyme solutions. The optimal reaction temperature for PRBL is around 30℃, while the optimal temperature for TRBL exceeds 100℃. This result indicates that lipase extracted solely with phosphate buffer is not heat-resistant, while lipase extracted with phosphate buffer and surfactant exhibits higher heat resistance, and its crude enzyme solution still displays high activity at 100℃.

[0054] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for extracting heat-resistant lipase from rice bran, characterized in that, Includes the following steps: (1) Stabilization of rice bran: Take rice bran and place it in a perforated rectangular container. Place it in a sterilizing kettle and stabilize the rice bran at 130℃ for 5 minutes. After stabilization, take the rice bran out of the sterilizing kettle and place it at room temperature for 4 hours. Stir it 3 times every hour. After cooling to room temperature, dry it to obtain stabilized rice bran and store it for later use. (2) Heat-resistant lipase extraction: Weigh the stabilized rice bran and place it in a sealed container with a lid. Add buffer solution at a ratio of 10 mL buffer solution per 1 g of rice bran. The buffer solution is a 50 mM Tris buffer solution with pH 8.

0. The buffer solution contains 5 mM of nonionic surfactant Triton X-100. After mixing the stabilized rice bran with the buffer solution, place it in a shaker for shaking treatment. The treatment conditions are a shaking rate of 250 r / min and a shaking time of 10 min. Subsequently, the mixture was centrifuged in an 8000 r / min centrifuge for 10 min, and the crude enzyme solution was collected to obtain the rice bran heat-resistant lipase.

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