Flavor Esters Research Articles

Immobilization and characterization of hormone-sensitive lipase (HSL) from Glaciozyma antarctica

HSL-like esterase (GlaEst12) was isolated from the psychrophilic yeast Glaciozyma antarctica, and has a broad substrate specificity hence allowing hydrolysis of a wide range of ester molecules. This characteristic makes it relevant in a variety of industries, including food processing, pharmaceutical, and bioenergy. Immobilization of HSL-like esterase from G. antarctica (GlaEst12) was achieved through physical adsorption onto different types of supports such as Amicogen LKZ118, Amicogen LKZ518, Accurel MP1000, Seplite LXC621, and Seplite LX120. Among these, LKZ518 and LX120 exhibited a high immobilization rate (95 - 100 %), surpassing the other tested supports. Nonetheless, their recorded enzyme activity was notably lower when contrasted with the activity observed on LKZ118. While LKZ118 displayed a 50 % immobilization rate, it demonstrated remarkably high enzyme activity compared to the other supports. The optimal enzyme concentration for immobilization was determined to be 5 mg per gram of LKZ118 support. The immobilized lipase (GlaEst12_LKZ118) showed an optimum temperature et 60 °C similar to free GlaEst12 when tested using pNP-octanoate substrate. However, it showed a shift in pH optimum from pH 8 for free GlaEst12 to pH 9 for GlaEst12_LKZ118. GlaEst12_LKZ118 demonstrated prolonged stability at 60 °C, with a half-life of 180 min. Furthermore, it exhibited stability in the presence of methanol when treated at 50 °C, and increased activity in the presence of DMSO. Operational stability tests revealed that GlaEst12_LKZ118 maintained 65.5 % of its relative activity after 7 cycles, indicating its robust operational stability. In summary, the study demonstrated the successful immobilization of HSL lipase derived from G. antarctica using various supports and the immobilized enzyme showed promising activity levels for potential applications in biodiesel production and flavour ester synthesis.

Biocatalyst immobilization on magnetic nano-architectures for potential applications in condensation reactions.

In this review article, a perspective on the immobilization of various hydrolytic enzymes onto magnetic nanoparticles for synthetic organic chemistry applications is presented. After a first part giving short overview on nanomagnetism and highlighting advantages and disadvantages of immobilizing enzymes on magnetic nanoparticles (MNPs), the most important hydrolytic enzymes and their applications were summarized. A section reviewing the immobilization techniques with a particular focus on supporting enzymes on MNPs introduces the reader to the final chapter describing synthetic organic chemistry applications of small molecules (flavour esters) and polymers (polyesters and polyamides). Finally, the conclusion and perspective section gives the author's personal view on further research discussing the new idea of a synergistic rational design of the magnetic and biocatalytic component to produce novel magnetic nano-architectures.

Immobilized lipase based on SBA-15 adsorption and gel embedding for catalytic synthesis of isoamyl acetate

In this study, Candida rugosa lipase (CRL) was immobilized on the silica carrier SBA-15 by physical adsorption, then mixed with hydrophobically modified sodium alginate and dropped into calcium chloride solution. Finally, the adsorption-embedding immobilized lipase (L-SBA-15-Mgel) was successfully prepared. The conformation of the carrier before and after lipase immobilization were analyzed by scanning electron microscopy (SEM), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR) and other characterization techniques. The molecular docking and molecular dynamics simulation of CRL and modifier dodecyl glycidyl ether (DGE) ligand were carried out to analyze the potential influence of modifier on enzyme characteristics. The basic enzymatic properties including the activity, stability and reaction kinetic parameters of the immobilized lipase L-SBA-15-Mgel were studied. L-SBA-15-Mgel showed good storage stability and reusability. After 16 days of storage, the activity retention was 47.85%, and there was still 63.20% of the residual activity after seven cycles of reuse. The immobilized lipase was applied to the efficient synthesis of food flavor ester isoamyl acetate. Under the reaction conditions of 50 °C and 200 rpm, the highest yield of isoamyl acetate catalyzed by L-SBA-15-Mgel was 85.19%.

