阅读说明：本技术 一种酶法酯交换的技术 (Technology for enzymatic transesterification ) 是由 王永华 何诗 戚穗坚 杨博 王卫飞 蓝东明 罗日明 于 2020-12-08 设计创作，主要内容包括：本发明涉及一种酶法酯交换方法,包括以下步骤：在脂肪酶的作用下,甘油三酯A与甘油三酯B进行酯交换反应；所述甘油三酯A为短链甘油三酯、中链甘油三酯和长链甘油三酯中的至少一种；所述甘油三酯B为长链甘油三酯；所述脂肪酶为脂肪酶MAS1或MAS1-H108A。本发明方法可以同时高收率地实现短/中/长甘油三酯与长链甘油三酯的酯交换。(The invention relates to an enzymatic transesterification method, which comprises the following steps: under the action of lipase, triglyceride A and triglyceride B are subjected to transesterification reaction; the triglyceride A is at least one of short chain triglyceride, medium chain triglyceride and long chain triglyceride; the triglyceride B is long-chain triglyceride; the lipase is a lipase MAS1 or MAS 1-H108A. The process of the present invention can simultaneously achieve transesterification of short/medium/long triglycerides with long triglycerides in high yield.)

1. An enzymatic transesterification process, comprising the steps of: under the action of lipase, triglyceride A and triglyceride B are subjected to transesterification reaction;

the triglyceride A is at least one of short chain triglyceride, medium chain triglyceride and long chain triglyceride;

the triglyceride B is long-chain triglyceride;

the lipase is a lipase MAS1 or MAS 1-H108A.

2. The enzymatic transesterification process of claim 1, wherein the lipase is MAS 1-H108A.

3. The enzymatic transesterification process of claim 1, wherein the mass ratio of triglyceride A to triglyceride B is 1: 2.5-5; further, the mass ratio of triglyceride a to triglyceride B is 1: 2.5 to 3.5.

4. The enzymatic transesterification method according to claim 1, wherein the temperature of the transesterification reaction is 55 to 75 ℃.

5. The enzymatic transesterification method according to claim 4, wherein the temperature of the transesterification reaction is 65 to 75 ℃.

6. The enzymatic transesterification method according to any one of claims 1 to 5, wherein the lipase is an immobilized lipase.

7. The enzymatic transesterification method according to any one of claims 1 to 5, wherein the lipase is added in an amount of 20 to 100U/g substrate.

8. The enzymatic transesterification method according to any one of claims 1 to 5, wherein the short-chain triglyceride in the triglyceride A is at least one of triacetin, tripropionin and tributyrin; and/or the medium chain triglyceride is at least one of tricaprylin, tricaprin and trilaurin; and/or the long-chain triglyceride is at least one of tripalmitin, tristearin, palm oil, olive oil, rapeseed oil, tea oil, soybean oil, walnut oil, corn oil, beef tallow and lard.

9. The enzymatic transesterification method according to any one of claims 1 to 5, wherein the long-chain triglyceride in the triglyceride B is at least one of glycerol trioleate, glycerol trilinoleate, palm oil, olive oil, rapeseed oil, tea oil, soybean oil, walnut oil, corn oil, beef tallow and lard.

10. The enzymatic transesterification method according to any one of claims 1 to 5, wherein the reaction system is a solvent reaction system or a solvent-free reaction system; further, when the reaction system is a solvent reaction system, the reaction solvent is tert-butyl alcohol.

Technical Field

The invention relates to the field of chemistry, in particular to an enzymatic transesterification method.

Background

Fats and oils are widely used in food and industrial applications. However, most edible fats and oils have limited technical applications because the natural triglyceride structure does not provide the desired properties to the product. In order to enhance the technical characteristics of natural edible oil and fat, the structure of triglyceride can be changed through the process of ester exchange, thereby changing the physicochemical properties of the triglyceride.

