Process for the production of diacylglycerol oil by enzymatic method in a solvent-free system

Abstract

The application discloses a process for producing diacylglycerol oil by an enzyme method in a solvent-free system, and comprises the following steps: taking low-cost lipase as a catalyst, preparing lipase A by immobilization of the enzyme, preparing lipase B by an ester exchange reaction, and then taking the lipase B as a catalyst to catalyze esterification of free fatty acid and monoglyceride in a solvent-free system to prepare diacylglycerol oil.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to an enzymatic production technology and process for functional oils. Background Technology

[0002] Diacylglycerol (DAG) is a molecule formed by the esterification of the glycerol backbone with only one free hydroxyl group, and the other two hydroxyl groups with fatty acids. In natural animal and vegetable oils, diglycerides are lipid byproducts, present in very low amounts (generally not exceeding 1%). Recent animal and human studies have shown that edible oils rich in diglycerides have a range of benefits, including weight control, lowering serum cholesterol levels, preventing coronary heart disease, and reducing uric acid levels in athletes. Therefore, it is a functional oil with high market development value and application potential.

[0003] In 2009, the former Ministry of Health approved diglyceride oil as a new food raw material, with enzymatic processing being the only production process that complies with regulations. Currently, enzymatic production processes for diglycerides include hydrolysis, glycerol hydrolysis, and esterification.Enzymatic hydrolysis produces a large amount of free fatty acids as a byproduct while generating diglycerides. Therefore, compared with enzymatic glycerol hydrolysis and esterification, the yield of diglyceride oil produced by enzymatic hydrolysis is low. Enzymatic esterification, on the other hand, utilizes the esterification reaction of free fatty acids and monoglycerides (or glycerol) from some oil processing byproducts to produce diglyceride oil, making efficient use of byproduct resources. Generally, the lipases used in enzymatic esterification are usually derived from Candida antarctica (Lipozyme 435 and Novozym 435), Rhizomucor miehei (Lipozyme RM IM and Lipozyme RM), and Rhizopus oryzae. Lipases derived from Thermomyces lanuginosus, such as Lipozyme TL 100L and Lipozyme TLIM (an immobilized version of Lipozyme TL 100L), have very low activity in the esterification reaction system of glycerol (or monoglycerides) and fatty acids (Guo and Sun, Solvent-free enzymatic synthesis of 1,3-diconjugatedlinoleoyl 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 preparation of diglyceride oils by lipases from *Lanuginosus* and *Aspergillus niger* via the catalytic esterification of monoglycerides and free fatty acids is an urgent problem that needs to be solved.

[0004] We still need to find an economical and efficient enzymatic production process for diglycerides, utilizing commercially available and inexpensive lipases to catalyze the esterification reaction of monoglycerides and free fatty acids, thereby separating functional oils rich in diglycerides. 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 an economical and efficient enzymatic production process for diglyceride oil in a solvent-free system. The process involves preparing diglyceride oil through enzymatic esterification, using relatively inexpensive commercial enzymes as catalysts to catalyze the esterification reaction of free fatty acids and monoglycerides, thereby producing functional oils rich in diglycerides. Before the enzymatic esterification reaction, the lipase is immobilized and subjected to random transesterification to improve the catalytic efficiency of the lipase in the esterification reaction system.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a solvent-free enzymatic process for producing diglyceride oil, the process comprising the following steps: Step (1) Immobilization of lipase: The lipase was immobilized on a weakly polar carrier to prepare immobilized lipase A; The lipase is derived from one or more of Thermomyces lanuginosus or Aspergillus niger; the weakly polar carrier is AB-8, BS-30 or DM-130. Step (2) Transesterification reaction: The immobilized lipase A obtained in step (1) is mixed with animal and vegetable oils and transesterification reaction is carried out at 35℃~65℃. This step is repeated until the acid value of the animal and vegetable oils after transesterification drops to 1.5 mgKOH / g or below. Immobilized lipase B is then separated from the animal and vegetable oils after the reaction; wherein the acid value of the animal and vegetable oils is less than 0.2 mgKOH / g. Step (3) Esterification reaction: Add the immobilized lipase B and adsorbent obtained in step (2) to the monoglyceride and free fatty acid, and carry out the esterification reaction at 40-65℃. After the crude product is deacidified by distillation, the purified diglyceride oil product is obtained.

[0009] As a preferred embodiment of the solventless enzymatic process for producing diglyceride oil of the present invention, step (1) specifically involves diluting the enzyme solution concentration of the immobilized carrier to 12 mg / mL with a buffer solution, setting the enzyme solution pH to 5.5, the temperature to 25°C, and adding 8 wt% maltodextrin (based on the carrier). After adsorption for 8 h, the unadsorbed free enzyme is removed with a buffer solution, and the immobilized lipase A is obtained after vacuum drying.