Insights into the enzymatic synthesis of alcoholic flavor esters with molecular docking analysis

The enzymatic synthesis is essential for the flavor esters in the food and fragrance industries. This paper introduces a novel preparation method for lipase microarrays (CALB@PMHOS-TEOS) with loadings up to 229 ± 1.4 mg/g. Using surfactant-free hydrophobic silica-hybridized mesoporous materials and Candida antarctica lipase, this resulted in the effective synthesis of flavor esters. Using CALB@PMHOS-TEOS a Pickering emulsion system was formed at the oil-water interface for the sustainable synthesis of flavor esters. This resulted in a 93.5 ± 0.5 % conversion of hexanoic acid within 2 h at an optimal temperature of 35 °C, which is the highest level recorded in the literature to date. Furthermore, the conversion of hexanoic acid was maintained at 63.9 ± 1.2 % after 9 cycles of CALB@PMHOS-TEOS reuse. The application of the enzyme to the synthesis in a variety of flavor esters achieved a new benchmark in the existing literature. A molecular docking model was evaluated to understand the molecular mechanism underpinning the immobilized lipase. This work introduces a novel method for the eco-friendly and efficient synthesis of flavor esters for applications across various fields including food and cosmetics.

Green Enzymatic Synthesis of Geranyl Butyrate: Process Optimization and Mechanistic Insights.

Flavor esters are organic compounds widely used in the food industry to enhance the aroma and taste of products. However, most chemical processes for the production of these flavoring compounds use toxic organic solvents. Some organic solvents derived from petroleum can leave behind residual traces in food products, which may raise concerns about potential health risks and contamination. In this study, we employ Eversa Transform 2.0, a commercial lipase derived from the lipase from Thermomyces lanuginosus, to produce geranyl butyrate in aqueous media. The chemical process was optimized using the Taguchi method, and a conversion of 93% was obtained at the optimal reaction conditions of: 1:5 molar ratio (v/v), 15% biocatalyst load (w/w), at 50 °C, in 6 h. Classic (molecular dynamics) and quantum (density functional theory) simulations unveiled amino acid residues involved in the stabilization of the enzyme-substrate complex. Detailed QM/MM mechanistic studies identified the nucleophilic attack of the deacylation reaction as the rate-limiting step of the entire mechanism, which has a free energy barrier of 14.0 kcal/mol.

Multi-walled carbon nanotubes as lipase carriers for organic synthesis: Current trends and recent update

Lipase-catalyzed organic reactions have been widely practiced in the past three decades. Especially interesting are insoluble/immobilized forms due to providing a possibility of facile use and recyclability, thus reducing process costs, and making the procedure more environmentally friendly. Carbon-based supports have been extensively exploited for this purpose, because of neutral and biodegradable nature and thermal and chemical stability. Their high specific surface area, characteristic surface morphology and lower mass transfer resistances play a vital role in the performance of the attached enzyme. This review paper presents an overview of the main aspects of lipase immobilized on multi-walled carbon nanotubes (MWCNTs). Moreover, different immobilization strategies to achieve a biocatalyst with improved performances are discussed. Furthermore, as lipases are considered to have high commercial worth for synthesis of valuable organic molecules, the second part of the paper is dedicated to the overview of the most important industrial sectors in which these nanobiocatalysts have been used. In specific, applications in biodiesel production, flavour ester synthesis and racemization are summarize

Controlled synthesis of distiller's grains biochar for turbidity removal in Baijiu

Resource utilization of distiller's grains (DGs) is crucial for realizing sustainable development of Baijiu industry. In the prevent investigation, a low-cost activated biochar (DGABC) suitable for removing turbidity from low-alcohol Baijiu was prepared by the controlled pyrolysis of DGs, followed by steam activation. The as-prepared biochar featured a large specific surface area (320–480 m2/g) and pore volume (0.45–0.47 cm3/g). Importantly, the DGABC possessed remarkable exterior hydrophily and interior lipophilicity, which guaranteed its good dispersion in alcohol-water system as well as an efficient adsorption to the components with long lipophilic chain. As a result, the DGABC could efficiently remove the turbidity in low-alcohol Baijiu, which was mainly derived from the long lipophilic chain components, such as ethyl palmitate. Meanwhile, most of the flavor esters that had a shorter lipophilic chain and lower hydrophobicity were well kept in the low-alcohol Baijiu. Therefore, this work provided a promising strategy for DGs recycling in Baijiu industry.