The transesterification includes two types of chemical transesterification and enzymatic transesterification, and the chemical transesterification modifies the structural composition of triglyceride through random transesterification and is widely used in industrial production. However, enzymatic transesterification has received much attention in recent years, and has various advantages over conventional chemical transesterification because an enzyme is reacted as a catalyst, the process is simple, flexible, involves no use of harmful chemicals and has less by-products, and degradation of heat-unstable bioactive compounds and fatty acids is inhibited because the reaction temperature of enzymatic transesterification is generally not high. Thus, a final product with better nutritional quality and shelf life may be obtained.

Because fatty acids with different carbon chains have different functions, different combinations and orientations of different types of fatty acids form different structural lipids. In the preparation of structural lipids, short chain fatty acids, medium chain fatty acids and long chain fatty acids are mainly included as functional fatty acid sources of structural lipids.

The long-chain and short-chain fatty acids in the short-long chain structure fat are randomly arranged on a glycerol skeleton, the short-chain fatty acids can be rapidly metabolized in the liver in the digestion process, and the heat quantity is only about half of that of common fat. Therefore, the preparation of the short-chain and long-chain triglyceride has wide application significance. Zhang Jing et al used glyceryl triacetate and glyceryl trioleate as raw materials, lipase Lipozyme TL IM as a catalyst, and the reaction temperature was 55 ℃, glyceryl triacetate: the yield of the short-long chain structured fat prepared by using triolein as 2:1(mol/mol), reacting for 18h, using enzyme as 25IUN/g substrate and organic solvent as n-hexane at a shaking rate of 50Hz is 76% (Zhang crystallon, Zhou Jia Chun, Xia quan Ming. low calorie fat Salatrim developed [ J ] grain and fat 2004(01): 22-24). The Lipozyme RM IM is used as a catalyst in the workmanship, 10 percent of enzyme is added, 1 percent of water content is added, the substrate molar ratio is 2.5:1, the reaction temperature is 55 ℃, the reaction is carried out for 12 hours, and the ester exchange reaction is carried out to prepare the structure fat containing unsaturated fatty acid (the workmanship of the enzymatic preparation and the characteristic research of the low-calorie structure fat [ D ] the university of compost industry, 2015 ]).

Medium chain fatty acids, because of their smaller volume and better solubility, can enter the liver directly through the portal vein. When the medium-chain fatty acid enters the villus cells of the small intestine, the medium-chain fatty acid can not be re-esterified to form triglyceride, so that fat accumulation can not be formed. Therefore, the prepared medium-long chain triglyceride has wide application significance. And the Yee Ying Lee and the like select lipase Lipozyme TL IM, and take palm kernel oil and palm oil as substrates to synthesize the MLCT structural lipid by enzymatic transesterification. Optimal reaction conditions are obtained through response surface method optimization: the mass ratio of the palm kernel oil to the palm oil substrate was 9:1, the reaction time was 7.26h, the temperature was 50 ℃ and the enzyme addition amount was 5%, and the content of the obtained MLCT-structured fat was 60% (Lee Yee-Ying etc., Journal of food science and technology,2015,52 (2)). Zhaomanli [6] takes camphor tree seed oil and tea oil as reaction substrates, lipase Lipozyme TL IM is selected to catalyze ester-ester exchange to prepare MLCT structure fat, the optimal conditions are that the reaction temperature is 60 ℃, the reaction is carried out for 3h, the molar ratio of the camphor tree seed oil to the tea oil substrate is 1:1.5, and the content of MLCT structure fat in the obtained product is 55.81% (research on preparing structure fat by catalyzing the camphor tree seed oil by Zhaomanli [ D ]. Nanchang university, 2014.).

In addition, part of the long-chain fatty acid has a plurality of beneficial effects on the human body. Such as n-3 polyunsaturated fatty acids, the source of n-3 polyunsaturated fatty acids is primarily the conversion of alpha-linolenic acid in the human body to small amounts of docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, obtained from marine fish or fish oil. The triglyceride type polyunsaturated fatty acid is one of the naturally occurring forms, and has good taste and stable properties. Therefore, the preparation of the functionalized triglyceride containing the n-3 polyunsaturated fatty acid has wide application significance.