[0010] As a preferred embodiment of the solvent-free enzymatic process for producing diglyceride oil according to the present invention, the transesterification reaction temperature in step (2) is 40-55°C and the time is 2-5 h.

[0011] As a preferred embodiment of the solvent-free enzymatic process for producing diglyceride oil according to the present invention, in step (3), the molar ratio of monoglyceride to free fatty acid is 1:(1-1.8).

[0012] As a preferred embodiment of the solvent-free enzymatic process for producing diglyceride oil according to the present invention, in step (3), the monoglyceride is 2-monoglyceride.

[0013] As a preferred embodiment of the solventless enzymatic process for producing diglyceride oil of the present invention, in step (3), the free fatty acids are C8 to C18 carbon chain fatty acids, but do not include stearic acid.

[0014] As a preferred embodiment of the solventless enzymatic process for producing diglyceride oil of the present invention, in step (3), the fatty acids are acetic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, linoleic acid or DHA (docosahexaenoic acid).

[0015] As a preferred embodiment of the solventless enzymatic process for producing diglyceride oil of the present invention, 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] As a preferred embodiment of the solvent-free enzymatic process for producing diglyceride oil according to the present invention, in step (3), the esterification reaction temperature is 45~60℃.

[0017] As a preferred embodiment of the solvent-free enzymatic process for producing diglyceride oil according to the present invention, in step (3), the adsorbent is a weakly polar adsorbent.

[0018] As a preferred embodiment of the solvent-free enzymatic process for producing diglyceride oil according to the present invention, the weakly polar adsorbent is AB-8, BS-30, or ADS-17.

[0019] As a preferred embodiment of the solvent-free enzymatic process for producing diglyceride oil according to the present invention, wherein:

[0020] Another object of the present invention is to provide the application of the solvent-free enzymatic process for producing diglyceride oil as described in any of the above claims in the food and pharmaceutical fields.

[0021] 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 esterification reaction system of free fatty acids and monoglycerides, solves the problem of high enzyme cost in the esterification production of diglycerides. In addition, this process avoids the use of solvents, is green and environmentally friendly, and has broad market development prospects. Detailed Implementation

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Unless otherwise specified, all raw materials used in the examples are commercially available.

[0026] Lipase immobilization: 0.5 g of immobilization carrier was diluted with buffer solution to a concentration of 12 mg / mL. The enzyme solution was kept at pH 5.5 and temperature 25℃. 8 wt% maltodextrin (based on the carrier) was added. After adsorption for 8 h, unadsorbed free enzyme was removed with buffer solution, and the mixture was vacuum dried to obtain immobilized lipase A. Immobilized lipase A, using Thermomyces lanuginosus Lipase (TLL) (or Aspergillus niger lipase, ANL) as the free enzyme, is abbreviated as TLL-AB8-A. Other immobilized lipases are processed similarly.

[0027] The method for detecting diglyceride content is as follows: refer to the method in GB / T 26636.

[0028] Molecular distillation conditions: main evaporation temperature 190℃, condensation temperature 30℃, distillation pressure <1 Pa.

[0029] Example 1 The effect of the acid value of the oil after transesterification on the esterification reaction catalyzed by lipase B: 100 g of rapeseed oil (acid value less than 0.2 mg / g) was weighed, and 12% (based on oil mass) of immobilized lipase TLL-AB8-A (lipase A) was added. The transesterification reaction was carried out at 45℃ under vacuum. After 3 h of reaction, the immobilized lipase and rapeseed oil were separated. A portion of the recovered TLL-AB8 was reserved for catalyzing the esterification reaction of monoglycerides and free fatty acids. The acid value of the recovered rapeseed oil was determined (Table 1). If the acid value was above 1.5 mg / g, a portion of the lipase was added to the rapeseed oil at a dosage of 12% for another 3 h of transesterification reaction until the acid value of the rapeseed oil after the reaction was less than 0.8 mg / g. The immobilized lipase B obtained at this point was used to catalyze the subsequent esterification reaction.

[0030] Esterification reaction: Oleic acid and 1-oleic acid monoglyceride were mixed in a 1:1 molar ratio, and ADS-17 was added to the mixture. The mass ratio of ADS-17 to 1-oleic acid monoglyceride was 0.6:1. The amount of lipase B added was 5% (based on the total substrate weight). The reaction temperature was 50℃, and the reaction was stopped after 8 h under vacuum conditions (50 Pa pressure). The content of diglycerides in the crude product was analyzed and the results are shown in the table below.