Covalent immobilization of lipase on an ionic liquid-functionalized magnetic Cu-based metal-organic framework with boosted catalytic performance in flavor ester synthesis.

Enzymatic esterification plays an important role in the fields of chemistry and biotechnology. In this study, lipase was immobilized on an ionic liquid (IL)-modified magnetic metal-organic framework (MOF) and used to synthesize isoamyl acetate. The immobilized lipase (PPL-ILs/Fe3O4@MOF) showed 2.1-fold and 1.8-fold higher activity compared to the free and immobilized lipase without ILs (PPL-Fe3O4@MOF), respectively. In addition, the anti-denaturant ability and reusability of the PPL-ILs/Fe3O4@MOF were also higher than those of other samples. The ester yield reached 75.1% when the biocatalyst was used to synthesize isoamyl acetate in hexane. The synthesized supports supplied a good microenvironment for the immobilized lipase through multiple interactions. Results of the structural analysis showed that the conformation state of lipase molecules changed after immobilization. The magnetism of the prepared biocatalyst makes it easy to recycle so that PPL-ILs/Fe3O4@MOF maintained 70.2% of the initial activity after eight cycles. The prepared composite materials exhibited good potential in lipase immobilization with enhanced catalytic ability and stability.

Efficient and sustainable preparation of cinnamic acid flavor esters by immobilized lipase microarray

Green and effective synthesis of flavor esters is a long-term interest in food, cosmetic and pharmaceutical industries. Here, an immobilized lipase microarray was prepared by immobilizing lipase CSL on hydrophobically modified ordered mesoporous silicon (CSL@OMS-C8) for the enzymatic production of cinnamic esters with different flavors. The results showed that the CSL@OMS-C8, with an immobilization capacity of 249.3 ± 11.1 mg/g, exhibited 1.6 times higher catalytic activity than that of free lipase at the optimum temperature of 70 °C. The maximum yield of benzyl cinnamate reached 97.3% in 2 h with the corresponding CE value of 650.5 μmol/g · h, leading to the highest record compared to the current literature. Besides, the CSL@OMS-C8 could maintain 81.4 ± 1.1% conversion for cinnamic acid after five-cycle reuse, thus revealing the high stability and reusability of immobilized lipase. Finally, the broad applicability of the immobilized lipase microarray was confirmed by its capacity to successfully catalyze the synthesis of methyl cinnamate, ethyl cinnamate, butyl cinnamate, geranyl cinnamate and isoamyl cinnamate. This study reveals new insights on the efficient and sustainable enzymatic approach to produce natural cinnamic acid flavor esters and, consequently, could pave the way for the natural and clean production in food industries.

Improved Catalytic Performance of Lipase Eversa® Transform 2.0 via Immobilization for the Sustainable Production of Flavor Esters—Adsorption Process and Environmental Assessment Studies

The aim of this study was to produce several flavor esters via esterification of octanoic acid with different commercial short-chain alcohols (methanol, propanol, isoamyl alcohol, hexanol and benzyl alcohol) and fusel oil in solvent-free systems. Lipase Eversa® Transform 2.0 immobilized via mechanism of interfacial activation on poly(styrenene-divinylbenzene) (PSty-DVB) beads was used as heterogeneous biocatalyst and its catalytic performance was compared with that of the soluble lipase. The heterogeneous biocatalyst was prepared by employing 5 mmol·L−1 buffer sodium acetate at pH 5.0 and 25 °C using an initial protein loading of 40 mg·g−1. The maximum amount of immobilized protein reached was 31 mg·g−1, corresponding to an immobilization yield of 80%. Mass transfer studies demonstrated that the lipase was preferentially adsorbed inside the pores of the support, which was confirmed by scanning electron microscopy analysis. Lipase immobilization can be described by a pseudo-first-order kinetic model via a physisorption process. When used as biocatalysts of the target reactions, the highest conversion percentage (between 65% and 85% of acid conversion after 60–90 min of reaction) values were achieved for esterification reactions catalyzed by immobilized lipase. Reusability tests revealed high retention of the original activity of the immobilized lipase after six successive batch reactions using isoamyl alcohol (47%) and fusel oil (72%). The proposed reaction systems can be considered green processes (EcoScale score above 80), with exception of methanol medium, classified as an acceptable green process (EcoScale score of 68). These results show that the heterogeneous biocatalyst prepared can be an economic and sustainable option for flavor esters production on an industrial scale.