However, according to the current research on the preparation of structured lipids by transesterification, there is no enzyme method that can simultaneously achieve high yield transesterification of short/medium/long triglycerides with long triglycerides. Even in the transesterification research of single short/medium/long triglyceride and long-chain triglyceride, the problems of low raw material conversion rate, low product yield and complex transesterification process generally exist, especially the problems of short-long chain glyceride, such as tributyrin, generated during the transesterification process, tributyrin which has adverse effects on the structure and activity of most lipase, so that the transesterification efficiency and the product yield of the short-long chain glyceride are not ideal, and the supply and the application of functional structural ester are greatly limited.

In addition, because most triglycerides have high viscosity and lipase activity therein is affected, many enzyme-catalyzed transesterification reactions often have to be carried out in a solvent system to achieve good catalytic yield, but the use of solvents often causes disadvantages such as increased production cost, environmental unfriendliness, increased steps, and the like.

Disclosure of Invention

Based on this, it was an object of the present invention to provide an enzymatic transesterification process which allows the transesterification of short/medium/long triglycerides with long triglycerides simultaneously and which allows good yields to be achieved in each case.

The specific technical scheme is as follows:

an enzymatic transesterification process comprising the steps of: under the action of lipase, triglyceride A and triglyceride B are subjected to transesterification reaction;

the triglyceride A is at least one of short chain triglyceride, medium chain triglyceride and long chain triglyceride;

the triglyceride B is long-chain triglyceride;

the lipase is a lipase MAS1 or MAS 1-H108A.

In some of these embodiments, the lipase is MAS 1-H108A.

In some of these embodiments, the lipase MAS1 or MAS1-H108A is derived from Streptomyces sp.

In some of these embodiments, the ratio of triglyceride a to triglyceride B by mass is 1: 2.5-5; further, the mass ratio of triglyceride a to triglyceride B is 1: 2.5 to 3.5.

In some of these embodiments, the temperature of the reaction is 55 to 75 ℃.

In some of these embodiments, the temperature of the reaction is 65 to 75 ℃.

In some of these embodiments, the lipase is an immobilized lipase.

In some embodiments, the lipase is added in an amount of 20-100U/g substrate.

In some of these embodiments, the short-chain triglyceride is at least one of triacetin, tripropionin, and tributyrin.

In some of these embodiments, the medium chain triglyceride is at least one of tricaprylin, tricaprin, and trilaurin.

In some of these embodiments, the long chain triglyceride in the triglyceride a is at least one of tripalmitin, tristearin, palm oil, olive oil, rapeseed oil, tea oil, soybean oil, walnut oil, corn oil, beef tallow, and lard.

In some of these embodiments, the long-chain triglyceride in the triglyceride B is at least one of triolein, trilinolein, palm oil, olive oil, rapeseed oil, tea oil, soybean oil, walnut oil, corn oil, tallow, and lard.

In some of these embodiments, the reaction system is a solvent-borne reaction system or a solventless reaction system.

In some embodiments, when the reaction system is a solvent reaction system, the reaction solvent is tert-butanol.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a method for simultaneously realizing the transesterification of short/medium/long triglyceride and long-chain triglyceride with high yield. Namely, by selecting the lipase MAS1 or MAS1-H108A as a catalytic enzyme, especially selecting MAS1-H108A as a catalytic enzyme, and combining proper reaction conditions, the transesterification of short/medium/long triglycerides and long-chain triglycerides is finally realized at the same time, and the yield is up to 65-90%.

In addition, the method does not use a solvent, does not obviously influence the reaction yield, can be compatible with a solvent system and a solvent-free system, and has the advantages of wide application range, simple and convenient operation, low cost and green and sustainable property.

Detailed Description

Experimental procedures according to the invention, in which no particular conditions are specified in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.

The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.

The present embodiment provides an enzymatic transesterification method, including the steps of: under the action of lipase, triglyceride A and triglyceride B are subjected to transesterification reaction;

the triglyceride A is at least one of short chain triglyceride, medium chain triglyceride and long chain triglyceride;

the triglyceride B is long-chain triglyceride;

the lipase is a lipase MAS1 or MAS 1-H108A.

