Enzymatic synthesis of ascorbyl oleate and evaluation of biological activities

https://doi.org/10.1016/j.foodres.2022.111851Get rights and content

Highlights

  • The use of a cost-effective enzyme was efficient for synthesis of ascorbyl oleate.

  • The synthesized showed interesting biological and non-toxic properties.

  • It provided effective protection against lipid oxidation to olive oil.

  • And offered interesting perspectives for nutraceutical application.

Abstract

Compounds that reduce or neutralize free radicals have been evaluated for use as nutraceutical or antioxidant additives in processed foods. This study aimed to enzymatically produce ascorbyl oleate and assess its biological properties. The synthesis was performed under previously maximized conditions (L-ascorbic acid/oleic acid 1:9 molar ratio, 70 °C, 1 h reaction). Immobilized commercial lipase from Candida antarctica (NS 88011) was used as biocatalyst. The reaction product was isolated, and its structure was confirmed by High-Performance Liquid Chromatography and Nuclear Magnetic Resonance. Ascorbyl oleate showed antioxidant and antimicrobial activity, besides no toxicity, did not influencing blood coagulation and also not presenting hemolytic profile. Better storage stability was achieved under refrigerated conditions, and the oxidative stability demonstrated free radicals fighting efficiency, increasing olive oil's shelf life. In vitro gastrointestinal simulation showed that ascorbyl oleate maintained antioxidant potential up to the duodenum stage during the digestive process. Therefore, the synthesized natural compound presented a high potential to be applied in the food and pharmaceutical industries.

Introduction

The consumption of processed foods has dramatically increased in recent years due to the needs imposed by modern life. The maintenance of processed foods oxidative stability has been widely studied due to the need to control free radical formation, which antioxidants addition could avoid. Synthetic antioxidants (BHA, BHT and TBHQ) are most commonly used due to their low cost and efficiency; however, they present low metabolizing capacity, do not add any nutraceutical value, as well as causing liver damage and carcinogenicity (Robledo et al., 2014, Sharma et al., 2019).

Natural antioxidants, also known as green antioxidants, have increasingly taken a prominent place in processed foods formulations. L-ascorbic acid (Vitamin C) is among the most widely used, and is available not exactly of a purely natural nature, but of an identical nature. This means that its structure is the same as that of natural substances, but it has been prepared by synthesis (Nanditha and Prabhasankar, 2009). However, its polar nature makes its application in oil and fat products rather limited. In addition, ascorbic acid is easily oxidized, especially under aerobic conditions and exposure to light. It is degraded in the first stage to dehydroascorbic acid and then irreversibly to oxalic acid (Kleszczewska, 2000).

To avoid these problems, ascorbic acid may undergo an esterification process generating a lipophilic product that preserves its precursor antioxidant properties (Ćorović et al., 2017). Commercially, palmitic acid has been widely used as an acylating agent (Santibáñez et al., 2014, Sharma et al., 2016, Tufiño et al., 2019, Zhang et al., 2021), however, some studies have shown that ascorbyl esters obtained from unsaturated fatty acids present improved antioxidant properties, for example, Stojanović et al. (2015) realized that ascorbyl oleate (monounsaturated acyl group), showed considerably higher antioxidant activity than esters obtained from saturated chain (myristic, palmitic and stearic). Viklund, Alander, and Hult (2003) confirmed the high free radical fighting potential of the ester obtained from oleic acid on the oxidative stability of rapeseed oil when compared to ascorbyl palmitate. Unsaturated chain esters present a lower melting point and fusion enthalpy and are more miscible with oils than esters with saturated chains (Song et al., 2004, Viklund et al., 2003, Karmee, 2009, Stojanović et al., 2015). It is worth noting that unsaturated fatty acids also exhibit nutraceutical characteristics, playing an essential role in many metabolic pathways and disease prevention (Lestari and Meiyanto, 2018). In addition, oleic acid, belonging to the omega family, develops a key role in the synthesis of hormones during human metabolism (Campos-Sales et al., 2013). The literature suggests its consumption due to its protective effect against some types of cancer, such as brain and breast cancer. Its main dietary source is olive oil (Simonsen et al., 1998, Pauwels, 2011, Kumar and Downie, 2017, Arsic et al., 2019) Its main dietary source is olive oil. (Pastor, Bouzas and Tur, 2021). Free radicals not only accelerate oxidative processes in foods but are also harmful to the human body. They are considered the main factor responsible for premature aging and development of various ones, such as degenerative diseases (Alzheimer and Parkinson), cardiovascular, cancer, and others (Poprac et al., 2017, de Torre et al., 2020). To evaluate the nutraceutical effect of antioxidants prior to their in vivo application, in vitro digestion models are widely used to simulate gastrointestinal conditions (Naissinger da Silva et al., 2021). At all stages of digestion process, the continued ability to capture free radicals is a crucial factor in proving the synthesized product's efficiency.