Amino functionalization of magnetic multiwalled carbon nanotubes with flexible hydrophobic spacer for immobilization of Candida rugosa lipase and application in biocatalytic production of fruit flavour esters ethyl butyrate and butyl butyrate

Amino functionalization of magnetic multiwalled carbon nanotubes with flexible hydrophobic spacer for immobilization of Candida rugosa lipase and application in biocatalytic production of fruit flavour esters ethyl butyrate and butyl butyrate

"Synthesis of Pine-Apple Flavor by Celite-Immobilized Lipase "

Alkaline-thermotolerant lipase from Bacillus licheniformis MTCC-10498 was purified and immobilized on Celite-545 matrix. The matrix shows a binding efficiency of 80% for lipase of B. licheniformis with a specific activity 1.02 U/mg of protein. The immobilized lipase shows 50% activity up to 70°C. This immobilized lipase was checked for its efficacy in the synthesis of butyl butyrate, a flavor ester. The formation of ester was highest when butyric acid and butyl alcohol were as in an equimolar ratio (100 mM each) in n-hexane under continuous shaking (120 rpm) along with 20 mg celite-bound lipase at 45°C in 5 h. The addition of molecular sieves (50 mg) to the reaction cocktail promoted the ester yield. The immobilized lipase was reused up to 8 th cycle. The immobilized lipase when used for the esterification of butyl alcohol and butyric acid under above-optimized conditions resulted in butyl butyrate (91.6%) conversion in n-hexane.

Development of a Tailored Sol-Gel Immobilized Biocatalyst for Sustainable Synthesis of the Food Aroma Ester n-Amyl Caproate in Continuous Solventless System.

This study reports the synthesis of a hybrid sol-gel material, based on organically modified silanes (ORMOSILs) with epoxy functional groups, and its application in the stabilization of lipase type B from Candida antarctica (CalB) through sol-gel entrapment. The key immobilization parameters in the sol-gel entrapment of lipase using epoxysilanes were optimized by the design of numerous experiments, demonstrating that glycidoxypropyl-trimethoxysilane can allow the formation of a matrix with excellent properties in view of the biocatalytic esterifications catalyzed by this lipase, at an enzyme loading of 25 g/mol of silane. The characterization of the immobilized biocatalyst and the correlation of its catalytic efficiency with the morphological and physicochemical properties of the sol-gel matrix was accomplished through scanning electron microscopy (SEM), fluorescence microscopy (FM), as well as thermogravimetric and differential thermal analysis (TGA/DTA). The operational and thermal stability of lipase were increased as a result of immobilization, with the entrapped lipase retaining 99% activity after 10 successive reaction cycles in the batch solventless synthesis of n-amyl caproate. A possible correlation of optimal productivity and yield was attempted for this immobilized lipase via the continuous flow synthesis of n-amyl caproate in a solventless system. The robustness and excellent biocatalytic efficiency of the optimized biocatalyst provide a promising solution for the synthesis of food-grade flavor esters, even at larger scales.

Bioremediation of degraded pit mud by indigenous microbes for Baijiu production

Microbes in pit mud play key roles in fermentation cellars for Chinese strong-flavor Baijiu (SFB) production. Pit mud, however, is frequently degraded during production, compromising the quality of the end product. In this study, a bioremediation method was used to restore degraded pit mud (DPM) using indigenous microbes derived from SFB production. Metabolomics and metagenomics were used to determine the dynamics of prokaryotes during DPM restoration and their link to SFB production. The composition of flavor compounds in SFB changed (P = 0.0001) before and after restoration of DPM. Consistent with the improved sensory quality, the ethyl caproate/ethyl lactate ratio, an SFB quality measure, increased after restoration (P < 0.001). The concentrations of humus, NH4+, available phosphorus, and available potassium in DPM increased during the restoration process (P < 0.05), which is consistent with high-quality pit mud. The relative abundance of microbes that are beneficial to SFB fermentation, such as Caproiciproducens, a bacterium that produces caproic acid, increased during the restoration process. Furthermore, a total of 18 metabolic pathways were enriched (P < 0.05) from DPM before and after restoration. This includes butanoate metabolism and pyruvate metabolism, which are related to the synthesis of key flavor esters in SFB.

