An Overview of Structured Lipid in Food Science: Synthesis Methods, Applications, and Future Prospects

Abstract

Structured lipids have attracted significant interest in industries, such as food, pharmaceuticals, and cosmetics, because they can be alternatives to traditional lipids. Enzymatic synthesis techniques utilizing lipases have gained attention for structured lipids synthesis because of their site and substrate specificity. However, most lipases discovered to date exhibit sn-1,3 regiospecificity or nonspecificity, limiting the selective range of natural oils and fats that can be used as raw materials. In this review, we provide an overview of the current synthesis methods and applications of structured lipids and the limitations of existing lipases in producing structured lipids. In addition, we discuss innovative approaches, including metagenomics, and machine learning, to discover, and classify new lipases and the use of gene editing technologies for lipase engineering. These efforts aim to overcome the limitations of existing lipases and expand the range of natural oils and fats that can be used for the production of structured lipids. Therefore, this study aims to promote a better understanding of structured lipids as a material with high-added value and provides insight into ongoing research efforts in the field.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

In the food, pharmaceutical, and cosmetic industries, materials are manufactured as goods based on natural products. However, the increasing demand for natural resources because of population upsurge, depletion of growing areas caused by environmental degradation, and extinction of plants caused by climate change necessitates a shift in strategic approaches to produce materials [1, 2]. As a response to these challenges, various new technologies have been developed to substitute natural products and ensure economic efficiency and widespread use of rare raw materials with high-added value [3–5]. Furthermore, progress is ongoing on the development of substitutable materials for the three major nutrients and functional substances.

Fat, one of the three major nutrients, provides necessary fatty acids and energy. It also plays a vital role in internal metabolic processes as a carrier that assists in the absorption of fat-soluble vitamins into the body [6], a precursor of hormones and neurotransmitters needed for the maintenance of body temperature, a buffering agent, and a component of intercellular communication [7–9]. Furthermore, microscopic levels of lipid derivatives are necessary components of brain function [10]. In the pharmaceutical and cosmetic sectors, lipids are also used as solvents for hydrophobic substances and emulsifiers for the homogenous mixing of water-soluble and fat-soluble substances [11, 12].

Structured lipids, as an alternative to conventional fats, have been receiving attention because of their customized functional properties and health benefits [13]. They can be synthesized through different methods such as chemical or enzymatic catalyses [14]. In the synthesis of structured lipids, chemical catalysis is generated by reactions at high temperatures using catalysts such as sodium methoxide, sodium hydroxide, and lithium hydroxide [15–17]. This approach has a low-processing cost but requires a complex process, including the formation of byproducts and their elimination. By contrast, the production of structured lipids using biological enzymes does not lead to side reactions because of the substrate and site specificity necessary for the targeted production of structured lipids. In addition, commercially available immobilized enzymes are ecologically benign, react at comparatively low temperatures, do not encourage side reactions, are simple to reuse and recover, and offer economic benefits [18]. Recently, a manufacturing method using biological enzymes that immobilizes diverse enzymes has become the standard [19].

This review will discuss the trends of currently produced and investigated structured lipids, the advantages and disadvantages of different synthesis methods, and the present state of lipase excavation for the enzymatic generation of valuable structured lipids. In addition, this review will delve into the challenges and future prospects in the field, hoping that the discovery and creation of lipases for existing enzyme production will establish the direction of future structured lipid development through novel approaches for creating more advanced structured lipids.

2. Structured Lipids: Definition, Properties, and Synthesis Methods

2.1. Definition of Structured Lipids

Structured lipids are modified fats and oils that are chemically or enzymatically altered to enhance their physicochemical properties, nutritional values, and functional attributes. These lipids have a specific arrangement of fatty acids on the glycerol backbone, which allows for designing lipids with customized properties suitable for applications in the food, pharmaceutical, and cosmetic industries.

2.2. Advantages and Disadvantages of Structured Lipids

2.2.1. Advantages

Structured lipids offer numerous advantages, such as improved physicochemical properties, enhanced nutritional values, and customized functionality [14, 20]. These advantages enable the development of lipids with specific melting points, viscosity, and oxidative stability, making them more suitable for various applications [13]. By incorporating desirable fatty acids such as medium-chain fatty acids or polyunsaturated fatty acids (PUFAs), structured lipids can provide health benefits, such as reducing the risk of cardiovascular diseases and improving energy metabolism [21, 22]. Furthermore, structured lipids can be engineered to demonstrate specific functionalities, such as enhanced emulsification, solubility, or bioavailability, which are advantageous in food, pharmaceutical, and cosmetic formulations [23].