The synthesis of ascorbyl esters catalyzed by lipases has already been described by many authors, as a study by Liu et al. (2011), Novozym 435 provided yields up to 78.2% for the synthesis of ascorbyl palmitate. Park et al. (2003) obtained a high conversion (83%) in the synthesis of ascorbyl oleate using CAL-B lipase. However, the high price of the enzyme is still the main obstacle in the implementation of the enzymatic synthesis process in industries. The new enzyme preparation NS 88011, taken from fraction B of C. antarctica and immobilized in a low-cost hydrophobic polymer resin (no further information described by the manufacturer), showed good catalytic properties, high activity, and stability, and can be applied to replace commercial lipases (Novozym 435, Lipozyme TL-IM and Lipozyme RM-IM) (Dalla Costa et al., 2021). In the literature, to date, most research is focused on optimizing the production of ascorbyl esters and only evaluating the antioxidant potential, presenting a gap in front of the other biological properties of the compound, such as, antimicrobial properties, toxicity, stability, among others. Furthermore, their safety evaluation for use as food additives is based mainly on the assumption of total hydrolysis into ascorbic acid and its respective fatty acids, thus requiring their evaluation and assurance before contact with the consumer.

In this context, this study aimed to synthesize ascorbyl oleate using an economically feasible lipase as a catalyst, differentiating from other studies found in the literature. It will also be performed its isolation for the evaluation of its properties, since, so far, few studies are focused on this. The antioxidant activity will be evaluated against two radicals and through the oxidative stability of olive oil, which is a real condition. Antimicrobial activity, toxicity, hemocompatibility and storage stability of ascorbyl oleate and its precursors will also be determined. Furthermore, to evaluate the possible presence of nutraceutical properties, ascorbyl oleate was evaluated for its antioxidant efficacy by simulating an in vitro gastrointestinal digestion process.

Section snippets

Substrates and enzymes

L-ascorbic acid (99% Sigma-Aldrich, San Luis, Missouri, USA), oleic acid (97% Sigma-Aldrich) and tert-butanol (P.A. Merk, Darmstadt, Germany) of the analytical grades were used, without further purification. The commercial lipase used from Candida antarctica NS 88011 immobilized on a hydrophobic polymer resin, kindly provided by Novozymes Brasil (Araucária, Paraná, Brazil) was used as catalyst.

Enzymatic synthesis of L-ascorbic acid and oleic acid

Ascorbyl oleate synthesis was performed by biocatalysis under the conditions maximized (Dalla Costa et

Synthesis of ascorbyl oleate

Ascorbyl oleate synthesis obtained a reaction yield of 50% using ascorbic acid/oleic acid molar ratio of 1:9, 70 °C, and 30% enzyme loading, conditions that were maximized and agreed upon by Dalla Costa et al. (2021).

Ascorbyl oleate identification

The HPLC-DAD-MS/MS data provided a higher relative abundance ion at 440 m/z, suggesting ascorbyl oleate (PM = 440 g/mol). Corroborating this hypothesis is the observed fragments of the reaction precursor's molecular ion, ascorbic acid (C6H7O6-), and oleic acid (C18H33O2-)

Conclusions

Ascorbyl oleate's enzymatic synthesis and antioxidant and antimicrobial properties were reported in this study. The synthesized compound's molecular structure was identified and confirmed after addition of the suitable spectroscopic characterization. Ascorbyl oleate showed satisfactory antioxidant potential, no toxicity, no influence on blood coagulation and hemolytic profile. It was more stable than its precursor (ascorbic acid), achieving better performance under refrigeration. Extra virgin

Credit Author Statement

K. A. Dalla Costa, A. Santa Catarina, I. C. R. Leal and P. C. Sathler conducted the experiments and performed the laboratory analyses. K. A. Dalla Costa wrote the manuscript with input from all authors. D. de Oliveira , R. L. C. and R. M. Dallago analysed the data and interpreted. J. Zeni and N. Paroul critically revised and gave the final approval of the article.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

The authors thank the National Council for Scientific and Technological Development (CNPq), the Coordination for the Improvement of Higher Education Personnel Brazil (CAPES), the Federal University of Rio de Janeiro (UFRJ) and the URI for the financial support of this work and scholarships.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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