Optimization and Purification of Terpenyl Flavor Esters Catalyzed by Black Cumin (Nigella sativa) Seedling Lipase in Organic Media

Geranyl butyrate and citronellyl butyrate esters are industrially important fruity flavors that are being used in food and as a fragrance in cosmetics. Previously terpenyl fruity flavors have been successfully synthesized in organic solvents using crude seedlings enzymes. The purpose of the current study was to standardize reaction parameters for the optimal synthesis of geranyl butyrate using the best chosen black cumin seedling lipase in an organic medium through direct esterification reactions. Geranyl butyrate and citronellyl butyrate esters were identified, quantified through gas chromatography, confirmed through GC-MS, and partiallypurified through the distillation process. Effect of organic solvents (acetonitrile, n-hexane, pentane, heptane, and toluene), alcohol and acid concentrations (0.125–0.3 M), temperature (20–50°C), incubation time (1–72 h), and enzyme concentrations (0.05–0.3 g) were studied on the synthesis of geranyl butyrate using black cumin seedling lipase. The highest conversion yields of ester (96%) were obtained when 0.25 M of geraniol and butyric acid were reacted at 37°C for 48 h in the presence of 0.25 g of crude seedling lipase enzyme in n-hexane. It was concluded that the germinated black cumin seedling lipase proved to be the best among the selected biocatalysts for the synthesis of geranyl butyrate in n-hexane.

Chemical and Flavor Characteristics of Enzyme-Modified Cheese Made by Two-Stage Processing.

(1) Background: to date, a clear description of the impact of specific enzymes on the enzyme-modified cheese (EMC) flavor is lacking. Moreover, comparative studies on the aroma compounds’ intensity of EMC have been rarely investigated. Therefore, this study was done to determine the influence of incubating substrates with proteases and different lipases on cheese ripening index and aroma compounds. (2) Methods: two-stage processing was adopted; proteolysis followed by lipolysis. (3) Results: results showed that the usage of Flavourzyme may improve the value of pH 4.6-WSN/TN%. Butanoic acid and hexanoic acid have a significant influence on the overall flavor of EMCs. In particular, the ethenyl acetate compound was detected in all products and was perceived as a fruity and sweet aroma, which has not been reported in previous literature. The concentration of short-chain fatty acids of EMCs made by Lipase MER was higher than EMCs made by Palatase, while the total content of medium and long-chain fatty acids of EMCs made by Lipase MER was lower than EMCs made by Palatase. The percentage of esters compounds in EMCs made by Lipase AY 30G was higher than the other two lipases, except EMC1. (4) Conclusions: Flavourzyme may be used to speed up the ripening of cheeses that need extensive proteolysis. The ability of Lipase MER to hydrolyze short-chain fatty acids was stronger than that of Palatase, while the ability of Lipase MER to hydrolyze medium and long-chain fatty acids was weaker than that of Palatase. The use of Lipase AY 30G was accompanied by the production of some other flavor esters, which made the final hydrolysates more fragrant and may be a good choice to produce fruity cheese flavor EMC. While Lipase MER may barely contain ester activity. This study may provide a reference for the selection of incubated enzymes for specific flavor EMC.

Process Intensification of Enzymatic Synthesis of Flavor Esters: A Review.

The conventional flavor synthesis method suffers from low yields, time inefficiency, and extreme reaction conditions. Therefore, there is a necessity for the green and novel synthesis approach to overcome these limitations. The current review presents a holistic insight into different aspects associated with the synthesis of flavor esters using the immobilized enzyme. The application of process intensification tools such as ultrasound and microwave irradiation can enhance the reaction efficiency because of higher product recovery, less formation of by-products, and decreased energy consumption. This review presents the process intensification of value-added flavor esters synthesis and the mechanism of ultrasound and microwave action on the enzyme to enhance the enzyme activity and increase the reaction rate. It also summarizes the role of process intensification in enzymatic flavor ester synthesis, followed by specific examples as reported in the literature.