2.2.2. Disadvantages

Structured lipids also have some disadvantages. The synthesis of structured lipids, particularly through enzymatic methods, can be costly because of expenses related to enzyme production and purification [24]. Moreover, the majority of lipases employed in enzymatic synthesis are sn-1,3 regiospecific or nonspecific, limiting the ability to produce lipids with the desired fatty acid distribution [25]. Finally, the complexity of structured lipid synthesis necessitates the precise control of reaction conditions and the use of various catalysts or enzymes [26].

2.3. Synthesis of Structured Lipids

Manufacturing techniques of structured lipids can be chemical or enzymatic (Figure 1). This section explores the principles, advantages, and disadvantages of chemical and enzymatic synthesis methods.

[figure(s) omitted; refer to PDF]

2.3.1. Chemical Synthesis of Structured Lipids

Chemical catalysis of structured lipids primarily synthesizes lipids for use in the chemical industry, including specific-purpose synthetic oils and biodiesel [27]. Production processes of structured lipids employing chemical catalysts involving thermal decomposition, catalytic decomposition, microemulsification, and esterification reactions are characteristically irreversible [28]. Furthermore, they can proceed in a single direction, contrary to biological enzymatic reactions. However, chemical synthesis is conducted using a strong acid or a strong base that requires high temperatures and energy.

In a recent study, researchers utilized chemical interesterification (CIE) to fabricate trans-free cocoa butter alternatives (CBAs) from a blend of palm kernel stearin (PKS), coconut oil (CNO), and fully hydrogenated palm stearin (FHPS) [29]. In another study, the same chemical interesterification process was applied to a combination of palm stearin (PS) and olive oil (OO), resulting in fats with various degrees of plasticity, thus expanding the possibilities for the commercial utilization of PS and OO [30].

Despite these successful examples, it should be noted that chemical synthesis has improved in recent years, involving environmentally friendly and efficient processes that can be produced at low temperatures using physical methods, including sonication and cavitation [31]. Nevertheless, the use of organic solvents for quenching chemical catalyst reactions and considering the generation and elimination steps of byproducts is a very unfavorable process compared with synthetic biological engineering methods [32].

2.3.2. Enzymatic Synthesis of Structured Lipids

Lipases (EC. 3.1.1.3) generally catalyze reversible processes that can hydrolyze and dehydrate condensation reactions to decompose and synthesize bonds between carboxyl and hydroxyl groups [33]. In addition to decomposing and synthesizing lipids, lipases can also degrade and synthesize substrate-specific compounds and can be applied in several scientific and industrial fields.

In lipolysis and synthesis, lipases exhibit regiospecificity, though most commercially available lipases display sn-1,3 nonspecificity in lipids rather than sn-2 regiospecificity, which is the more desirable one. As a result, modifying the composition and distribution of fat are difficult.

Approximately four reactions exist when synthesizing structured lipids employing lipase as an enzyme, depending on the substrates used in the reaction mixture. Free fatty acids and glycerol are directly esterified, acylglycerol is transesterified intramolecularly or intermolecularly, triacylglyceride (TAG) is acidolyzed and transesterified with free fatty acids, and TAG is transesterified to produce monoacylglyceride (MAG) and diacylglyceride (DAG) [34–37] (Figure 2). In each of these methods, the water concentration, reaction temperature, and substrate are critical parameters that determine the direction of the lipase reaction.

[figure(s) omitted; refer to PDF]

Lipases, free and immobilized ones, are different based on the characteristics shown in the production process of structured lipids. First, free lipases are often inexpensive and can lower process costs; nevertheless, they are sensitive to extreme temperatures and pH, leading to decreased yields. In addition, the recovery of enzymes for reuse requires an additional complex procedure, and the activity of the recovered enzymes cannot be assured. By contrast, immobilized lipases alleviate the problem of enzyme yield loss and reaction stability induced by environmental changes such as pH, temperature, and organic solvents, allowing faster enzyme recovery and reuse [38].

Techniques for immobilizing enzymes include adsorption in a porous material, covalent binding between the enzyme and support, and enzyme conjugation through cross-linking and interenzyme coupling [39]. Furthermore, this method immobilizes enzymes through several physical binding interactions, including van der Waals forces, hydrophobic interactions, hydrogen bonds, and ionic connections [40].

