A method for producing medium and long carbon chain triglyceride oil by enzyme in a solvent-free system

Abstract

The application discloses a process for producing medium and long carbon chain triglyceride (MLCT) oil and fat in a solvent-free system, and belongs to the field of oil and fat. In the process, a low-cost lipase is used as a catalyst, and lipase A is prepared through immobilization of the enzyme on a carrier; then, lipase B is prepared through an enzymatic transesterification reaction; and finally, the lipase B is used as a biological catalyst to catalyze the enzymatic reaction of free fatty acid and glycerol in a solvent-free system to prepare MLCT oil. The process realizes green and efficient production of MLCT through a series of methods by using low-cost free lipase, and the production cost is reduced, so the process has an industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a technology and process for producing functional weight-loss oils using lipase. Background Technology

[0002] Medium- and long-chain triglycerides (MLCTs) are molecules whose glycerol backbone contains both medium-chain fatty acids (C6-C12) and long-chain fatty acids (>C12). Only a few natural animal and vegetable oils contain MLCTs. Recent animal and human studies have shown that edible oils rich in MLCTs have a range of benefits, including weight control, lowering blood lipid levels, improving nitrogen balance, and preventing coronary heart disease and atherosclerosis. Therefore, they are functional edible oils with significant development value and market application potential.

[0003] In 2012, the former Ministry of Health approved medium- and long-chain fatty acid edible oils (MLCT) as a new food ingredient, with enzymatic preparation being the only production process that complies with regulations. Currently, enzymatic production processes for MLCT include transesterification and esterification.Enzymatic transesterification utilizes natural oils such as soybean oil and rapeseed oil, along with triglycerides rich in medium-chain fatty acids, as substrates to prepare MLCT through enzymatic reactions. Enzymatic esterification, on the other hand, utilizes some oil processing byproducts, such as free fatty acids, in the esterification reaction with glycerol to produce MLCT oil, thus making efficient use of byproduct resources. Generally, the lipases used in enzymatic esterification are typically Lipozyme 435, Novozym 435, and Lipase CL IM from Candida antarctica; Lipozyme RM IM and Lipozyme RM from Rhizomucor miehei; and Lipase DF IM from Rhizopus oryzae. Lipases from Thermomyces lanuginosus, such as Lipozyme TL 100L and Lipozyme TL IM (an immobilized version of Lipozyme TL 100L), exhibit low activity in glycerol and fatty acid esterification systems (Guo and Sun, Solvent-free). Enzymatic synthesis of 1,3-diconjugated linoleoyl glycerol optimized by response surface methodology, Biotechnology Progress, 2004, 20, 619−622; Phuah et al. Review on the current state of diacylglycerol production using enzymatic approach, Food and Bioprocess Technology, 2015, 8, 1169-1186; Lu Shan, Preparation of high-purity 1,3-dioleoyl glycerol, Master's thesis, Jiangnan University, 2013). However, lipases derived from Thermomyces lanuginosus are generally very inexpensive. For example, Lipozyme TL IM costs about 10% of Lipozyme RM and 5% of Lipozyme 435; Lipozyme TL100L costs about 5% of Lipozyme RM and 2% of Lipozyme 435. Therefore, how to utilize an inexpensive lipase (such as one derived from Thermomyces lanuginosus) is a key question. The lipases of *Lanuginosus* and *Aspergillus niger* are of great significance in preparing medium- and long-chain triglyceride oils by catalyzing the reaction of glycerol and free fatty acids or their derivatives.

