Lipases ( EC3111113, glycerol ester hydrolases ) are a special class of ester-bond hydrolases that are classified according to substrate specificity as non-specific, fatty acid-specific and specific lipases. Lipases are found in animals, plants and microorganisms. In the organic phase, lipase can catalyze ester synthesis, ester exchange reaction, ester polymerization reaction, peptide synthesis and amide synthesis, etc. Therefore, lipase has been widely used in the food industry in recent years.

Properties of lipase

Since the discovery of lipase in 1834, there have been more than one hundred years of research history, lipase is an important class of metabolic enzymes in organisms, its natural substrate for long-chain fatty acid esters (such as various fats and oils, etc.), can play a role in the heterogeneous system (oil-water interface) or the organic phase, and has a certain position specificity.

Although lipase comes from different sources, and the amino acid composition of lipase from different sources is different, its molecular weight is between 20,000-60,000, and its active center has the same or similar structural composition, and its active center, except for a few exceptions, is generally a triplex composed of serine, aspartic acid, histidine, and the central part of the spatial structure of the enzyme molecule is a hydrophobic β-folding surrounded by an amphiphilic α-helix. The spatial structure of the enzyme molecule has a hydrophobic β-fold in the center, surrounded by an amphiphilic α-helix, and the three amino acids are located in the “ring” on the side of the central β-fold in a highly conserved geometric orientation. Most lipases also have a mobile structure, the “lid”.

This “lid” covers the active catalytic site in the “ring” when the lipase is not activated. When the lipase molecule is activated, the “lid” opens, allowing the substrate for the enzyme action to be The activation of the lipase molecule opens the lid, allowing the substrate of the enzyme to bind to the “substrate binding site” of the enzyme molecule.
Lipase can only work in heterogeneous systems, i.e., at the interface of oil and water, and does not work on uniformly dispersed or water-soluble substrates, and even if it does work, it does so very slowly, and lipase works on the hydrophilic and hydrophobic interfaces of the system.

Application of lipase in food industry

1. Application in oil processing

Lipase can catalyze the generation of fatty acids and glycerol from fats and oil hydrolysis reaction, which is widely used in fatty acid and soap industry.

Because of the general hydrolysis reaction, solid fats and oils are extremely difficult to disperse in the reaction system, and the reaction speed is slow. Tetsuo Kobayashi et al. carried out lipase hydrolysis in a water-organic solvent two-phase reaction system, and dissolved tallow in a suitable organic solvent, so that the organic solvent containing the substrate was fully dispersed in the aqueous phase to increase the reaction speed, and the fatty acids and glycerol of the reaction products were allocated to the organic phase and the aqueous phase for separation and recovery, respectively, and the decomposition of tallow could reach 100% after 48 h. The lipase can hydrolyze an ester with another ester, and it is widely used in the fatty acid and soap industries.

Lipase can mix one kind of ester with another kind of fatty acid or alcohol or ester and generate a new ester with acyl exchange, transesterification reaction occurs. Through the transesterification reaction, the properties of fats and oils can be changed.

ChangM K et al. catalyzed the transesterification reaction of hydrogenated cottonseed oil and a certain proportion of rapeseed oil by immobilized lipase with n-hexane as the solvent, and the melting point of the product was 36℃ higher than that of the natural cocoa butter, which can be used as a substitute for cocoa butter; Lin Zhiyong carried out a study on the production of cocoa butter from sebiferum oil, and obtained better conditions for the production of cocoa butter analogue through the conditions of transesterification reaction.

Under certain conditions, lipase can catalyze the esterification reaction between fatty acid and glycerol, so as to convert a large number of free fatty acids in the oil into neutral glycerol esters, which not only reduces the acid value, but also increases the amount of neutral glycerol esters, and realizes the biorefinery and deacidification of fats and oils. In addition, lipase can also be used in the strengthening of polyunsaturated fatty acids, synthesizing phospholipids and so on.

2. Application in dairy industry

Lipase in dairy production will have a double effect, on the one hand, due to the lipase on the decomposition of milk fat, will cause fresh milk in the storage process to produce a bitter taste, caused by the milk powder in the preservation process of quality deterioration, will make the cheese products produce unpleasant flavor.