[0031] Table 1. Effects of transesterification number and recovered rapeseed oil acid value on the performance of lipase B-catalyzed esterification. As can be seen from the table above, when the immobilized TLL-AB8-A enzyme does not undergo transesterification, the DAG content in the esterification reaction product is low. As the number of transesterifications increases, the acid value of the recovered rapeseed oil gradually decreases. When the acid value in the oil decreases to 1.5 mgKOH / g oil, the immobilized lipase B obtained at this time has high esterification activity and the DAG content in the product is high.

[0032] Example 2 Influence of Immobilization Materials: Immobilized lipase A was prepared by immobilizing TLL enzyme (or ANL) on carriers of different polarities. The nonpolar carriers selected were BS-55, X-5, and D101; the moderately polar carriers were BS-30, DM-130, and AB-8; and the polar carriers were silica gel and NKA9. Immobilized lipase A underwent transesterification in rapeseed oil for 3 hours. This transesterification process was repeated until the acid value of the recovered rapeseed oil dropped below 1.5 mg / g, at which point immobilized lipase B was obtained and used to catalyze the esterification reaction of oleic acid and 1-monoglycerate. Other transesterification and esterification reaction conditions were the same as in Example 1. After the reaction, the diglyceride content in the crude product was analyzed, and the results are shown in the table below.

[0033] Table 2. Effect of immobilized support on lipase B-catalyzed esterification reaction 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 the esterification reaction of oleic acid and 1-monoglycerate, the results showed that lipase B immobilized on a weakly polar material had the highest activity in the esterification reaction and the highest DAG content in the crude product. In addition, when lipase ANL from Aspergillus niger was immobilized on a weakly polar carrier, its catalytic esterification reaction of oleic acid and 1-monoglycerate also had a high DAG content.

[0034] Example 3 Effect of transesterification temperature: ANL was immobilized on AB-8 to obtain lipase ANL-AB8, and then rapeseed oil transesterification reaction was catalyzed at 35℃~65℃ for 3 h. This process was repeated until the acid value of the recovered rapeseed oil dropped below 1.5 mgKOH / g to prepare lipase B under different temperature conditions. Other transesterification and esterification reaction conditions were the same as in Example 1. The DAG 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 40-55℃.

[0036] Example 4 Effect of esterification temperature: ANL was immobilized on AB-8 to obtain lipase ANL-AB8, which was then used to catalyze the transesterification reaction of rapeseed oil at 45℃ for 3 h. This process was repeated until the acid value of the recovered rapeseed oil dropped below 1.5 mgKOH / g to prepare lipase ANL-AB8-B. This enzyme was used to catalyze the esterification reaction at a temperature of 40℃~65℃. Other transesterification and esterification reaction conditions were the same as in Example 1. The DAG 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 in esterification reaction: The immobilized enzyme ANL-AB8-B obtained in Example 4 was used to catalyze the esterification reaction of oleic acid and monoglyceride. The molar ratio of 1-oleic monoglyceride to oleic acid was 1:0.8, 1:1, 1:1.2, 1:1.4, 1:1.6 and 1:1.8, respectively. Other conditions were the same as in Example 1. The DAG 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 1-monoglycerate to oleic acid is within the range of 1:(1–1.8). The crude DAG products obtained under molar ratios of 1:0.8 and 1:1, after two-stage molecular distillation purification, showed a DAG content of over 90%, while the crude DAG products obtained under other molar ratios, after two-stage molecular distillation purification, showed a DAG content of over 95%.

[0040] Example 6 Effect of fatty acid type on esterification reaction: The immobilized enzyme ANL-AB8-B obtained in Example 4 was used to catalyze the esterification reaction of 1-oleic acid monoglyceride and different fatty acids. The selected fatty acids were acetic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, linoleic acid, and DHA (docosahexaenoic acid). Other conditions were the same as in Example 1. The DAG content in the crude product is shown in the table below.

[0041] Table 6. Effect of fatty acid type on lipase B-catalyzed esterification reaction in esterification reaction. The results in the table above show that the best lipases are those for C8-C18 fatty acids, except for C18:0. Among them, the enzyme has the best specificity for myristic acid (C14:0).

[0042] Example 7 The effect of the type of monoglyceride in the esterification reaction: The immobilized enzyme TLL-AB8 was obtained according to the method of Example 2, and then the rapeseed oil transesterification reaction was catalyzed at 45°C for 3 h. This process was repeated until the acid value of the recovered rapeseed oil dropped below 1.5 mgKOH / g, catalyzing the esterification reaction of oleic acid and monoglycerides. The monoglycerides investigated were 1-oleic acid monoglyceride and 2-oleic acid monoglyceride. Other conditions were the same as in Example 1. The DAG content in the crude product is shown in the table below.

[0043] Table 7. Effect of monoglyceride type on lipase B-catalyzed esterification reaction in esterification reaction. As shown in the table above, when 2-monoglyceride is used as a substrate, the DAG content in the crude esterification product is significantly higher than that of 1-monoglyceride.

