KDN Biotech (Shanghai) Co., Ltd.
KDN Biotech (Shanghai) Co., Ltd. Benefit the earth by biotechnology!
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Short chain esters often have a characteristic pleasant, fruity odour. Consequently, these esters have notable commercial significance in the fragrance, cosmetics, food, and pharmaceutical industries [1]. Flavour esters produced by extraction from plant and animal sources are not viable due to their presence in minor quantities. Chemical production of flavour esters is not eco-friendly and has some toxic effects on the customer’s health. Nowadays, many researchers and industries have switched to biocatalytic flavour synthesis due to consumer’s inclination towards natural flavours over chemical ones. These reactions use mild operating conditions, have high specificity with reduced side reactions, and produce high purity flavour compounds by avoiding the expensive separation techniques [2]. Among three different major biotechnological methods (through enzymes, plant cell cultures, and plant tissue cultures), processes employing enzymes are the most common techniques [3].
Methyl butyrate (MB) or methyl ester of butyric acid is an ester with a fruity odour of pineapple, apple, and strawberry. Availing small amounts in plant sources, usually pineapple flavour is produced by distillation of vegetable based essential oils on small scale for utilization as perfumes or food flavours. Octyl acetate (OA) or octyl ethanoate is a flavour ester that is formed from octanol and acetic acid with a fruity orange flavour used in food and beverage industries [4]. Lipase catalyzed esterification and transesterification reactions for flavour esters have numerous food applications such as in the synthesis of modified triacylglycerols, emulsifiers, peptides, and oligosaccharides. Lipases, which are considered to be natural by the food legislation agencies, have been widely investigated for ester synthesis, mainly in organic solvents, due to their enhanced solubility in hydrophobic substrates and elimination of side reactions caused by water [5]. Lipase mediated synthesis of flavour esters under solvent-free conditions (in which the reaction medium involves a reactant itself (i.e., an alcohol) as a solvent) has significant importance in different food and pharmaceutical industries due to the avoidance of toxic solvent and elimination of its recovery during the operation [6]. Lipase catalyzed production of flavour esters by transesterification reactions is influenced by a number of transesterification variables such as molarity of alcohol, reaction time, addition of water, temperature, agitation speed, and amount of immobilized enzyme. Several researchers reported the application of immobilized lipases for the flavour ester synthesis. Lipases were employed for transesterification in organic solvent to produce flavour esters such as isoamyl acetate [7, 8], isoamyl butyrate [9], geranyl acetate [10], citronellyl acetate [11], octyl acetate [12], and methyl butyrate [13]. Akoh and Yee [14] studied the lipase catalyzed transesterification of primary terpene alcohols with vinyl esters in organic media as a solvent. Many works were performed by using immobilized lipases and solvent-free conditions for the synthesis of flavour esters to overcome the problems associated with free enzyme separation and solvent toxicity. Immobilized lipase from C. rugosa and porcine pancreatic lipase were employed for the synthesis of isoamyl acetate (banana flavour), ethyl valerate (green apple flavour), and butyl acetate (pineapple flavour) in n-hexane [15]. Several authors assessed the immobilized lipases for transesterification ability to produce various flavour esters [16, 17]. Solvent-free synthesis of ethyl oleate reported by Foresti et al. [18], results in a 78.6% conversion in 7 h using Candida antarctica B lipase adsorbed on polypropylene powder. In another study Ye et al. [19] synthesized saccharide fatty acid esters in solvent-free conditions and reported 88% yield of fructose oleate.
Based on the present demand and inclination of customers towards natural flavours, the present study has intended to synthesize the flavour esters, namely, methyl butyrate and octyl acetate, through immobilized lipase mediated transesterification under solvent-free conditions.
Lipase from Rhizopus oryzae NRRL 3562 was produced and covalently immobilized on activated silica [20]. p-Nitrophenyl palmitate (p-NPP), methyl butyrate, and octyl acetate standard were purchased from Sigma (USA). All chemicals used were of AR grade and were procured from Merck, Qualigens, and Himedia, India.
Lipase assay was done spectrophotometrically using p-NPP as the substrate [21], and total protein was estimated using modified Lowry method using bovine serum albumin (BSA) as standard [22]. One unit (U) of lipase activity was defined as the amount of lipase that liberates one micromole of p-nitrophenol per minute under the standard assay conditions.
Methyl butyrate synthesis was carried out in screw-capped vials containing 3 mL of different molar concentrations (0.2–10 M) of methanol in vinyl butyrate with different ratios (0.1–10%) of additional water. Reaction was initiated by addition of different units (20−120 U) of immobilized R. oryzae NRRL 3562 lipase. Samples were placed for different reaction times (2−20 h) in an orbital shaker at different rpm (100−200 rpm) and temperatures (28−40°C), along with the respective controls (without immobilized lipase). From the reaction mixture, samples were withdrawn at specified time intervals and centrifuged at 1747 g for 10 min to remove the immobilized enzyme. The samples were diluted with n-hexane (10 times) and analyzed by gas chromatography.
Octyl acetate synthesis was carried out in screw-capped vials containing 3 mL of varying molar concentrations (0.2−10 M) of octanol in vinyl acetate with different ratios (0.1−10%) of additional water. Reaction was initiated by addition of different units (20−120 U) of immobilized R. oryzae NRRL 3562 lipase. Samples were placed for a reaction time of 2−20 h in an orbital shaker at different rpm (100−200) and temperatures (28−40°C) along with the respective controls (without immobilized lipase). The reaction samples were collected at specified time intervals and centrifuged at 1747 g for 10 min to remove the immobilized enzyme. The centrifuged samples were diluted with n-hexane (10 times) and analyzed by gas chromatography.