Despite technological advancements, the practical use of immobilized lipases in structured lipids is largely limited by most lipases being sn-1,3 regiospecific or nonspecific [41] (Table 1). Two illustrative examples, however, show the potential applications of lipases. In one study, medium- and long-chain triglycerides (MLCTs) were synthesized by incorporating lauric acid into flaxseed oil via lipase-catalyzed acidolysis, demonstrating improved thermo-oxidative decomposition behavior [42]. Another study prepared structured TAGs rich in 1,3-dioleoyl-2-palmitoylglycerol (OPO) and 1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) through enzymatic acidolysis of fractionated palm stearin with free fatty acids, presenting high product yields and the potential for use in infant formulas [43].

Table 1

Current types and forms of immobilized enzymes manufactured commercially.

Product names	Source	Immobilization material	Development company
Novozyme 435	Candida antarctica	Resin	Novozymes
Lipozyme 435	Candida antarctica	Resin	Novozymes
Lipozyme TL IM	Thermomyces lanuginosa	Silica gel	Novozymes
Lipozyme RM IM	Rhizomucor miehei	Resin	Novozymes
Lipase Rd	Rhizopus delemar	MP 1000	Tanabe Seiyaku Co. Ltd.
Lipase QLM	Alcaligenes sp.	MP 1000	Meito Sangyo Co.
Lipase AK	Pseudomonas fluorescens	MP 1000	Amano Pharmaceutical Co.
Lipase D	Rhizopus oryzae	MP 1000	Amano Pharmaceutical Co.
Lipase DF	Rhizopus oryzae	MP 1000	Amano Pharmaceutical Co.

Even though some lipases may extract fatty acids from the sn-2 position of TAG, these enzymes are quite uncommon for commercial immobilized lipases. Although specificity and selectivity for substrates, low energy consumption, and the creation of immobilized enzymes enable reusable successive processes, the enzymatic synthesis of structured lipids is limited(Table 2).

Table 2

Overview of current structured lipids synthesized via enzymatic and chemical approaches.

No.	Enzymatic/chemical	Catalyst/enzyme	Backbone	Fatty acids	Products	Ref.
1	Enzymatic	Lipozyme TL IMLipozyme RM IM	Crude olive pomace oil	Caprylic acid (C8 : 0)Capric acid (C10 : 0)	Low-calorie structured triacylglycerols	[44]
2	Enzymatic	Lipozyme 435Lipozyme RMLipozyme TL IM	Medium-chain triglyceride	EPA-enriched fish oil (60% EPA, 15% DHA)	EPA-enriched medium- and long-chain triacylglycerol (MLCT)	[45]
3	Enzymatic	Lipozyme TL IMLipase from Candida sp.	Palm stearin monopalmitoylglycerol	Oleic acid linoleic acid	Human milk fat substitutes (HMFS; OPL, OPO, and LPL)	[46]
4	Enzymatic	Lipozyme 435	Trioctanoate	n-Propyl gallate	1,2-Dioctanoylgalloylglycerol (DOGG)	[47]
5	Enzymatic	Novozyme 435	Linseed oil	Caprylic acid (C8 : 0)Capric acid (C10 : 0)	1,3-Dicapryl-2-linolenoylglycerol1,3-Dicapril-2-linolenoylglycerol	[48]
6	Enzymatic	Lipase MAJ1	Tricaprylin	Methyl palmitate	LML type TAG	[49]
7	Enzymatic	Lipozyme RM IM	Grape seed oil	Capric acid (C10 : 0)	MLM type TAG	[50]
8	Enzymatic	Lipozyme RM IM	Palm kernel stearin	Coconut oilFully-hydrogenated palm stearin	Cocoa butter alternatives (CBA)	[51]
9	Enzymatic	Lipozyme TL IM	Tripalmitin	Oleic acidLinoleic acid	Human milk fat substitutes (HMFS; OPL and LPL)	[52]
10	Enzymatic	Lipozyme RM IM	Coconut oil	Soybean oil	Improved bioavailability carrier oil	[53]
11	Enzymatic	Lipase TL100Lipase RMLLipase CAL-A	Palm oil	Linolenic acid	Functional fatty acids in oil (ω-3)	[54]
12	Enzymatic	Lipozyme RM IM	Pomegranate seed oil	Conjugated linoleic acidConjugated linolenic acid	Functional fatty acids in oil	[55]
13	Enzymatic	Lipozyme RM IM	Illipe butter	Palm midfraction	Cocoa butter equivalent (CBE)	[56]
14	Chemical	Sodium silicate (Na2SiO3)	Glycerol	Palm oleinPalm stearin	MAG, DAG	[57]
15	Chemical	Sulfuric acid (H2SO4)HND-6p-Toluenesulfonic acid (p-TSA)	Coconut oil	Caprylic acid (C8 : 0)Capric acid (C10 : 0)	Medium-chain triacylglycerols (MCTs)	[58]
16	Chemical	Sodium methoxide	Palm olein	Palm kernel oilPalm stearin	Trans-free plastic fats	[59]
17	Chemical	Sulfonated Zn-dopedSBA-15 (Ph-SO3H-Zn-SBA-15)	Soybean lecithin	Methyl butyrate	SCFA (short-chain fatty acid)-lecithin	[60]