[0004] We still need to find an economical and efficient enzymatic production process for MLCT oil, which utilizes commercially available and inexpensive lipases to catalyze the reaction of glycerol and free fatty acids or their derivatives, thereby separating functional edible oils rich in MLCT. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] One objective of this invention is to provide a process for producing MLCT oil in a solvent-free system via enzymatic esterification. This process involves using inexpensive liquid enzymes as raw materials, and after steps such as immobilization and transesterification, obtaining lipases with high catalytic activity in the reaction system of free fatty acids (or their derivatives) and glycerol, thereby preparing functional oils rich in MLCT.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for producing medium- and long-chain triglyceride oils using a solvent-free enzymatic system, comprising the following steps: Step 1, Immobilization of lipase: Lipase is immobilized on a nonpolar carrier to prepare immobilized lipase A; The lipases include one or more derived from Thermomyces lanuginosus and Aspergillus niger; the nonpolar carriers include macroporous adsorption resins of type ADS, D101, or BS. Step 2, transesterification reaction: Immobilized lipase A is mixed with animal and vegetable oils with an oleic acid value of less than 0.2 mg KOH / g, and transesterification reaction is carried out at 35-55℃ until the acid value of the animal and vegetable oils after the reaction is less than 1.5 mg KOH / g. After the reaction, the lipase and animal and vegetable oils are separated to obtain immobilized lipase B. Step 3, reaction for producing medium- and long-chain triglyceride oil: Immobilized lipase B catalyzes the esterification reaction of glycerol and fatty acids or their derivatives at 45-60℃. A polar adsorbent is also added to the esterification reaction system. After distillation and deacidification, the crude product is purified into medium- and long-chain triglyceride oil. The molar ratio of fatty acids or fatty acid derivatives to glycerol is greater than or equal to 3.5:1, and the free fatty acids or their derivatives contain both C6-C12 medium-chain fatty acids or their derivatives and C12 or longer-chain fatty acids or their derivatives.

[0009] A preferred method, wherein step 1, the immobilization of lipase, is as follows: the enzyme solution concentration is diluted to 10-20 mg / mL with phosphate buffer solution to maintain the enzyme solution at pH 5-7.0; then 0.4-0.6 g of a nonpolar immobilization carrier that has been washed with alcohol, acid, alkali and water is added to the buffer solution containing lipase; 6-10% maltodextrin is added; after adsorption for 4-10 h, the unadsorbed free enzyme is removed with buffer solution; and the immobilized lipase A is obtained after vacuum drying.

[0010] In a preferred embodiment, the immobilization method of lipase in step 1 is as follows: the enzyme concentration is diluted to 15 mg / mL with phosphate buffer solution to maintain the enzyme solution at pH 6.0. Then, 0.5 g of nonpolar immobilization carrier that has been washed with alcohol, acid, alkali and water is added to the buffer solution containing lipase. The temperature of the buffer system is maintained at 25°C, and 8% maltodextrin is added. After adsorption for 8 h, the unadsorbed free enzyme is removed with buffer solution, and the immobilized lipase A is obtained after vacuum drying.

[0011] In a preferred embodiment, the total transesterification time in step 2 is 6 h to 12 h.

[0012] In a preferred embodiment, step 2 specifically involves mixing immobilized lipase A with animal and vegetable oils having an oleic acid value of less than 0.2 mg KOH / g, and conducting a transesterification reaction at 35–55°C. After one transesterification reaction, the acid value of the animal and vegetable oils is measured. When the acid value is greater than 1.5 mg KOH / g, fresh animal and vegetable oils are mixed with the immobilized lipase A that has been used once for another transesterification reaction. The acid value is measured after the reaction until the acid value of the animal and vegetable oils after transesterification drops below 1.5 mg KOH / g.

[0013] In a preferred embodiment, in step 3, the fatty acid derivative includes fatty acid ethyl ester.

[0014] In a preferred embodiment, in step 3, the molar ratio of medium-chain fatty acids or their derivatives to long-chain fatty acids or their derivatives is 0.8 to 1.5.

[0015] In a preferred embodiment, in step 3, the esterification reaction is carried out under vacuum conditions and the pressure of the reaction system is less than 100 Pa.

[0016] In a preferred embodiment, the polar adsorbent added in step 3 includes silica gel, Amberlite FPA54, and activated carbon.

[0017] The second objective of this invention is to provide a medium- to long-chain triglyceride oil prepared by the above method.

