Preparation of astaxanthin by lipase-catalyzed hydrolysis from its esters in a slug-flow microchannel reactor

Highlights

• A slug-flow microchannel reactor was constructed for lipase-catalyzed hydrolysis of astaxanthin esters.

• The low pressure reactor was suitable for two-phase biochemical reactions.

• 75.4 % of astaxanthin ester in H. pluvialis oil was converted to free astaxanthin in 200 min.

• The content of free astaxanthin in H. pluvialis oil increased from 2.13 mg/L to 18.8 mg/L after hydrolysis.

Abstract

Natural astaxanthin is widely used as a food and cosmetics additive because of its multiple biological activities. However, astaxanthin produced by Haematococcus pluvialis is generally esterified, and its activity is far less than that of free astaxanthin. Hydrolysis of astaxanthin esters to free astaxanthin by enzymes can overcome the drawbacks of chemical saponification methods. In this paper, a slug-flow microchannel reactor was constructed and tested in enzymatic hydrolysis of astaxanthin esters. The reactor consists of a “T” slug-flow generator, a stainless-steel microchannel, two constant-flow pumps, and a temperature controller. The reactor has the advantages of simple configuration and easy scale-up, and is suitable for two-phase biochemical reactions. Using the microchannel reactor, astaxanthin esters in H. pluvialis oil were efficiently hydrolyzed to free astaxanthin by lipase from Aspergillus niger. After hydrolysis, the content of free astaxanthin in H. pluvialis oil was 18.8 mg/L, 7.83-times higher than that before hydrolysis (2.13 mg/L). The hydrolysis rate reached 75.4 %. These results indicate that the microchannel reactor can be useful for the production of free astaxanthin from its esters.

Introduction

Astaxanthin (3,3′-dihydroxy-β,β′-carotene-4,4′-dione) is an oxygen-containing carotenoid derivative, widely distributed in aquatic animals, birds, microalgae, and yeast [1,2]. Studies have shown that astaxanthin has very strong antioxidant activity, >100-times that of vitamin E [3]. It can also inhibit the occurrence of tumors, delay the oxidation of polyunsaturated fatty acids, resist ultraviolet rays, enhance human immunity, improve eyesight, and so on. Astaxanthin has broad applications in medicine, cosmetics, food, and aquaculture [[4], [5], [6]]. The astaxanthin market is projected to reach a value of USD 2.57 billion per year by 2025 because of increasing awareness about its health benefits, safety, and multi-functionality [7].

Although astaxanthin can be synthesized by chemical methods, the synthesized product is racemic, and its stability, biological activity, and safety are much lower than those of natural astaxanthin [2,8]. Natural astaxanthin is mainly obtained from Phaffia rhodozyma and Haematococcus pluvialis. Astaxanthin produced by P. rhodozyma fermentation mostly exists in free form [9,10], but the low fermentation yield impedes industrial application of this method. H. pluvialis is able to accumulate astaxanthin to 5 % of dry cell weight [[11], [12], [13]], but the astaxanthin mostly exists as esters (5 % free form, 95 % esterified) [14,15]. Studies have shown that the bioactivity of astaxanthin esters is much lower than that of free astaxanthin, and the absorption and utilization of astaxanthin esters were also poor in vivo [16]. Therefore, how to effectively convert astaxanthin esters into free astaxanthin has become the subject of research.

At present, the main way to produce natural astaxanthin is the saponification of astaxanthin esters. However, in alkaline conditions, the hydroxyl group in astaxanthin can be dehydrogenated to form astacene. Biological functions of astacene are different from those of astaxanthin [[16], [17], [18]]. Many efforts have been made, including chemically controlled transesterification, to avoid the formation of astacene. Unfortunately, there are no reports of successful industrial application of de-esterification of astaxanthin esters by chemical methods.

Lipase is a type of enzyme that can catalyze the hydrolysis of esters [19]. Astaxanthin esters can be hydrolyzed to free astaxanthin. There are many reports on the conversion of astaxanthin esters to free astaxanthin by lipase in batch reactions [16,20,21]. However, it is generally necessary to add emulsifier to the reaction system to increase the contact interface between lipid-soluble substrates and water. The addition of emulsifier creates difficulties in the subsequent purification of the product, resulting in low efficiency of recovery of free astaxanthin by extraction.

In recent years, microchannel (or microfluidic) reactor technology has been widely developed. These channels/reactors are generally on the micrometer, or even nanometer, scale. Compared with traditional reactors, microchannel reactors have the characteristics of a fast reaction rate, high conversion rate, and lower chemical consumption [22]. With these advantages, microreactor technology has gradually been applied in many enzyme-catalyzed reactions [23]. There are many types of microreactor. Among them, the slug-flow microreactor is characterized as moving discrete volumes of dispersed phases (slugs, droplets) in the microchannel. The resulting flow pattern approximates the uniform residence time of all droplets (slugs). The contact area of the two immiscible liquids (or gas/liquid) in the slug-flow system is large, and the diffusion distance between the interface and the slug core is short. Internal circulation in the slugs further increases the interfacial mass and heat transfer intensity, thus accelerating the chemical reaction [[24], [25], [26]]. Because the substrate of lipase-catalyzed reactions is often insoluble in water, but soluble in organic solvents, the reaction can be completed by lipase in a two-phase system in a slug-flow microchannel reactor, thus avoiding the use of emulsifiers. Indeed, the slug-flow microreactor is particularly suitable for lipase-catalyzed biochemical reactions.

In this paper, an experimental microchannel reactor was established for two-phase bioreaction. The reactor allows the generation of slug-flow, bioconversion, and separation of the oil and water phases. The developed reactor was tested in lipase-catalyzed hydrolysis of H. pluvialis oil to produce free astaxanthin.