3. Functional Food Application of Structured Lipids

3.1. Cocoa Butter Equivalents (CBEs)

Cocoa butter is a lipid obtained from cacao beans, and it has many commercial applications in chocolate, drinks, and baking. Regarding composition, cocoa butter differs from other lipids because it contains comparatively few TAGs. That is, >80% of the triglycerides that make up cocoa butter are composed of 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, 13.8%–21.8%), 1-palmitoyl-2-oleoyl-3-stearoyl-rac-glycerol (POS, 26.3%–44.8%), and 1,3-distearoyl-2-oleoyl-glycerol (SOS, 20.0%–29.4%) with oleic acid bound to the sn-2 position [61]. The unique triglyceride fatty acid composition of cocoa butter reveals remarkable physical qualities in the final culinary product, including hard physical qualities at ≤25°C, soft physical properties, and a texture that melts at ≥30°C [62]. Given these properties, cocoa butter is more expensive than edible oils and fats. Owing to the increased international demand and lower yields caused by environmental problems, artificial lipid synthesis is being actively explored to obtain raw materials.

CBE is a structured lipid with a triglyceride and fatty acid profile comparable to cocoa butter, and it is manufactured from inexpensive vegetable oils [63]. Recent research on CBE synthesis has focused on POS-rich lipid synthesis, which accounts for approximately 40% of cocoa butter’s triglycerides [64] (Table 3). Commercial CBE POS is synthesized using lipids rich in POP and SOS [71]. Palm oil midfraction, which can be derived through the fractional crystallization of palm olein, is the raw material of CBE for POP [72]. Fractional crystallization of SOS from tropical plants, including shea butter or a catalyst, is used to produce SOS.

Table 3

Types and triacylglyceride formulations of cocoa butter equivalent currently researched and developed.

	Product name	POP (%, w/w)	POS (%, w/w)	SOS (%, w/w)	Ref.
Natural product	Cocoa butter	13.8∼21.8	26.3∼44.8	20.0∼29.4	[65]
CBE research	CBE from PMF${}^{\ast }$/PKO${}^{\ast \ast }$/PS${}^{\ast \ast \ast }$ by Thermomyces lanuginose lipase	17.7	28.4	19.5	[66]
	CBE from PMF by Rhizomucor miehei lipase	30.7	40.1	14.5	[67]
	CBE from PO${}^{\ast \ast \ast \ast }$ by Rhizomucor miehei lipase	26.6	42.1	18.0	[68]
	CBE from PMF by Lypozyme lipase	21	40	27	[69]
Commercialized CBE	ILLEXAO CB 40	34.2	13.8	31.4	[70]
	ILLEXAO SC 70	31.6	13.6	33.7
	PALMY 50R	36.9	12.6	30.8
	PALMY 20G	47.1	12.0	21.0
	Coberine 507	43.8	12.9	21.7
	Coberine 608	37.3	13.2	31.7
	Chocovit 230	48.0	13.0	18.8
	Chocovit 270	39.0	12.1	29.1

${}^{\ast }$PMF: palm oil midfraction. ${}^{\ast \ast }$PKO: palm kernel oil. ${}^{\ast \ast \ast }$PS: palm stearin. ${}^{\ast \ast \ast \ast }$PO: palm olein.

CBE synthesis is limited by the requirement for oleic acid-rich lipids at the sn-2 position of the lipids. Most commercially available lipases are sn-1,3 regiospecific enzymes because they lack sn-2 site-specific reactions. Therefore, a considerable difference exists between the composition of natural cocoa butter and triglycerides, and substituting up to 5% of cocoa butter in food with triglycerides is acceptable.