Synthesis of methyl butyrate was analyzed by injecting the diluted aliquots of the reaction mixture in an Agilent 6820 Gas Chromatograph with a flame-ionization detector (USA). The capillary column (length: 30 m, internal diameter: 0.25 mm) with nitrogen as the carrier gas at a constant pressure of 4 kg cm2 was used. Column temperature was kept at 60°C for 1 min and then raised to 220°C at the rate of 10°C. Thereafter, it was raised to 240°C at the rate of 10°C and finally maintained at this temperature for 5 min. The temperatures of the injector and detector were set at 200°C and 265°C, respectively. The retention time of methyl butyrate was 21.2 min. The % molar conversion of product was identified and calculated by comparing the peak areas of standard methyl butyrate at the particular retention time.
The diluted reaction mixture was analyzed for synthesis of octyl acetate by Agilent 6820 Gas Chromatograph with a flame-ionization detector (USA). Nitrogen was used as the carrier gas at a constant pressure of 4 kg cm2. The capillary column (length: 30 m, internal diameter: 0.25 mm) was kept at 45°C for 2 min, thereafter raised to 260°C, and maintained at this temperature for 1.63 min. The temperatures of the injector and detector were set at 250°C and 280°C, respectively. The retention time of octyl acetate was 6.7 min. The % molar conversion of product was identified and calculated by comparing the peak areas of standard octyl acetate at the particular retention time.
The effect of alcohol molar concentration on molar conversion was investigated in a solvent-free system. As shown in Figure 1, a maximum molar conversion of methyl butyrate and octyl acetate was observed at 0.6 M methanol in vinyl butyrate (1 M theoretical alcohol molarity) and 2 M Octanol in vinyl acetate, respectively. The difference in alcohol molarity towards different products may be attributed to either the steric hindrance or electronic effects of substrates on the immobilized lipase or specificity of immobilized lipase towards the substrates. However, the lower molar conversion at higher molar ratio has been attributed to the inhibitory effect of vinyl acetate and vinyl butyrate on enzyme activity [12, 23]. Increasing the nucleophile (alcohol) concentration is one way of pushing the equilibrium in a forward direction. However, at higher concentrations of alcohol, reaction rate may slow down due to slower diffusion rates of alcohols into the support. Hence, it is necessary to optimize the actual excess nucleophile concentration to be employed in a given reaction [24]. Esterification activity gradually decreased upon increasing the alcohol to acid molar ratio beyond 2 M and 0.6 M in case of octyl acetate and methyl butyrate, respectively, which indicate the inhibitory effect of alcohols on enzyme activity beyond those concentrations. The inhibitory effect of alcohol was also reported by Ghamgui et al. [7], where 64% molar conversion of isoamyl acetate was obtained with 2 M alcohol/acid molar ratio. Further increase in the acid/alcohol molar concentration of S. simulans lipase activity was inhibited. In another study by Claon and Akoh [11], molar conversion of citronellyl acetate has been decreased by usage of more than 0.3 M acetic acid.
| Usage | Daily |
| Fragrance | Unscented |
| Product Type | Flavors And Fragrances |
| Source | Biological Fermentation-based |
| Stability | Stable At Room Temperature |
| Skin Type | All Skin Types |
| Size | 1kg |
| Enzyme Type | Lipase |
| Ph Range | 5.5-7.5 |
| Function | Used To Make Flavors And Fragrances |
One of the primary application occasions for Cosmetic Immobilized Lipase is in daily skincare routines. As a cruelty-free product suitable for all skin types, it can be used by individuals looking to enhance their facial care regimen. Whether you have dry, oily, combination, or sensitive skin, this product can help improve the overall health and appearance of your complexion.
Another scenario where Cosmetic Immobilized Lipase can be beneficial is for targeted treatments on the face. Its gentle formula makes it ideal for addressing specific skin concerns such as fine lines, wrinkles, or uneven texture. By incorporating this product into your beauty routine, you can achieve a more radiant and youthful-looking complexion.
Furthermore, Cosmetic Immobilized Lipase can also be used in professional settings, such as spas or beauty salons. Its unscented fragrance makes it suitable for use on clients with sensitivities to strong scents, ensuring a comfortable and relaxing experience during treatments.
Whether you are looking to improve your daily skincare routine, target specific areas on your face, or offer professional treatments to clients, Cosmetic Immobilized Lipase is a versatile product that can meet your needs. With its origins in China and its model number IM-100, this high-quality product is a must-have for anyone seeking effective and gentle skincare solutions.
Cosmetic Immobilized Lipase Product Customization Services:
Brand Name: Immobilized lipase
Model Number: IM-100
Place of Origin: China
Ph Range: 5.5-7.5
Size: 1kg
Cruelty-Free: Yes
Skin Type: All Skin Types
Stability: Stable At Room Temperature
The Product Technical Support and Services for Cosmetics Enzymes include:
- Troubleshooting assistance for any issues related to the use of the product
- Guidance on proper storage and handling of the enzymes
- Recommendations for optimal usage and application of the product
- Information on the compatibility of the enzymes with other cosmetic ingredients
- Training and educational resources on the benefits and applications of enzymes in cosmetics
Product Name: Cosmetics Enzymes
Description: Our Cosmetics Enzymes are a natural and effective way to rejuvenate your skin and enhance your beauty.
Ingredients: Enzymes extracted from fruits and plants.
Directions: Apply a small amount of Cosmetics Enzymes on clean skin and massage gently until fully absorbed.
Storage: Store in a cool, dry place away from direct sunlight.
Shipping: Your order of Cosmetics Enzymes will be carefully packaged and shipped within 1-2 business days. We offer standard and expedited shipping options for your convenience.