[0018] A third objective of this invention is to provide the application of the medium- and long-chain triglyceride oils described above in the food and pharmaceutical fields.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention obtains immobilized lipase B by immobilizing and transesterifying lipases derived from Thermomyces lanuginosus and Aspergillus niger. This improves the catalytic activity of economical and practical lipases in the reaction system of free fatty acids or their derivatives with glycerol, and solves the problem of high enzyme cost in the esterification production of MLCT oils. In addition, this process avoids the use of solvents, is green and environmentally friendly, and has broad market development prospects. Detailed Implementation

[0020] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0021] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0022] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0023] Unless otherwise specified, all raw materials used in the examples are commercially or laboratory-made.

[0024] Lipase Immobilization: The nonpolar immobilization carrier was sequentially washed with alcohol, acid, alkali, and water, and then stored for later use. The enzyme concentration was diluted to 15 mg / mL with phosphate buffer solution to maintain the enzyme solution at approximately pH 6.0. Subsequently, 0.5 g of the nonpolar immobilization carrier was added to the buffer solution containing lipase, the buffer system temperature was maintained at 25℃, and 8% maltodextrin was added. After adsorption for 8 h, the unadsorbed free enzyme was removed with buffer solution, and the product was vacuum dried to obtain immobilized lipase A. Immobilized lipase A, abbreviated as TLL-ADS-A, was immobilized on macroporous adsorption resin ADS using Thermomyces lanuginosus Lipase (TLL) (or Aspergillus niger lipase, ANL) as the free enzyme. Other immobilized lipases were processed similarly.

[0025] The method for detecting MLCT content is as follows: refer to the method in standard JX20070100.

[0026] Molecular distillation conditions: main evaporation temperature 190℃, condensation temperature 30℃, distillation pressure <1 Pa.

[0027] Example 1 Effects of transesterification number and acid value on MLCT synthesis by lipase B: 300 g of Grade I camellia seed oil (acid value less than 0.2 mg / g) was weighed and 12% (based on oil weight) of immobilized lipase TLL-ADS8-A (lipase A) was added. A transesterification reaction was carried out at 45℃ under vacuum (<500 Pa). After 3 h of reaction, the immobilized lipase and camellia seed oil were separated. The immobilized lipase TLL-ADS-A after one transesterification reaction was stored for later use. The acid value of the camellia seed oil recovered after one transesterification reaction was determined (Table 1). If the acid value was above 1.5 mg / g, 12% of the immobilized lipase TLL-ADS-A after one transesterification reaction was added to fresh camellia seed oil for another 3 h of transesterification reaction until the acid value of the camellia seed oil after the reaction was less than 1.5 mg / g. The resulting immobilized lipase B was used to synthesize MLCT.

[0028] Synthesis of MLCT oil: Medium-chain free fatty acids C10:0 and soybean oil-based long-chain free fatty acids were mixed in a 1:1 molar ratio. The molar ratio of the mixed fatty acids to glycerol was 3.5:1. 20% (based on total substrate weight) of Amberlite FPA54 and 8% (based on total substrate weight) of lipase B were added to the mixture. The reaction was carried out at 50℃ under vacuum (50 Pa) for 16 h before the reaction was stopped. The content of MLCT in the crude product was analyzed and the results are shown in the table below.

[0029] Table 1. Effects of transesterification number and recovered tea seed oil acid value on the performance of lipase B-catalyzed esterification. As shown in the table above, the immobilized TLL-AB8-A enzyme, without transesterification, produces a low MLCT content in its esterification product. With increasing transesterification cycles, the acid value of the recovered tea seed oil gradually decreases. When the acid value drops below 1.5 mg KOH / g oil, the resulting immobilized lipase B exhibits high esterification activity and a high MLCT content in the product. Some lipases derived from *Thermomyces lanuginosus* possess high hydrolytic activity. Transesterification can reduce their hydrolytic activity and enhance their enzyme activity in esterification or transesterification reactions. Therefore, transesterification increases the activity of lipases in the preparation of MLCT.