3.2. Human Milk Fat Substitutes (HMFSs)

Human milk fat is the primary energy source for babies, supplying 50% of their daily caloric intake [73], and is an essential source of vital fatty acids and fat-soluble vitamins. Oleic acid and palmitic acid are the main fatty acids in human milk fat, making up approximately 40% and 25%, respectively, and they exist as 1,3-dioleoyl-2-palmitoyl-glycerol [15]. Palmitic acid, a hydrolyzed free fatty acid, is used as an energy source. However, it forms an insoluble salt with the calcium and minerals needed for the growth of newborns and excretes them in the feces, preventing calcium absorption and causing constipation [74]. Therefore, in formulating HMFSs, the amount and composition of palmitic acid at the sn-2 position are important.

Commercial HMFSs are synthetic lipids with fatty acid contents and distribution comparable to human milk fat. Therefore, it is an important raw material in synthesizing infant food products (formula). Generally, HMFSs are manufactured by synthesizing free fatty acids obtained from hydrolyzing palmitic acid-rich fats and oils, tripalmitin, and oleic acid-rich vegetable oils, such as olive oil and sunflower oil [75]. In addition, they provide PUFAs that are important for the development of newborns and critical fatty acids are not found in human milk fat [76].

3.3. Trans-Free Plastic Fats

Many techniques such as margarine and shortening were created to ensure that vegetable fats, when used as animal fat substitutes, have the same physical qualities and texture as animal fat [77]. The liquid plant fat was converted into a solid with physical properties similar to animal fat. However, these vegetable fats that substitute animal fats have disadvantages [78]. The plastic fat used in margarine is produced through a hardening reaction involving the hydrogenation of vegetable oils, the primary raw materials [79]. The cis-type double-bond chain of unsaturated fatty acids is opened, and hydrogen is introduced during the curing process. Transfatty acids produced at this time increase the levels of total cholesterol and low-density lipoprotein cholesterol in the blood while lowering the levels of beneficial high-density lipoprotein cholesterol, causing cardiovascular and metabolic diseases [80]. Because of these problems, the level of transfatty acids in foods is closely limited, constraining the usage of hydrogenated oils [81].

Structured lipids can be practically produced through the enzymatic esterification of vegetable and solid fats to reduce transfatty acids while maintaining their physical qualities [82]. Saturated and unsaturated fats can be compared with partially hydrogenated oils in terms of their physical properties through esterification reactions that take place in the presence of lipases. Furthermore, margarine and shortening are produced by including helpful fatty acids, such as conjugated linoleic acid, without structured lipids, simply lowering transfat.

3.4. Low-Calorie Fats and Medium-Chain TAG

The calorific value of fat, a high-energy source, varies depending on the number of carbon atoms in fatty acids. Specifically, structured lipids with short- or long-chain fatty acids can be used as low-calorie fats. Short-chain fatty acids with 2–4 carbon atoms have a substantially lower caloric content than typical fat (9 kcal/g) [83]. Edible oils and fats, which are familiar sources of essential fatty acids, are mainly composed of long-chain fatty acids (L), which are stored in the body because of prolonged metabolic processes [84]. By contrast, medium-chain fatty acids (M), which include 8–12 carbon chains with hydrophilic properties, are easily oxidized and can be rapidly used by the body as an energy source. Unlike typical fat metabolism, medium-chain fatty acids are delivered directly to the liver through the hepatic portal vein and accumulate less frequently in tissues [85]. Therefore, they are efficient lipid substances for preventing metabolic diseases such as obesity, diabetes, and hyperlipidemia.

In addition, several edible oils (soybean oil, corn oil, olive oil, and canola oil) mainly comprise long-chain TAG- and medium-chain TAG-rich edible oils through enzymatic degradation and synthesis [86]. By reacting edible oils with heavy chain fatty acids in the presence of sn-1,3 stereospecificity lipase, other edible oils, including medium-long-medium (MLM) and medium-long-long (MLL), can be produced. Specifically, the MLM culinary sustenance provides better absorption of long-chain fatty acids, particularly essential ones, than other edible oils.

3.5. Functional Fatty Acids (PUFAs)

Omega-3 fatty acids, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), efficiently improve brain and cardiovascular health; gamma-linolenic acid efficiently enhances skin health and blood circulation; and conjugated linolenic acid efficiently lowers body fat [87]. As the demand for functionally structured lipids containing high quantities of these helpful fatty acids increases, several studies are being conducted. By using the esterification process of perilla oil containing alpha-linolenic acid, linseed oil, and fish oils containing EPA and DHA in conjunction with lipase, studies have reported the synthesis of structured lipids containing large amounts of beneficial fatty acids [88–90]. These are beneficial because they are pure and contain many valuable fatty acids. In addition, fish oil can eliminate refusal to take it because of its objectionable odor .