The lipase MAS1 is a wild type and is derived from actinomycetes Streptomyces sp.Strain W007, and the lipase MAS1-H108A is a mutant of a wild type lipase MAS 1. The lipase MAS1 or MAS1-H108A (immobilized) used in the process of the invention can be purchased or prepared according to the conventional method in the field.

In some of these embodiments, the lipase is MAS 1-H108A. The method provided by the invention has the advantages that the MAS1-H108A is selected to catalyze the transesterification reaction, and the yield is higher.

The inventor of the invention unexpectedly finds that the lipase MAS1 or MAS1-H108A only has the MASs ratio of 1: 2.5-5, especially 1: 2.5-3.5 times, the catalyst can be well catalyzed to generate ester exchange reaction, and good yield is obtained.

Further, the mass ratio of the triglyceride A to the triglyceride B is 1: 2.6-3.4, 1: 2.7-3.3, 1: 2.8-3.2, 1: 2.9-3.1, or 1: 3.

According to the method, the lipase is MAS1-H108A, and the MASs ratio of triglyceride A to triglyceride B is 1: 2.5-3.5, the reaction temperature is 65-75 ℃, higher ester exchange yield can be obtained, and the ester exchange yield aiming at the short/medium/long triglyceride and the long-chain triglyceride is up to 86.9-89.7%.

The present invention will be described in further detail with reference to specific examples.

Immobilized lipase MAS1-H108A mutant: the preparation reference of the preparation reference, namely "Lilin" immobilized lipase MAS1-H108A, and the application research of the preparation reference in the catalytic synthesis of triglyceride rich in alpha-linolenic acid [ D ]. university of southern China, 2019 ].

Immobilized lipase MAS1 wild type: the preparation reference, wanxiames lipase MAS1 immobilization and its study on catalytic synthesis of PUFA-type functional lipids [ D ]. university of southern China, 2017 ].

Lipozyme tl IM: purchased from novicent (tianjin) ltd;

glyceryl triacetate: purchased from Shanghai Aladdin, Inc.;

glyceryl tripropionate: purchased from Shanghai Aladdin, Inc.;

glycerol tributyrate: purchased from Shanghai Aladdin, Inc.;

tricaprylin: purchased from Shanghai Aladdin, Inc.;

tricaprin: purchased from Shanghai Aladdin, Inc.;

glycerol trilaurate: purchased from Shanghai Aladdin, Inc.;

glycerol trioleate: purchased from Shanghai Aladdin, Inc.;

glycerol trilinoleate: purchased from Shanghai Aladdin, Inc.;

glyceryl trilinoleate: purchased from chemical reagents, Inc. of Kyoto Kaisha;

tripalmitin: purchased from Shanghai Aladdin, Inc.;

glyceryl tristearate: purchased from Shanghai Aladdin, Inc.;

tea oil: purchased in local market

Rapeseed oil: purchased in local market

Palm oil: purchased in local market

Olive oil: purchased in a local market.

Example 1

Immobilized lipase MAS1-H108A mutant is used for catalyzing the reaction of tributyrin and triolein, the enzyme adding amount is 50U/g substrate, no solvent is added, and ester exchange reaction is carried out under different substrate MASs ratios and reaction temperatures. After the reaction is finished, taking reaction liquid, measuring the content of short-chain and long-chain triglyceride through LC-MS, and taking the yield of the short-chain and long-chain triglyceride as an index of the ester exchange degree, wherein the specific data is shown in Table 1.

TABLE 1

Example 2

The method is characterized in that immobilized lipase MAS1 wild type is used for catalyzing the reaction of tributyrin and triolein, the enzyme adding amount is 50U/g substrate, no solvent is added, and ester exchange reaction is carried out at different substrate MASs ratios and reaction temperatures. After the reaction is finished, taking reaction liquid, measuring the content of short-chain and long-chain triglyceride through LC-MS, and taking the yield of the short-chain and long-chain triglyceride as an index of the ester exchange degree, wherein the specific data is shown in Table 2.