[0030] Example 2 Influence of Immobilization Materials: Immobilized lipase A was prepared by immobilizing TLL enzyme (or ANL) on carriers of different polarities. The non-polar carriers selected were resins BS-55, ADS, and D101; the medium-polar carriers were DM-130 and AB-8; and the polar carriers were silica gel and NKA9. Immobilized lipase A underwent transesterification in tea seed oil for 3 hours. This transesterification process was repeated until the acid value of the recovered tea seed oil dropped below 1.5 mg / g, at which point immobilized lipase B was obtained and used to catalyze the esterification reaction of mixed fatty acids and glycerol. Other transesterification and esterification reaction conditions were the same as in Example 1. After the reaction, the content of MLCT in the crude product was analyzed, and the results are shown in the table below.

[0031] Table 2. Effect of immobilized support on lipase B-catalyzed esterification reaction Using the commercially available immobilized lipase Lipozyme TL IM derived from Thermomyces lanuginosus as the selected catalyst, the activity differences between Lipozyme TL IM and the prepared immobilized lipase BTLL-D101 in catalyzing the esterification reaction of free fatty acids and glycerol were compared. The results showed that the crude product of the esterification reaction catalyzed by Lipozyme TL IM contained only 5.6% MLCT, while the crude product of the esterification reaction catalyzed by the TLL-D101 immobilized lipase B prepared by the method of this invention contained as much as 31.7% MLCT.

[0032] The results in the table above show that when lipase TLL from Thermomyces lanuginosus was immobilized on carriers of different polarities to obtain lipase A, and then after multiple transesterification reactions, lipase B immobilized on different carriers was obtained. When lipase B was used to catalyze esterification reactions to synthesize MLCT, the results showed that lipase B immobilized on nonpolar materials had the highest activity in the esterification reaction and the highest MLCT content in the crude product. In addition, when lipase ANL from Aspergillus niger was immobilized on a nonpolar carrier, its catalyzed esterification reaction also had a high MLCT content.

[0033] Commercially available immobilized lipase Lipozyme TL IM uses polar silica gel as a carrier. When a polar material is used as the enzyme immobilization carrier, polar substances such as glycerol are easily adsorbed onto the enzyme surface during the reaction. Glycerol is a strongly polar molecule that readily removes bound water from the enzyme molecule, leading to enzyme inactivation. In contrast, when a non-polar carrier is used as the enzyme immobilization material, the interaction between the immobilized material and glycerol is weak, and glycerol's ability to remove bound water from the enzyme is limited, resulting in relatively higher catalytic activity of the immobilized enzyme.

[0034] Example 3 Effect of transesterification temperature: TLL was immobilized on ADS to obtain lipase TLL-ADS-A, which was then used to catalyze the transesterification reaction of tea seed oil at 35℃~65℃ for 3 h. This process was repeated until the acid value of the recovered oil dropped below 1.5 mgKOH / g, thus preparing lipase B under different transesterification temperature conditions. Other transesterification and esterification reaction conditions were the same as in Example 1. The MLCT content in the crude product is shown in the table below.

[0035] Table 3 Effect of transesterification temperature on lipase B-catalyzed esterification reaction The results in the table above show that the optimal transesterification temperature range is 35-55℃.

[0036] Example 4 Effect of esterification temperature on the synthesis of MLCT: ANL was immobilized on D101 to obtain lipase ANL-D101-A, which was then used to catalyze the transesterification reaction of tea seed oil at 45°C for 3 h. This process was repeated until the acid value of the recovered tea seed oil dropped below 1.5 mgKOH / g to prepare lipase ANL-D101-B. This enzyme was then used to catalyze the esterification reaction of mixed fatty acids and glycerol. The esterification reaction temperature was 40°C to 65°C. Other transesterification and esterification reaction conditions were the same as in Example 1. The MLCT content in the crude product is shown in the table below.

