Elsevier

Food Chemistry

Volume 281, 30 May 2019, Pages 124-129
Food Chemistry

Determination of vitamin B12 in four edible insect species by immunoaffinity and ultra-high performance liquid chromatography

https://doi.org/10.1016/j.foodchem.2018.12.039 Get rights and content

Highlights

  • A new UHPLC method for the analysis of vitamin B12 in edible insects is proposed.

  • Vitamin B12 was studied in mealworm, cricket, grasshopper and cockroach.

  • Possible occurrence of pseudovitamin B12 was reported in all four species.

  • Risk of overestimation of vitamin B12 content in edible insects.

Abstract

Insects are rich in major nutrients, such as protein and fat. Recently, minor nutrients like vitamins have become the subjects of interest in insects. Hence, this study reports on the development and validation of a method for the determination of vitamin B12 in mealworm (Tenebrio molitor larvae), cricket (Gryllus assimilis), grasshopper (Locusta migratoria) and cockroach (Shelfordella lateralis), using an ultra-high performance liquid chromatography approach with preliminary immunoaffinity chromatography sample preparation. The method was validated regarding linearity, specificity, accuracy and precision, as well as limits of detection/quantification, and was found to be satisfactory for the desired application. Found levels of vitamin B12 were 1.08 µg/100 g for mealworm, 2.88 µg/100 g for cricket, 0.84 µg/100 g for grasshopper, and 13.2 µg/100 g dry weight for cockroach, representing the first validated report on the content of vitamin B12 in edible insects. Observed interferences are likely caused by the presence of pseudovitamin B12.

Introduction

By 2050 up to 10 billion people will inhabit earth (Cohen, 2003). Meeting their demands for a secure food supply will constitute a major challenge. In order to meet this challenge, insects have been proposed as a promising source of food and feed. Compared to other food of animal origin, insects have multiple advantages, such as lower CO2 and methane emission, a much lower demand of water, and the possibility to be reared on feed of low quality and limited space (van Huis & Oonincx, 2017).

Although a significant part of the world population already consumes insects on a regular basis, a habit called entomophagy (van Huis, 2013), this practice is not common in Europe and the US. Legislation in the EU has recently opened up for insects or insect-based foods and feeds (Council Regulation, 2015), following a trend of public awareness and interest in the topic. Since the beginning of 2018, edible insects fall into the novel food regulation of the EU (Council Regulation, 2018), although already being on the market in several countries of the EU. Multiple studies report on the nutritional value of insects, especially regarding macronutrients, such as fat and protein (Rumpold and Schlüter, 2013, van Huis, 2013). With the advance of the topic, minor nutrients such as vitamins are now in the focus of the consumer. Among them, vitamin B12 is of particular interest. Although it is needed in very small amounts, meeting the daily required intake is still challenging today. Being almost exclusively found in food of animal origin (Bito et al., 2016, Chamlagain et al., 2015, Lenaerts et al., 2018, Watanabe et al., 1999), this is not only of great importance for vegetarians and vegans, but also for the part of the world population that struggles to meet their nutritional intake. It is noteworthy that consumer acceptance studies suggest, that vegetarians might be more open to the consumption of insects than to other food of animal origin (Tan et al., 2015, Wilkinson et al., 2018). Although non-scientific sources repeatedly claim that insects are rich in vitamin B12, original research data on this topic is extremely limited (Finke, 2002, Finke, 2008, Finke, 2013, Finke, 2015, Jones et al., 1972, Lenaerts et al., 2018).

Due to a complex structure and multiple possible vitamers, vitamin B12 is a particular challenging compound for analysis. Being only synthesized by a few bacteria and archaea species (Fang, Kang, & Zhang, 2017), it accumulates within the food-chain, and is thus mostly found in food of animal origin, with the highest known concentrations (up to 70 µg/100 g) in the livers of mammals (Souci, Fachmann, & Kraut, 2008). Besides low concentrations in food matrices, the accurate determination of vitamin B12 is hindered by the fact that it can occur in multiple different bioactive forms, known as vitamers (Burk & Winzler, 1943). The 5′-adenosylcobalamin (AdoCbl) and methylcobalamin (CH3Cbl) are the two vitamers that are the actual cofactors for enzymatic reactions in the human body, namely AdoCbl for methylmalonyl-CoA mutase and CH3Cbl for methionine synthase (Nielsen, Rasmussen, Andersen, Nexø, & Moestrup, 2012). Hydroxocobalamin (OHCbl) does not directly act as an enzymatic cofactor, but it can be converted to the two active forms and is mostly found in blood and the liver (Froese & Gravel, 2010). Finally, cyanocobalamin (CNCbl) can also be converted like OHCbl, but is rarely found in nature. Nonetheless, for quantitation of vitamin B12, CNCbl is of great importance as it is the most stable B12 vitamer, which facilitates sample preparation (Chamlagain et al., 2018).