TABLE 2

Example 3

The immobilized lipase MAS1-H108A mutant is used for catalyzing the reaction of tributyrin and tea oil, and the ester exchange reaction is carried out at the enzyme adding amount of 50U/g substrate, no solvent is added, the substrate MASs ratio of 1:3 (tributyrin/tea oil (w/w)) and the reaction temperature of 70 ℃. After 6 hours of reaction, reaction liquid is taken, the content of short-chain and long-chain triglyceride is determined by LC-MS, and the yield of the short-chain and long-chain triglyceride is 81.9 percent.

Example 4

Immobilized lipase MAS1-H108A mutant is used for catalyzing the reaction of tricaprylin and triolein, and ester exchange reaction is carried out under the conditions of enzyme addition amount of 50U/g substrate, no solvent, different substrate MASs ratios and different reaction temperatures. After the reaction is finished, taking the reaction liquid, measuring the content of medium-long chain triglyceride by LC-MS, and taking the medium-long chain triglyceride as an index of ester exchange degree, wherein the specific data is shown in Table 3.

TABLE 3

Example 5

The method is characterized in that immobilized lipase MAS1 wild type is used for catalyzing the reaction of tricaprylin and triolein, and ester exchange reaction is carried out under the conditions of enzyme addition amount of 50U/g substrate, no solvent addition, different substrate MASs ratios and different reaction temperatures. After the reaction, the reaction solution is taken, the content of medium-long chain triglyceride is measured by LC-MS, and the specific data taking the medium-long chain triglyceride as the index of the ester exchange degree are shown in Table 4.

TABLE 4

Example 6

The immobilized lipase MAS1-H108A mutant is used for catalyzing the reaction of tricaprylin and rapeseed oil, the enzyme adding amount is 50U/g substrate, no solvent is added, the substrate MASs ratio is 1:3 (tricaprylin/rapeseed oil (w/w)), and the reaction temperature is 70 ℃ for ester exchange reaction. After 6 hours of reaction, reaction liquid is taken, the content of medium-long chain triglyceride is determined by LC-MS, and the yield of the medium-long chain triglyceride is 84.7 percent.

Example 7

Immobilized lipase MAS1-H108A mutant is used for catalyzing the reaction of tripalmitin and triolein, and ester exchange reaction is carried out under the conditions that the enzyme adding amount is 50U/g substrate, no solvent is added, and the substrate MASs ratio and the reaction temperature are different. After the reaction, the reaction solution was taken, the content of triglyceride which is not a single fatty acid was measured by LC-MS, and the yield of triglyceride which is not a single fatty acid on the glycerol backbone was used as an index of the degree of transesterification, and the specific data is shown in table 5.

TABLE 5

Example 8

The method is characterized in that a wild type immobilized lipase MAS1 is used for catalyzing the reaction of tripalmitin and triolein, and ester exchange reaction is carried out under the conditions of enzyme addition amount of 50U/g substrate, no solvent addition, different substrate MASs ratios and different reaction temperatures. After the reaction, the reaction solution was taken, the content of triglyceride which is not a single fatty acid was measured by LC-MS, and the yield of triglyceride which is not a single fatty acid on the glycerol backbone was used as an index of the degree of transesterification, and the specific data is shown in table 6.

TABLE 6

Example 9

The immobilized lipase MAS1-H108A mutant is used for catalyzing the reaction of palm oil and olive oil, and the ester exchange reaction is carried out at the enzyme adding amount of 50U/g substrate, no solvent is added, the substrate MASs ratio is 1:3 (palm oil/olive oil (w/w)), and the reaction temperature is 70 ℃. After 6 hours of reaction, reaction liquid is taken out, the content of 1, 3-dioleic acid-2-palmitic acid glyceride (OPO) is determined by LC-MS, and the yield of OPO is 80.6%.

Example 10

Immobilized lipase MAS1-H108A mutant is used for catalyzing the reaction of tricaprylin and triolein, the enzyme adding amount is 50U/g substrate, the solvent is tert-butyl alcohol, and ester exchange reaction is carried out under different substrate MASs ratios and reaction temperatures. After the reaction is finished, taking the reaction liquid, measuring the content of medium-long chain triglyceride by LC-MS, and taking the medium-long chain triglyceride as an index of the ester exchange degree, wherein the specific data is shown in Table 7.