[0037] Table 4. Effect of esterification temperature on lipase B-catalyzed esterification reaction The results in the table above show that the optimal esterification temperature is in the range of 45–60℃.

[0038] Example 5 Effect of substrate molar ratio on esterification reaction: The immobilized enzyme ANL-D101-B obtained in Example 4 was used to catalyze the esterification reaction of mixed fatty acids and glycerol at 50°C. The molar ratio of mixed fatty acids to glycerol was 2.5:1, 3:1, 3.5:1, and 4:1, and other conditions were the same as in Example 1. The MLCT content in the crude product is shown in the table below.

[0039] Table 5. Effect of substrate molar ratio on lipase B-catalyzed esterification reaction The results in the table above show that the optimal molar ratio of free fatty acids to glycerol is greater than or equal to 3.5:1.

[0040] Example 6 Effect of acyl donor type in esterification reaction: The immobilized enzyme ANL-D101-B obtained in Example 4 was used to catalyze the reaction of free fatty acids or fatty acid ethyl esters and glycerol at 50°C to prepare MLCT. Other conditions were the same as in Example 4. The MLCT content in the crude product is shown in the table below.

[0041] Table 6. Effect of acyl donor type on lipase B catalysis in esterification reaction. The results in the table above show that ANL-D101-B can catalyze the reaction of free fatty acids with ethyl esters and glycerol, with the effect being better on free fatty acids.

[0042] Example 7 The effect of the ratio of medium-chain fatty acids to long-chain fatty acids in free fatty acids: The immobilized enzyme ANL-D101-B was obtained according to Example 4. MLCT was prepared by catalyzing the reaction of free fatty acids and glycerol at 50°C. The ratio of medium-chain fatty acids to long-chain fatty acids in the free fatty acids was 0.5:1, 0.8:1, 1:1, 1.2:1, 1.5:1, and 2:1. Other conditions were the same as in Example 4. The MLCT content in the crude product is shown in the table below.

[0043] Table 7. Effect of the ratio of medium-chain to long-chain fatty acids on the esterification reaction catalyzed by lipase B. The results in the table above show that the optimal ratio range is 0.8:1 to 1.5:1.

[0044] Example 8 The effect of the type and polarity of the adsorbent in the esterification reaction: Immobilized lipase B, namely lipase ANL-D101-B, was prepared according to the method in Example 4. The esterification reaction of free fatty acids and glycerol was catalyzed by this enzyme at 50°C. Different adsorbents were added to the solvent-free esterification reaction system to investigate their effects on the esterification reaction. The types of adsorbents investigated included: polar adsorbents (silica gel, Amberlite FPA54, S-8), moderately polar adsorbents (ADS-17, AB-8, BS-30), and non-polar adsorbents (D101, X-5, ADS-8). Other esterification conditions were the same as in Example 4. The MLCT content in the crude product is shown in the table below.

[0045] Table 8. Effect of Adsorbent Type on Lipase B-Catalyzed Esterification Reaction in Esterification. The results in the table above show that adding a polar adsorbent to the esterification reaction yields the best results. When a polar material is present in the reaction system, its adsorption force on glycerol is strongest. Glycerol is more readily adsorbed onto the surface of the polar material, rather than the surface of the enzyme immobilization carrier, further reducing the ability of glycerol to remove water molecules from the enzyme and thus increasing enzyme activity.

[0046] This invention employs enzymatic esterification to prepare MLCT oil in a solvent-free system. Specifically, it uses a relatively inexpensive lipase as a biocatalyst to react free fatty acids and glycerol in a solvent-free system, producing functional oils rich in MLCT. Prior to the enzymatic esterification reaction, the lipase undergoes immobilization and transesterification treatment to improve its catalytic efficiency in the subsequent esterification reaction, thereby reducing production costs and increasing lipase activity during the esterification process. This invention provides a green, economical, and efficient enzymatic catalytic method for preparing MLCT, with broad prospects for industrial application.