3.6. Functional Emulsifiers

Emulsifiers are additives that bridge the combination of hydrophilic and hydrophobic chemicals and are employed to ensure the stability of several ingredients in the final food product. Characteristically, emulsifiers are composed of hydrophilic and hydrophobic molecules. In general, hydrophobic molecules have a long-chain structure similar to the molecular structure of fatty acids. Therefore, various emulsifiers use fatty acids as the hydrophobic molecular moiety. In particular, MAG and DAG are used in many industries as emulsifiers.

Lipases employed in reconstituted lipogenesis can synthesize emulsifiers by linking fatty acid chains to existing MAG, DAG, and other hydrophilic compounds with hydroxy groups. The emulsifier generated in this approach can be a multifunctional emulsifier with both hydrophilic molecules and fatty acid functionalities. Based on previous studies, multifunctional emulsifiers include sucrose palmitate, which is made of sucrose and palmitic acid, and emulsifiers that are made of fatty acids bound to ascorbic acid and erythorbic acid (Table 4) [96, 97]. These emulsifiers provide functions beyond those of existing emulsifiers. For example, they possess antibacterial and antioxidant characteristics. In addition, they are ecofriendly emulsifiers produced by enzymatic synthesis using natural materials and have low toxicity to the body [98].

Table 4

Types of functional emulsifiers.

Product names	Enzyme	Properties	Ref.
Erythorbyl laurate	Novozyme 435	Antioxidant, antibacterial	[91]
Ascorbyl palmitate	Novozyme 435	Antioxidant	[92]
Sucrose palmitate	Lipolase 100 L	Nontoxicity	[93]
Sucrose laurate	Lipolase 100 L	Antibacterial	[93]
Fructose oleate	Lipozyme	Nontoxicity	[94]
Lauric arginate	Novozyme 435	Antibacterial	[95]

Moreover, whether it is possible to maintain the functional features of hydrophilic and hydrophobic molecules while performing the role of emulsifiers to produce functional emulsifiers from structured lipids must be analyzed and assessed. Lipases react through dehydration condensation between hydrophilic head molecules and hydrophobic tail molecules; however, if they possess unique functionality at the binding site, they may lose their usefulness during synthesis. In addition, specifying the binding site when there are numerous binding sites for hydrophilic molecules other than lipids is impossible.

Briefly, structured lipids can be used in various food applications by customizing their composition and qualities. These lipids can create CBEs, HMFSs, trans-free plastic fats, low-calorie fats, medium-chain TAGs, and functional emulsifiers. Developing these functionally structured lipids could significantly affect the food industry and consumers by providing healthier and more diverse options. Research and innovation in this field will continue to expand the range of applications and benefits that structured lipids provide in functional foods (Figure 3).

[figure(s) omitted; refer to PDF]

4. Future Prospects

The market for extremophilic enzymes, such as lipases and esterases, isolated from microorganisms in extreme conditions, encompasses various applications in the food, pharmaceutical, and environmental industries, amounting to an enormous market size of approximately $600 million [99]. While the majority of these biocatalysts are discovered through traditional research methods such as metagenomics, innovative approaches are being developed to overcome cost and time constraints. Specifically, data science-based methods are being employed for classifying and systematizing the characteristics of known lipases and esterases and predicting the properties of new enzymes through phylogeny [100] and artificial intelligence and machine learning for predicting the properties of enzymes with specific amino acid sequences based on their three-dimensional (3D) structures [101]. From a genetic engineering perspective, third-generation gene editing technology [102], CRISPR/Cas9, offers a direction to address the challenges (e.g., reversibility/irreversibility, substrate specificity, temperature, and environmental adaptability) that existing enzymes possess through genetic manipulation [103].

4.1. Lipase Engineering Database (LED)

Lipases (EC 3.1.1.3) and esterases (EC 3.1.1.1) can be categorized based on amino acid sequence similarity and physiological characteristics related to their ability to hydrolyze fats. However, classifying lipases accurately requires relying more than just on sequence similarity. The LED (https://led.biocatnet.de/) was developed to systematically organize information on lipases’ sequences, structures, and functions and related proteins sharing the same α/β hydrolase fold. In particular, all lipases of the α/β hydrolase family consist of a catalytic triad and an oxyanion hole-containing core domain, a lid domain that protects the active site, a domain that supports the affinity of the active site, and an N- or C-terminal domain [104, 105]. These structural features can be utilized to classify lipases. In the latest version of the LED (ver. 4.1.0), 283,672 sequences and 1,590 structures have been categorized into 13 superfamilies. Although the LED remains a valuable tool for lipase classification, it requires regular database management and updates.