Possible methods for the determination of the content of vitamin B12 in food are microbiological assays (AOAC, 2006; Chamlagain et al., 2018) and chromatographic approaches (Chamlagain et al., 2015, Nakos et al., 2017, Schimpf et al., 2012, Zironi et al., 2013). Microbiological assays (MBAs) were the first methods to be standardized and applied for the quantification of vitamin B12 in food. They are based on the principle of growth of a vitamin B12 dependent microorganism. MBAs are still in use today, but suffer from severe drawbacks. Besides being time-consuming, they lack selectivity, as they are not capable of discriminating between possible vitamers, and further may report overestimations when pseudovitamin B12 is present in the sample (Chamlagain et al., 2015). Pseudovitamin B12 is a structural analogue to vitamin B12, with a changed lower ligand (5,6-dimethylbenzimidazole to adenine), which makes it almost biologically inactive for humans (Santos et al., 2007, Taga and Walker, 2008). Chromatographic approaches such as (ultra-)high performance liquid chromatography (U)HPLC are capable of distinguishing between active and inactive forms of vitamin B12, and are thus more selective than MBAs (Chamlagain et al., 2015).

Most chromatographic approaches employ an immunoaffinity step during sample preparation, in order to remove matrix components and to enrich the targeted analyte to ease quantification. For common food sources (e.g., milk, dairy products, meat), this has been proposed multiple times (Schimpf et al., 2012, Zironi et al., 2013). Furthermore, the strong affinity of all B12 vitamers to cyanide is used to convert the three possible vitamers (OHCbl, AdoCbl and CH3Cbl) to one vitamer, namely CNCbl (Chamlagain et al., 2015, Nakos et al., 2017), in order to improve method robustness, due to its increased stability to light and chemical conditions, as well as detection limits.

The number of scientific studies on the content of vitamin B12 in insects is extremely limited (Finke, 2002, Finke, 2013, Finke, 2015, Jones et al., 1972, Lenaerts et al., 2018) and do not meet contemporary demands for method validation. Thus, it was the aim of this study to adapt an unpublished in-house method for the determination of vitamin B12 in foods, based on CNCbl, preparative immunoaffinity chromatography and UHPLC, for the study of four of the most promising insect species as novel food sources of vitamin B12.

Section snippets

Reagents and standards

All chemicals and reagents were of analytical grade, and solvents for chromatography of HPLC grade. Ultra-pure (UHQ) water was used for all preparations and was provided by an SG Ultra Clear UC system from Sigma-Aldrich (St. Louis, MO). For enzymatic extraction, sodium acetate trihydrate (99.5–100.5%) from Merck (Darmstadt, Germany), acetic acid glacial (99.7%) and pepsin (No. A4289) from AppliChem (Saxony-Anhalt, Germany), and Taka-Diastase from Aspergillus oryzae (No. 86247; Sigma-Aldrich)

Chromatographic separation of B12 vitamers

Although only separation of CNCbl would have been necessary, this study aimed at separating all four possible B12 vitamers (Fig. 1), in order to assess the effectiveness of the CNCbl conversion and selectivity of the proposed method. Separation of B12 vitamers has been reported before (Frenkel et al., 1979, Kelly et al., 2006, Szterk et al., 2012, Viñas et al., 2003), but separation-time was drastically reduced by employing UHPLC technology, which allowed the separation of all four targeted

Conclusion

In this work, an ultra-high-performance liquid chromatography method, with prior preparative immunoaffinity chromatography, was proposed for the determination of vitamin B12 in edible insects, based on the conversion of possible vitamers to cyanocobalamin. The method was consequently validated and proved appropriate for the intended purpose. Mealworm, grasshopper, cricket and cockroach were studied regarding their content of vitamin B12 and exhibited vast variations of their suitability as

Acknowledgement

Financial support for this project was provided by the City of Vienna, grant “Hochschuljubiläumsstiftung der Stadt Wien”, project H-274639/2018. The authors want to thank Iris Biedermann for her skilful technical assistance and patience.

Conflict of interest statement

We, the authors of the presented paper hereby declare that we have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript.

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