Biosynthesis of ascorbyl benzoate in organic solvents and study of its antioxygenic and antimicrobial properties

doi:10.1016/j.foodchem.2006.04.013

Food Chemistry

Volume 101, Issue 4, 2007, Pages 1626-1632

https://doi.org/10.1016/j.foodchem.2006.04.013 Get rights and content

Abstract

Ascorbyl benzoate was synthesized through lipase-catalyzed esterification in organic media, and its properties studied. A series of organic solvents with a log P from −1.30 to 2.50 were investigated, in which cylcohexanone (log P = 0.96) was found to be the most suitable. The optimum reaction conditions in cylcohexanone were pH 6.0, a_w 0.33, a substrate concentration form 0.06 M to 0.1 M, 65 °C, and above 150 rpm speeds of shaking. Experimental results also demonstrated that benzoic acid was not an ideal substrate of lipase, which led to low conversion rates, but its limitation could be overcome by excess l-ascorbic acid. Schaal oven test illustrated that the antioxidant activity of ascorbyl benzoate was comparable to that of ascorbyl palmitate, and minimal inhibitory concentration (MIC) data showed that its antimicrobial activity was weaker than that of benzoic acid.

Introduction

Enzymatic reactions in organic media offer numerous possibilities for biotechnological production of useful chemicals (Klibanov, 2001), and lipase-catalyzed esterification plays a very important role in this process (Hari Krishna, 2002). Since natural substrates of lipase are long-chain fatty acid triacylglycerols, few data are available on lipase-catalyzed esterification of aliphatic alcohols with aromatic acids (Kobayashi & Adachi, 2004), although their esters find wide applications in areas such as perfumery, cosmetics and food.

Ascorbyl benzoate to be studied here is an aromatic acid ester of l-ascorbic acid. More interestingly, it is a potential bifunctional compound, because l-ascorbic acid usually works as a natural antioxidant, while benzoic acid is a traditional antimicrobial agent. Chemical synthesis of this kind of esters usually has many disadvantages such as heat sensitivity, and poor reaction selectivity leading to undesirable side reactions. In contrast, the enzymatic synthesis offers the advantages of milder reaction conditions, a selective specificity, fewer intermediary and purification steps, and a more environmentally friendly process (Hills, 2003, Villeneuve et al., 2000). Moreover, Compared with those produced by chemical catalysis, esters such as flavors produced through biocatalysis can be considered close to ‘natural’ and potentially satisfy the recent consumer demand.

The aim of this work was to study the lipase-catalyzed esterification of ascorbyl benzoate in organic media, and to explore its antioxidant and antimicrobial activity.

Section snippets

Enzyme

A commercially immobilized lipase (triacylglycerol hydrolase, EC. 1.3.1.3, Novozym435) from Novo Nordisk Industri (Bagsvaerd, Denmark) was used in this work, which is a preparation of lipase B from Candida antarctica immobilized on macroporous acrylic resin (bead size 0.3–0.9 mm, bulk density 430 kg m⁻³, activity 10,000 propyl laurate units) and displays a non-specific activity towards triaglycerols.

Chemicals

The purity of substrate is over 99.7% for l-ascorbic acid (Guangdong Guanghua Chemical Factory Co.,

Optimization of organic solvents

Hydrophobicity of an organic solvent is important for the enzyme activity. Log P, where P is the partition coefficient of a given solvent between n-octanol and water (Lyman, 1990), is a widely used parameter to describe solvent hydrophobicity and their possible effects on enzyme activity. Generally, solvents with a log P > 4.0 are recommended in enzyme-catalyzed reactions.

Nine organic solvents listed in Table 1 were selected in a log P range from −1.30 to 2.50. Only in four of them with a log P < 2.0,

Conclusions

Organic solvents with log P < 2.0, slightly acidic environments, low water activities, excess l-ascorbic acid, an appropriate substrate and lipase concentration were found favorable for production of ascorbyl benzoate. The hydrophilicity and solubility of l-ascorbic acid, the essential water and equilibrium theory of enzyme reaction, mass transfer, the activation and deactivation of temperature on the enzyme were supposed to explain the observed results. At the end of reaction, the product was

References (16)

S. Hari Krishna
Developments and trends in enzyme catalysis in nonconventional media
Biotechnology Advances
(2002)
J.Y.V.A.K. Kamil et al.
Antioxidative activity of chitosans of different viscosity in cooked comminuted flesh of herring (Clupea harengus)
Food Chemistry
(2002)
P. Villeneuve et al.
Customizing lipases for biocatalysis: a survey of chemical, physical and molecular biological approaches
Journal of Molecular Catalysis B: Enzymatic
(2000)
C.X. Zhang et al.
Two novel synthetic antioxidants for deep frying oils
Food chemistry
(2004)
AOCS
Official methods and recommended practices of the American Oil Chemists’ Society
(2004)
K. Faber
Biotransformations in organic chemistry
(2000)
O.R. Fennema
Principles of food science, Part 1, Food chemistry
(1976)
H.L. Goderis et al.
Lipase-catalyzed ester exchange reactions in organic media with controlled humidity
Biotechnology and Bioengineering
(1987)

There are more references available in the full text version of this article.

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In these studies, a dead-end complex formatted by one of the substrate and lipase was introduced to explain substrate inhibition phenomenon. So far, many ascorbyl esters such as ascorbyl oleate (Reyes-Duarte et al., 2011), ascorbyl palmitate (Burham et al., 2009), ascorbyl linoleate (Watanabe et al., 2008) and ascorbyl benzoate (Lv et al., 2007) have been synthesized by enzymatic esterification in organic solvent. However, the studies on the enzymatic kinetics and thermodynamics were rarely reported.
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Biosynthesis of ascorbyl benzoate in organic solvents and study of its antioxygenic and antimicrobial properties

Abstract

Introduction

Section snippets

Enzyme

Chemicals

Optimization of organic solvents

Conclusions

Biotechnology Advances

Food Chemistry

Journal of Molecular Catalysis B: Enzymatic

Food chemistry

Official methods and recommended practices of the American Oil Chemists’ Society

Biotransformations in organic chemistry

Principles of food science, Part 1, Food chemistry

Lipase-catalyzed ester exchange reactions in organic media with controlled humidity

Biotechnology and Bioengineering