In sour milk products, the free fatty acids produced by enzymatic digestion also inhibit the production of some fermentation agents. On the other hand, lactide hydrolysis in dairy products through the application of lipase can further enhance the flavor of cheese, milk powder and cream, promote the ripening of cheese and improve the quality of dairy products.

For example, through specific lipolysis, cream can have a very strong flavor. In the cream added a certain amount of lipase soda solution, and then homogenization, insulation enzyme, and then heat the method of enzyme inactivation, remove the lower layer of enzyme solution, filtration, can be obtained to enhance the aroma of cream products, its aroma and flavor greatly improved.

3. Application in food additive industry

L-ascorbyl palmitate is widely used as ester-soluble antioxidant and nutritional fortifier. Ascorbyl palmitate is esterified from L-ascorbic acid, compared with L-ascorbic acid, firstly, its antioxidant property has been significantly improved; secondly, due to the implantation of palmitic acid group, it has both hydrophilic ascorbic acid and lipophilic palmitic acid group, which makes it a kind of excellent surfactant; moreover, it also has strong anticancer and antitumor effects.

Luhong Tang et al. conducted a systematic study on the effects of several reaction media such as water, heptane and tert-amyl alcohol, and several lipases such as NOVO435 (Candida antartica), MML (Mucormiehei), LIPOLASE, PPL (Porcine pancreas), etc. on the synthesizing reaction of L-ascorbyl palmitate. The results showed that the reaction media and lipase species had great influence on the reaction. Among the several reaction media studied, tert-amyl alcohol was the only one suitable for the reaction, and among the lipases studied, NO2VO435 showed good catalytic activity.

Sucrose laurate has the functions of emulsification and antibacterial. It has attracted more and more attention. More and more researchers focus on the application of enzymes as catalysts to synthesize sucrose lauric acid selectively. For example, Pedersen et al. catalyzed the synthesis of sucrose 2-laurate monoester by metalloprotease thermolysin, Bacillus p seudofirmus AL-89.

Usually, aromatic and flavor components are chemically synthesized or extracted from natural sources, the amount of aromatic substances extracted from plants is limited to meet the needs of the people, therefore, the current shift to the production of biotechnological methods, the current domestic and foreign microbial enzymatic synthesis of aromatic compounds.

For example, Shieh CJ studied the optimized conditions for the transesterification of hexanol and triacylglycerol esters in n-hexadecane catalyzed by Trichoderma immobilized lipase through response surface methodology.Gandolfi R reported the synthesis of different aromatic esters (hexyl acetate, hexyl butyrate, geranyl acetate, and geranyl butyrate) catalyzed by the selective use of Rhizopus oryzae dry mycelium in the organic phase.

4.Application in food waste treatment

The fat-containing wastes and waste restaurant oils produced in the process of oil and fat processing are mainly composed of fatty acid triglycerides. Not only the free fatty acid content is high, but also contains aldehydes, ketones and polymers and other oxidized products. Wang Yong et al. studied the preparation of biodiesel from waste restaurant oil by enzyme-catalyzed transesterification in a three-step batch process with a reaction time of 48 h and a total conversion rate of 90.4%.

Comparing with the process using refined vegetable oil as feedstock, the conversion rate of transesterification was 97.3% under the same reaction conditions.

Wa tanabe Y et al. investigated the continuous enzyme-catalyzed transesterification of waste restaurant oil with a conversion of 90%, compared with that of refined vegetable oil, which was 93% under the same reaction conditions. yuji shimada et al. developed a reaction system for the stepwise alcoholysis of waste restaurant oil using immobilized lipase, and the conversion of transesterification was more than 90%. The conversion rate of esterification was more than 90%.

Prospects

There have been many reports on lipase synthesis technology in food and related fields, but most of them are still in the stage of applied basic research, and there are not many attempts to industrialize lipase-catalyzed synthesis. With the development of genetic engineering and protein engineering, the application of lipase in food will be more developed, which is of great significance to promote the rapid development of some fields of food industry.