In addition to databases such as LED, various methods are employed to identify and classify the amino acid sequences of new lipases. These methods include metagenomic screening and reconstructing ancestral sequence approaches [106, 107]; however, these methods often involve costly and time-consuming validation processes. To overcome these drawbacks, automated tools for systematic classification are needed, typically relying on sequence similarity search tools such as BLAST. The functionality of these tools is limited by the quality of the search algorithm and the database scale used for searching. They may also involve some inherent errors within the program. Consequently, they need to provide a perfect solution for discovering and predicting new lipases.

4.2. Discovery of Lipases Using Machine Learning and Artificial Intelligence

Recent advances in machine learning and artificial intelligence have opened new opportunities for the classification of lipases and esterases. Techniques such as deep learning and artificial neural networks facilitate accurate classification and functional prediction of new enzymes based on sequence and structural information. This allows for overcoming the time and cost constraints associated with traditional approaches.

4.2.1. Prediction of Lipase 3D Structure

DeepMind’s AlphaFold, the protein 3D structure prediction system developed by the creators of AlphaGo, has introduced new possibilities [101]. It employs artificial intelligence to predict amino acid sequences into 3D structures, improving accuracy through continuous iterative learning. After the first software release in 2018, AlphaFold2 was developed in 2020 [108]. AlphaFold contains approximately 3,000 lipase amino acid sequences; however, expanding to a more extensive database is necessary. While the 3D structure prediction of independent proteins is highly accurate, limitations in the prediction model arise from the need for more information on variables such as environmental conditions and proximity of other proteins, oligomers, metal ions, and cofactors.

RoseTTAFold, another protein 3D structure prediction system, provides more accurate predictions for specific regions, such as membrane proteins, by considering some of the variables mentioned above based on amino acid sequences [109]. However, similar to AlphaFold, the absence of machine learning for various variables results in reduced accuracy in predicting protein 3D structures under specific conditions [110].

Modeling methods for neural network-based protein 3D structure prediction have made significant progress in recent years, offering a new paradigm for protein structure prediction. Although systems such as AlphaFold and RoseTTAFold have many areas requiring improvement, their utility in various fields remains high. In discovering new lipases, clarifying structural features and phylogenetic classifications through prediction model systems can significantly shorten time constraints.

4.2.2. Prediction of Lipase Regiospecificity

Identifying novel lipases is essential for producing structured lipids with various functional properties. In addition to sn-1 regiospecific and nonspecific lipases, the discovery of sn-1(3) and sn-2 regiospecific lipases is significant. Despite several studies aiming to recognize lipases with sn-1(3) regiospecificity and sn-2 regiospecificity, obtaining experimental data, and research results takes time and effort. Various solutions can be proposed to overcome these limitations. For example, to determine enzyme inhibitors, Durai et al. [111] developed an in silico prediction model, predicting inhibitory potential based on structural compatibility and binding strength between natural substances and tyrosinase substrate interaction. These findings can be highly beneficial for identifying lipases that can synthesize structured lipids. Novel regiospecificity can be predicted based on structural compatibility and binding strength with each substrate after structural prediction for the active lipase site with known positional specificity to produce 1,3-DAG and 2,3-DAG with TAG and synthesize various structured lipids such as POS and POP. The outcomes of this prediction modeling may enable faster discovery of novel regiospecific lipases.

In addition to developing lipases with novel regiospecificity, ongoing research is needed to reduce the cost of commercially available lipases. Most commercially available lipases are expensive and derived from microorganisms [112]. Identifying new lipases, which are essential components of enzymatic synthesis methods for structured lipid production, is necessary. Despite active research on lipase extraction from diverse natural sources such as plants and fungi, only some industrial applications have been identified [113]. This is explained by the challenging mass production of lipases isolated from natural sources. Genetic engineering studies have offered potential solutions to these issues, such as finding novel lipases and producing lipases through genetic replication in various cultural environments, including bacteria and yeast, where mass production is more feasible. With active collaboration from discovery to mass production, further research on lipases that can synthesize structured lipids is expected.

