What are the applications of immobilized lipases in food?

Enzymes are a class of biocatalysts characterized by high efficiency and specificity. Enzymes can be biotransformed in and out of organisms and have excellent stereoselectivity, regioselectivity and chemoselectivity. Lipase as a green biocatalyst is widely used in chemical, food, pharmaceutical, energy, environment and other fields.
This paper focuses on the application of immobilized lipase in food industry.
Synthesis of aromatic compounds

Short chain esters with fruity flavors are popular as aroma agents in the food industry and these aroma compounds can be chemically synthesized or obtained from natural sources.
Flavor compounds are usually short chain fatty acids and alcohols such as methyl butyrate, butyl butyrate, isoamyl isobutyrate for pineapple or apple-like flavors, ethyl butyrate for pineapple or strawberry-like flavors, and isoamyl acetate/isoamyl butyrate for banana-like flavors.
Immobilized lipases catalyze the synthesis of natural flavors under mild conditions and they are safer and more reliable than chemical synthesis, thus showing a wide range of applications in the synthesis of aromatic compounds.
Garlapati et al. investigated the synthesis of methyl butyrate and octyl acetate by immobilized Aspergillus oryzae NRRL3562 lipase by catalytic esterification under solvent-free conditions. The effect of different esterification reaction parameters such as alcohol molar ratio, reaction time and temperature on molar conversion (%) was investigated. The results showed that the immobilized enzyme maintained more than 95% relative activity for methyl butyrate and octyl acetate up to 5 and 6 times, respectively, with high lipase activity.
Ghamgui et al. catalyzed the synthesis of isoamyl acetate (banana flavor) from the esterification reaction of acetic acid and isoamyl alcohol under pure substrate conditions using non-commercial Staphylococcus similars immobilized lipase, and examined the effect of reaction parameters such as lipase dosage and the molar ratio of acetic acid to isoamyl alcohol on the reaction. The results showed that the conversion of acetic acid and isoamyl alcohol could reach 64% in 8h. The immobilized enzyme preparation did not show any significant decrease in the immobilized enzyme activity after 4 cycles of use, and the immobilized enzyme stability and activity were high.
Matte et al. immobilized Thermomyces lanuginosus lipase (TLL) on natural and modified Immobead 150 to synthesize butyl butyrate and isoamyl butyrate by multipoint covalent linkage using ethylenediamine. The results showed that the multipoint covalently immobilized lipase on natural Immobead 150 (EMULTI) had a half-life of 5.32 h at 70 °C, which was about 30-fold more stable than its TLL solution, and exhibited high stability in acetone, n-hexane and iso-octane. The esterification reaction could reach more than 60% esterification rate within 24 h. Among all the immobilization methods, EMULI showed the best thermal stability, solvent stability and ionic liquid stability.
Oil and fat modification processing
Immobilized lipase has a broad application prospect in the oil and fat industry. It is mainly used in fat processing, and fat modification is a very critical part in food processing.
Immobilized lipase is superior to free enzyme because immobilization can improve the stability and activity of the enzyme. In the immobilized form, the enzyme can be reused. Most of the immobilization methods use non-covalent interactions.
Natural fats and oils are less stable due to the drawbacks of long branched chains and different saturations of fatty acids, and lipases can be used as biocatalysts to modify fats and oils by utilizing their position specificity and fatty acid specificity.
Lipase-modified fats and oils have higher nutritional value, stability, and quality, and have greater market potential in food processing.
Paula et al. immobilized commercial non-regioselective Pseudohyphomyces lipase (Novozym 435) and 1,3-regioselective Mycobacterium mycenae lipase in an organo-inorganic polysiloxane-poly(vinyl alcohol) heterogeneous matrix, which acted as biocatalysts in the reactor, to modulate the physical properties of milk fats by enzymatic transesterification reactions, obtaining a healthy transesterified fat mixture suitable for industrial production.
Tecelão et al. synthesized human milk fats (HMFs) by combining tripalmitin with oleic acid or omega-3 polyunsaturated fatty acids under conditions of enzyme-catalyzed acidolysis in a solvent-free medium at 60°C. Four immobilized lipases, Lipozyme RM IM, Theromyces Lanuginosa lipase, Lipozyme TLIM, and Novozym 435 were tested and the results showed that the activity and operational stability of the biocatalysts depended on the acyl donor used.