TABLE 7

Example 11

Immobilized lipase MAS1-H108A mutant is used for catalyzing the reaction of tricaprylin and rapeseed oil, tertiary butanol is added into a substrate with the enzyme adding amount of 50U/g, and the transesterification reaction is carried out at the substrate MASs ratio of 1:3 (tricaprylin/rapeseed oil (w/w)) and the reaction temperature of 70 ℃. After 6 hours of reaction, reaction liquid is taken, and the content of medium-long chain triglyceride is determined by LC-MS, wherein the yield of the medium-long chain triglyceride is 82.7 percent.

Comparative example 1

Immobilized lipase lipozyme TL IM is used for catalyzing the reaction of tributyrin and triolein, the enzyme adding amount is 50U/g substrate, no solvent is added, and the ester exchange reaction is carried out under different substrate mass ratios and reaction temperatures. After the reaction, the reaction solution is taken, the content of short-chain and long-chain triglyceride is measured by LC-MS, the short-chain and long-chain triglyceride is taken as an index of the transesterification degree, and the specific data are shown in Table 8.

TABLE 8

Comparative example 2

Immobilized lipase lipozyme TL IM is used for catalyzing the reaction of tributyrin and tea-seed oil, the enzyme adding amount is 50U/g substrate, no solvent is added, and the ester exchange reaction is carried out at the substrate mass ratio of 1:2 (tributyrin/tea-seed oil (w/w)) and the reaction temperature of 60 ℃. After reacting for 18h, taking reaction liquid, and determining the content of the short-chain and long-chain triglyceride through LC-MS, wherein the yield of the short-chain and long-chain triglyceride is 61.9%.

Comparative example 3

Immobilized lipase lipozyme TL IM is used for catalyzing the reaction of tricaprylin and triolein, and ester exchange reaction is carried out under the conditions that the enzyme adding amount is 50U/g of substrate, no solvent is added, the substrate mass ratio is different, and the reaction temperature is different. After the reaction is finished, taking the reaction liquid, measuring the content of medium-long chain triglyceride by LC-MS, and taking the medium-long chain triglyceride as an index of the ester exchange degree, wherein the specific data is shown in Table 9.

TABLE 9

Comparative example 4

Immobilized lipase lipozyme TL IM is used for catalyzing the reaction of tripalmitin and triolein, and ester exchange reaction is carried out under the conditions that the enzyme adding amount is 50U/g of substrate, no solvent is added, the substrate mass ratio is different, and the reaction temperature is different. After the reaction, the reaction solution was taken, the content of triglyceride which is not a single fatty acid was measured by LC-MS, and the specific data using the yield of triglyceride which is not a single fatty acid on the glycerol backbone as an index of the degree of transesterification are shown in table 10.

Watch 10

From the above results, it is understood that the method of the present invention selects MAS1-H108A mutant or MAS1 wild type immobilized lipase as a transesterification catalytic enzyme, especially MAS1-H108A mutant, and can simultaneously realize transesterification of short/medium/long triglycerides and long-chain triglycerides in high yield by combining suitable reaction conditions. The short-chain triglycerides achieved a maximum yield of 88.3% in example 1, 89.7% in example 4, and 86.9% in example 7.

In comparative examples 1 to 4, selecting immobilized lipase Lipozyme TL IM instead of immobilized lipase MAS1-H108A mutant or MAS1 wild type in examples 1 to 11, found that the yield for the transesterification of short, medium and long chain triglycerides with long chain triglycerides was lower than in examples 1 to 11, respectively, and that the catalytic yield for the transesterification of short chain triglycerides with long chain triglycerides was only 62.5% at the highest, the catalytic effect was significantly lower than that for the transesterification of medium and long chain triglycerides with long chain triglycerides (72.4% and 73.0%), and the transesterification of short/medium/long triglycerides with long chain triglycerides could not be achieved at the same time with high yield.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.