In addition to discovering novel lipases, extensive research is ongoing on the multiple roles of lipids, anticipating the production of structured lipids with additional functionalities. As lipids with diverse properties such as antioxidant, antibacterial, and anti-inflammatory capabilities have been identified, producing multifunctional structured lipids by enzymatically synthesizing lipids with these properties and functions can add significant value. This can increase the availability of structured lipids for widespread use in medicine, cosmetics, and food as dietary ingredients.

In summary, machine learning and artificial intelligence hold great potential for discovering and classifying lipases and esterases. Techniques such as deep learning and artificial neural networks can facilitate accurate predictions of enzyme structures and functions, leading to more efficient discovery and development of novel lipases for the synthesis of structured lipids. Furthermore, ongoing research on the multiple roles of lipids and the development of in silico prediction models can contribute to the production of structured lipids with additional functionalities and expanded applications across industries.

4.3. Improvement of Lipases through Gene Editing Technologies

Gene editing technologies, particularly the CRISPR/Cas9 system derived from the bacterial immune system, are emerging as potential tools in various fields as third-generation gene scissors [102]. CRISPR/Cas9 comprises gRNA, which binds to a particular base sequence, and Cas9 nuclease, which acts as the scissors [103]. Compared with the first- and second-generation gene scissors, the CRISPR/Cas9 system allows for highly precise gene manipulation. It has been widely utilized because of its simplicity, ability to cut at any site, and cost-effectiveness. Moreover, many studies have tried to improve the function of lipases and esterases, and gene editing technologies can be employed to enhance the activity of specific enzymes or improve their stability in specific environments [114, 115]. This can lead to the development of improved enzymes through genetic manipulation, resulting in more cost-effective industrial products.

Despite the advantages of CRISPR/Cas9, concerns about the safety and ethical issues of gene manipulation are always raised [116]. The simplicity and precision of this technology have led to recent research findings presenting the risk of carcinogenesis because a portion of the genes is lost within cells [117]. In addition, a case in China where the technology was directly applied to humans faced ethical issues, and the research results were not recognized [118]. Consequently, CRISPR/Cas9, although a superior genetic engineering technology, has limited application because of ethical and legal concerns.

Various methods are available for discovering and developing biological enzymes to produce structured lipids, each with advantages and disadvantages. Traditional research methods, such as metagenomic screening and ancestral sequence reconstruction, may be more accurate but require significant time and costs. On the contrary, new paradigms such as machine learning, artificial intelligence, and CRISPR/Cas9 may present some potential for errors. Each technique can only be considered the best approach. Therefore, to derive and validate lipases that can synthesize structured lipids, combining various methods is necessary to complement the advantages and disadvantages of each approach in terms of research accuracy and efficiency.

5. Conclusions

Structured lipids produced using biological enzymes, particularly lipases and esterases, have become essential elements of food, pharmaceuticals, and cosmetics because of their numerous benefits over traditional chemical catalysts. The regiospecificity and substrate specificity of these enzymes, including the ability to selectively modify the composition of fatty acids and distribution of lipids, make them useful for the synthesis of structured lipids. In particular, the utilization of immobilized lipases as catalysts offers many advantages, such as reusability, high selectivity, and low environmental effects, making them more attractive in commercial applications. However, the current range of sn-1,3 regiospecific lipases and available natural oils and fats for use in the synthesis of structured lipids is limited.

Therefore, further research and development of new lipases that can synthesize structured lipids with diverse functionalities and improvement of existing enzyme properties through genetic engineering and innovative screening methods are essential. Furthermore, the use of machine learning and artificial intelligence tools and the development of in silico prediction models can greatly aid in the efficient and accurate discovery of new lipases. With continued collaboration between academia and industry, the development and commercialization of structured lipids using biological enzymes will undoubtedly expand, offering wide-ranging benefits for various industries. The utilization of new technologies such as data science, machine learning, artificial intelligence, and CRISPR/Cas9 in discovering and enhancing lipases and esterases can promote the development of more efficient and cost-effective production methods for structured lipids. However, these novel technologies should be used responsibly, considering their potential ethical, legal, and safety issues.

In summary, structured lipids and their production using biological enzymes present numerous opportunities for improving human health and the environment. Future research should focus on discovering and enhancing novel enzymes, improving structured lipid production processes, and exploring new applications of structured lipids. The utilization of innovative technologies will accelerate the development of more effective and sustainable solutions for the synthesis of structured lipids.

Acknowledgments

The authors would like to acknowledge the financial support of the Fisheries Science Institute of Kunsan National University.

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