[0044] Example 8 Effects of Adsorbent Type and Polarity in Esterification: Immobilized lipase B, namely lipase TLL-AB8-B, was prepared according to the method in Example 2. This enzyme was used to catalyze the esterification reaction of 1-oleic acid monoglyceride and oleic acid. The effects of different adsorbents on the esterification reaction were investigated. 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 1. The DAG 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 weakly polar adsorbent to the esterification reaction yields the best results.

[0046] Comparative Example 1: Transesterification and Esterification The esterification reaction of oleic acid and 1-oleic acid monoglyceride was catalyzed using the commercially available immobilized lipase Lipozyme TL IM derived from Thermomyces lanuginosus as a catalyst. The activity difference between Lipozyme TL IM and the TLL-AB8-B enzyme prepared by the method in Example 2 in catalyzing the esterification reaction of oleic acid and 1-oleic acid monoglyceride was compared. The esterification reaction conditions were the same as in Example 1. After the reaction was completed, the DAG content in the crude product was measured.

[0047] The results showed that the crude esterification product catalyzed by Lipozyme TL IM contained only 34.5% DAG, while the crude esterification product catalyzed by TLL-AB8-B lipase prepared by the method of this invention contained as much as 44.6% DAG. After two-stage molecular distillation to remove free fatty acids and monoglycerides, the DAG content in the purified product was greater than 92%.

[0048] This invention employs enzymatic esterification to prepare diglyceride oil in a solvent-free system. Specifically, it uses a relatively inexpensive lipase as a biocatalyst to react free fatty acids and monoglycerides in a solvent-free system, producing an oil rich in diglycerides. Before the enzymatic esterification reaction, the lipase undergoes immobilization and random transesterification to improve its catalytic efficiency in the esterification reaction system, thereby reducing production costs and increasing the diglyceride content of the product. This invention provides a green, economical, and efficient method for the enzymatic esterification of diglycerides, reducing the cost of enzyme preparations for diglyceride preparation and showing promising prospects for industrial application.

[0049] 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 process for producing diglyceride oil, characterized in that: The process includes the following steps: Step (1) Immobilization of lipase: The lipase was immobilized on a weakly polar carrier to prepare immobilized lipase A; The lipase is derived from one or more of Thermomyces lanuginosus or Aspergillus niger; the weakly polar carrier is AB-8, BS-30 or DM-130. Step (2) Transesterification reaction: The immobilized lipase A obtained in step (1) is mixed with animal and vegetable oils and transesterification reaction is carried out at 35℃~65℃. This step is repeated until the acid value of the animal and vegetable oils after transesterification drops to 1.5 mgKOH / g or below. Immobilized lipase B is then separated from the animal and vegetable oils after the reaction; wherein the acid value of the animal and vegetable oils is less than 0.2 mgKOH / g. Step (3) Esterification reaction: Add the immobilized lipase B and adsorbent obtained in step (2) to the monoglyceride and free fatty acid, and carry out the esterification reaction at 40-65℃. After the crude product is deacidified by distillation, the purified diglyceride oil product is obtained.

2. The process for producing diglyceride oil using a solvent-free enzymatic system as described in claim 1, characterized in that: The specific step (1) is as follows: the enzyme concentration of the immobilized carrier is diluted with buffer solution to 12 mg / mL, the enzyme solution pH is 5.5, the temperature is 25℃, and 8 wt% maltodextrin based on the immobilized carrier 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.

3. The process for producing diglyceride oil using a solvent-free enzymatic system as described in claim 1, characterized in that: In step (2), the transesterification reaction temperature is 40–55 °C.

4. The process for producing diglyceride oil using a solvent-free enzymatic system as described in claim 1, characterized in that: In step (3), the molar ratio of monoglyceride to free fatty acid is 1:(1 to 1.8).

5. The process for producing diglyceride oil using a solvent-free enzymatic system as described in claim 1, characterized in that: In step (3), the monoglyceride is 2-monoglyceride.

6. The process for producing diglyceride oil using a solvent-free enzymatic system as described in claim 1, characterized in that: In step (3), the free fatty acid is a C8 to C18 carbon chain fatty acid, but does not include stearic acid.

7. The process for producing diglyceride oil 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.

8. The process for producing diglyceride oil using a solvent-free enzymatic system as described in claim 1, characterized in that: In step (3), the esterification reaction temperature is 45~60℃.

9. The process for producing diglyceride oil using a solvent-free enzymatic system as described in claim 1, characterized in that: In step (3), the adsorbent is a weakly polar adsorbent, preferably AB-8, BS-30 or ADS-17.

10. The application of the solvent-free enzymatic process for producing diglyceride oil according to any one of claims 1 to 9 in the food and pharmaceutical fields.

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