Enhancing the fat solubility of food additives
Isoascorbic acid is widely used as an antioxidant in the food industry, but it is difficult to be applied in lipid-based foods due to its high hydrophilicity.
The conversion of ascorbic acid to ascorbate esters catalyzed by immobilized lipase can effectively improve the lipophilicity of the product, which can be better used in fatty foods.
Santibáñez et al. used different carriers to immobilize Pseudomonas aeruginosa lipase TL for the enzymatic esterification of ascorbyl palmitate synthesis from palmitic acid and ascorbic acid in an organic medium, and compared its performance with that of commercial Novozym 435 lipase. The results showed that the conversion rate of Pseudomonas lipase TL reached 57% at 55 ℃, which was higher than the substrate conversion rate of commercial Novozym 435 lipase at 70 ℃.
Sun et al. converted isoascorbic acid to D-isoascorbyl palmitate by immobilized lipase, which improved the oil solubility of isoascorbic acid in organic medium and had a high conversion rate, with a yield of 95.32%.
Tang Luhong et al. will heptane and tertiary amyl alcohol and several kinds of reaction media and several kinds of lipase on the synthesis of L-ascorbyl palmitate reaction, the results show that tert-butanol is suitable for ester synthesis reaction, and Novozym 435 lipase has good catalytic activity.
Synthesis of Sugar Esters for Food Emulsifiers
Sugar esters are nonionic surfactants with a hydrophilic group of a sugar group and a hydrophobic group of a fatty acid, as well as amphiphilic properties. They can be synthesized in a single enzymatic reaction step using lipases based on the use of renewable, cheap and readily available raw materials wherever possible.Sugar esters food emulsifiers are widely used in the food industry due to their biodegradability and non-toxicity and non-hazardous nature to the environment.
Zaidan et al. catalyzed the synthesis of lactose esters by cross-linking lipase to a nanoreactor (i.e., NER-CRL) via covalent bonding and physical adsorption, and immobilizing crumpled pseudoligactomyces lactis (CRL) lipase on amino-activated mica. The results showed that NER-CRL and Amino-CRL had high operational stability with half-lives of more than 13 and 10 times, respectively, and 2.4 and 2.6-fold increase in specific activity over the free enzyme, respectively.
Adnani et al. mimicked the fat-catalyzed esterification reaction of xylitol and stearic acid, and catalyzed the synthesis of xylitol fatty acid esters by Novozym 435 (macroporous resin-immobilized Antarctic Pseudomalleiomyces yeast lipase) in n-hexane. The results showed that the actual yield of fatty acid esters was 96.10%.
Kapoor et al. catalyzed the esterification of glycerol with palmitic acid under low water conditions using cross-linked enzyme aggregates (CLEAS) of Antarctic pseudomalleye lipase B (CALB). The results showed that the reaction could reach 90.3% conversion for 24 h. The yields of mono- and diglycerides were 87% and 3.3%, respectively.
Synthetic cocoa butter
Cocoa butter has a melting point of 37℃, has the melting property of melt in the mouth, contains palmitic acid and stearic acid, and is an important raw material for processing chocolate in the food industry.
However, the production of natural cocoa butter is low and relatively expensive, so immobilized lipases have been used to catalyze the transesterification of fats and oils to produce cocoa butter substitutes, which are more widely used in the food industry.Dutt et al. used the Bacillus RK-3 strain isolated from soil to produce 1,3-region-specific lipase for transesterification reactions using palm oil and methyl stearate as the raw materials. The results showed that the final product was similar to CB and had a conversion of 83.17% within 24 h. The results of the reaction showed that the final product was similar to CB.
Gong Xin et al. investigated the catalytic preparation of low-calorie Sapium sebiferum cocoa butter using immobilized lipase Lipozyme TLIM and found that the highest exchange rate of 34.9% was achieved at a temperature of 65 ℃, an Aw of 0.06, and a catalytic time of 15.5 h, and the product had an SI value of 0.55 and a melting point of 37 ℃, which indicated that low-calorie cocoa butter could be prepared from Sapium sebiferum lipids.
Hu Fang et al. used lipase Lipozyme TLIM to catalyze the transesterification reaction to synthesize cocoa butter, and the result was that the yield of cocoa butter was as high as 85.586%, and the advanced structural analysis showed that the composition and structure of triglycerides in the product were similar to that of natural cocoa butter.