[0047] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A solvent-free enzymatic method for producing medium- and long-chain triglyceride oils, characterized in that: The enzymatic production process includes the following steps: Step 1, Immobilization of lipase: Lipase is immobilized on a nonpolar carrier to prepare immobilized lipase A; The lipases include one or more derived from Thermomyces lanuginosus and Aspergillus niger; the nonpolar carriers include macroporous adsorption resins of type ADS, D101, or BS. Step 2, transesterification reaction: Immobilized lipase A is mixed with animal and vegetable oils with an oleic acid value of less than 0.2 mg KOH / g, and transesterification reaction is carried out at 35-55℃ until the acid value of the animal and vegetable oils after the reaction is less than 1.5 mg KOH / g. After the reaction, the lipase and animal and vegetable oils are separated to obtain immobilized lipase B. Step 3, reaction for producing medium- and long-chain triglyceride oil: Immobilized lipase B catalyzes the esterification reaction of glycerol and fatty acids or their derivatives at 45-60℃. A polar adsorbent is also added to the esterification reaction system. After distillation and deacidification, the crude product is purified into medium- and long-chain triglyceride oil. The molar ratio of fatty acids or fatty acid derivatives to glycerol is greater than or equal to 3.5:1, and the fatty acids or their derivatives contain both C6-C12 medium-chain fatty acids or their derivatives and C12 or longer-chain fatty acids or their derivatives.

2. The method for producing medium- and long-chain triglyceride oils using a solvent-free enzymatic system as described in claim 1, characterized in that: The method for immobilizing lipase in step 1 is as follows: dilute the enzyme solution concentration to 10-20 mg / mL with phosphate buffer solution to maintain the enzyme solution at pH 5-7.

0. Then, add 0.4-0.6 g of nonpolar immobilization carrier that has been washed with alcohol, acid, alkali and water to the buffer solution containing lipase, add 6-10% maltodextrin, and after adsorption for 4-10 h, remove the unadsorbed free enzyme with buffer solution, and then vacuum dry to obtain immobilized lipase A.

3. The method for producing medium- and long-chain triglyceride oils using a solvent-free enzymatic system as described in claim 1, characterized in that: Step 2 specifically involves mixing immobilized lipase A with animal and vegetable oils with an oleic acid value of less than 0.2 mg KOH / g, and conducting a transesterification reaction at 35–55°C. After one transesterification reaction, the acid value of the animal and vegetable oils is measured. When the acid value is greater than 1.5 mg KOH / g, fresh animal and vegetable oils are used as raw materials and the immobilized lipase A that has been used once is mixed again for transesterification reaction. The acid value is measured after the reaction until the acid value of the animal and vegetable oils after transesterification drops below 1.5 mg KOH / g.

4. The method for producing medium- and long-chain triglyceride oils using a solvent-free enzymatic system as described in claim 1, characterized in that: In step 3, the fatty acid derivative includes fatty acid ethyl esters.

5. The method for producing medium- and long-chain triglyceride oils using a solvent-free enzymatic system as described in claim 1, characterized in that: In step 3, the molar ratio of medium-chain fatty acids or their derivatives to long-chain fatty acids or their derivatives is 0.8 to 1.

5.

6. The method for producing medium- and long-chain triglyceride oils using a solvent-free enzymatic system as described in claim 1, characterized in that: In step 3, the esterification reaction is carried out under vacuum conditions and the pressure of the reaction system is less than 100 Pa.

7. The method for producing medium- and long-chain triglyceride oils using a solvent-free enzymatic system as described in claim 1, characterized in that: In step 3, the polar adsorbent includes silica gel, Amberlite FPA54, or activated carbon.

8. The medium- and long-chain triglyceride oil prepared by the solventless enzymatic production method for medium- and long-chain triglyceride oil according to any one of claims 1 to 7.

9. The application of the medium- and long-chain triglyceride oil as described in claim 8 in the food and pharmaceutical fields.

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