Correlation between microbial communities and flavor compounds during the fifth and sixth rounds of sauce-flavor baijiu fermentation

Sauce-flavor baijiu is a representative of Chinese traditional fermented baijiu using grains as the raw materials through the co-fermentation of microorganisms. The whole manufacturing process includes 7 times of distillation and generates 7 kinds of base baijius. The final product is a mixture of the 7 kinds of base baijius. Thus the base baijius greatly affect the quality of the final product. The quality of the base baijiu obtained by the sixth distillation is obviously poorer than that of the fifth one. However, the reason is still unclear and limits the quality control of baijiu fermentation. In this study, the flavor substances and microbiota in the up, middle and bottom layers of fermented grains in the fifth and sixth rounds were compared. Some flavor esters showed obviously decreased concentrations in the sixth round, including ethyl benzoneacetic acid, ethyl hexanoic acid, ethyl dodecanoic acid, diethyl butanedioic acid, and ethyl 2-hydroxyl-propanoic acid. Meanwhile, an off-flavor p-cresol was detected in the sixth round. Correlation analysis of flavor chemicals and microbiota indicated that fungi in the fifth round played an important role for ester synthesis. Some bacterial and fungal species were both positively correlated with p-cresol synthesis, and the related p-cresol metabolic pathways were proposed for the first time. These results revealed flavor divergences of fermented grains between the fifth and sixth rounds, and will ultimately help to improve baijiu quality.

Biocatalyzed Synthesis of Flavor Esters and Polyesters: A Design of Experiments (DoE) Approach.

In the present work, different hydrolases were adsorbed onto polypropylene beads to investigate their activity both in short-esters and polyesters synthesis. The software MODDE® Pro 13 (Sartorius) was used to develop a full-factorial design of experiments (DoE) to analyse the thermostability and selectivity of the immobilized enzyme towards alcohols and acids with different chain lengths in short-esters synthesis reactions. The temperature optima of Candida antarctica lipase B (CaLB), Humicola insolens cutinase (HiC), and Thermobifida cellulosilytica cutinase 1 (Thc_Cut1) were 85 °C, 70 °C, and 50 °C. CaLB and HiC preferred long-chain alcohols and acids as substrate in contrast to Thc_Cut1, which was more active on short-chain monomers. Polymerization of different esters as building blocks was carried out to confirm the applicability of the obtained model on larger macromolecules. The selectivity of both CaLB and HiC was investigated and best results were obtained for dimethyl sebacate (DMSe), leading to polyesters with a Mw of 18 kDa and 6 kDa. For the polymerization of dimethyl adipate (DMA) with BDO and ODO, higher molecular masses were obtained when using CaLB onto polypropylene beads (CaLB_PP) as compared with CaLB immobilized on macroporous acrylic resin beads (i.e., Novozym 435). Namely, for BDO the Mn were 7500 and 4300 Da and for ODO 8100 and 5000 Da for CaLB_PP and for the commercial enzymes, respectively. Thc_Cut1 led to polymers with lower molecular masses, with Mn < 1 kDa. This enzyme showed a temperature optimum of 50 °C with 63% of DMA and BDO when compared to 54% and 27%, at 70 °C and at 85 °C, respectively.

Immobilization of Candida rugosa lipase on magnetic chitosan beads and application in flavor esters synthesis

In this work, magnetic chitosan (MCH) beads were synthesized by phase-inversion method, and grafted with polydopamine (PDA) and then used for direct immobilization of Candida rugosa lipase by Schiff base reaction. The amount of immobilized enzyme and the retained activity were found to be 47.3 mg/g and 72.8%, respectively, at pH 7.0, and at 25 °C. The apparent Km (9.7 mmol/L), and Vmax (384 U/mg) values of the immobilized lipase were significantly changed compared to the free lipase. The MCH@PDA-lipase was better thermal and storage stability at different temperatures than those of the free lipase. In hexane medium, the esterification reaction results showed that the maximum conversions of isoamylalcohol and isopentyl alcohol to isoamyl acetate and isopentyl acetate using the MCH@PDA-lipase were found to be 98.4 ± 1.3% and 73.7 ± 0.7%, respectively. These results showed that the MCH@PDA-lipase can be used as an operative immobilized enzyme system for many biotechnological applications.