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

Accepted Manuscript Determination of vitamin B12 in four edible insect species by immunoaffinity and ultra-high performance liquid chromatography Anatol Schmidt, Lisa-Maria Call, Lukas Macheiner, Helmut K. Mayer PII: DOI: Reference:

S0308-8146(18)32143-5 https://doi.org/10.1016/j.foodchem.2018.12.039 FOCH 23998

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

2 August 2018 6 December 2018 12 December 2018

Please cite this article as: Schmidt, A., Call, L-M., Macheiner, L., Mayer, H.K., Determination of vitamin B12 in four edible insect species by immunoaffinity and ultra-high performance liquid chromatography, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.12.039

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Research paper

Determination of vitamin B12 in four edible insect species by immunoaffinity and ultrahigh performance liquid chromatography

Anatol Schmidt1, Lisa-Maria Call, Lukas Macheiner and Helmut K. Mayer*

Department of Food Science and Technology, Food Chemistry Laboratory BOKU – University of Natural Resources and Life Sciences, Vienna Muthgasse 11, A-1190 Vienna, Austria

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https://orcid.org/0000-0003-2424-5582

*Corresponding author: Prof. Helmut K. Mayer Department of Food Science and Technology, Food Chemistry Laboratory, BOKU – University of Natural Resources and Life Sciences Vienna, Muthgasse 11, A-1190 Vienna, Austria Phone: +43-1-47654-75412 Fax: +43-1-47654-75436 E-mail: [email protected]

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Abstract Insects are rich in major nutrients, such as protein and fat. Recently, minor nutrients such as 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.

Keywords: Vitamin B12, Insects, Pseudovitamin B12, UHPLC, Mealworm, Cricket, Grasshopper, Cockroach

Chemical compounds studied in this article: Cyanocobalamin (PubChem CID: 5311498); Hydroxocobalamin (PubChem CID: 45357193);

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1. 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 & 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, Bito, Asai, Takenaka, Yabuta, Tago, et al., 2016; Chamlagain, Edelmann, Kariluoto, Ollilainen, & Piironen, 2015; Lenaerts et al., 2018, 2015; Watanabe, Katsura, Takenaka, Fujita, Abe, Tamura, 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, Fischer, Tinchan, Stieger, Steenbekkers, & van Trijp, 2015; Wilkinson, Muhlhausler, Motley, Crump, Bray, & Ankeny, 2018). Although non-scientific sources repeatedly claim that insects are rich in vitamin B 12, original research data on this topic is extremely limited (Finke, 2002, 2008, 2013, 2015; Jones, Cooper, & Harding, 1972; Lenaerts, Van Der Borght, Callens, & Van Campenhout, 2018).

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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 B 12, CNCbl is of great importance as it 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, Pepelanova, Beutel, Krings, Berger, & Scheper, 2017; Schimpf, Spiegel, Thompson, & Dowell, 2012; Zironi, Gazzotti, Barbarossa, Devicienti, Scardilli, & Pagliuca, 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 (5,6dimethylbenzimidazole to adenine), which makes it almost biologically inactive for humans (Santos, Vera, Lamosa, de Valdez, de Vos, Santos, et al., 2007; Taga & Walker, 2008). Chromatographic

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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, Spiegel, Thompson, & Dowell, 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, 2013, 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.

2. Material and Methods 2.1. 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) were used. Potassium cyanide (97%) for the conversion into cyanocobalamin was obtained from Acros Organics (Fair Lawn, NJ). Mobile phases of the chromatographic system were blends of trifluoroacetic acid (99%) from Sigma-Aldrich with UHQ

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water or methanol (≥99.9%) from VWR (Fontenay-sous-Bois, France), and UHQ water. Standard substances

of

hydroxocobalamin

(OHCbl)

≥95%,

cyanocobalamin

(CNCbl)

≥98%,

5´-

adenosylcobalamin (AdoCbl), ≥97% and methylcobalamin (CH3Cbl) ≥97% were purchased from Sigma-Aldrich. AdoCbl and CH3Cbl were only handled under subdued light. Thiourea ≥99% was purchased from Sigma-Aldrich.

2.2. Sample acquisition Mealworm (Tenebrio molitor, larvae), grasshopper (Locusta migratoria, adult), cricket (Gryllus assimilis, adult) and cockroach (Shelfordella lateralis, adult) were purchased frozen (−18 °C) from a pet food vendor (Reptiles Vienna, Vienna, Austria). As per the information provided by the vendor, the insects were reared on a diet of carrots and feed bran for mealworm and cockroach, and carrots, feed bran and potatoes for cricket, as well as hay and Chinese cabbage for grasshopper. Insects were killed by exposure to −18 °C for 24 h, without a preceding fasting period. Certified reference material issued by the Institute for Reference Materials and Measurements of the Joint Research Centre of the European Commission (CRM, ERM®-BD600, dried milk powder) was obtained from Sigma-Aldrich.

2.3. Sample preparation The protocol for vitamin B12 extraction from sample matrices was adopted from the manufacturer’s instructions (R-Biopharm, Glasgow, UK) and modified for insects. Preparation of samples was performed as follows. Obtained insect samples were freeze-dried using a freeze dryer (Edwards, Crawley, UK) at −60 °C and 10−1 mbar. Freeze-dried samples (2 g) were weighed in 50-mL polypropylene tubes (Sarstedt, Nümbrecht, Germany) and dispersed in 50 mL of 50 mM sodium acetate buffer (pH 4.0) using an Ultra Turrax (IKA, Staufen, Germany) under subdued light, using a safelight (KFB 247, hama®, Monheim, Germany) as sole light source. To ensure light protection during all consecutive manipulation steps, aluminium foil and brown flasks were used. In order to free protein-bound vitamin B12, 1 g of pepsin and 0.25 g of Taka Diastase were added. Samples were placed on a magnetic stirrer for 10 min. To convert all possible vitamers into cyanocobalamin

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(CNCbl), 1 mL of 1% potassium cyanide solution was added to the samples, which were left stirring for a further 5 min. Samples were then incubated for 30 min at 37 °C under continuous shaking on a Heidolph Unimax 1000 (Walpersdorfer, Germany), and another 30 min at 100 °C in a Heraeus Oven (Hanau, Germany). After cooling to room temperature, samples were transferred into 100-mL volumetric flasks and brought to volume with 50 mM sodium acetate buffer, before they were filtered through a Whatman S&S 595 ½ filter paper (Maidstone, UK). Ten millilitres of filtrate were passed through EASI EXTRACT® immunoaffinity columns (RBiopharm, Glasgow, UK). The columns were washed by passing 10 mL of UHQ water through, before CNCbl was eluted from the column, using 3 mL of 100% methanol while performing several backflushes. The eluate was evaporated to dryness on a Reacti-Therm Heating Module (Thermo Fisher Scientific, Waltham, MA) using a stream of nitrogen. Finally, the residue was reconstituted with 300 µL of solvent A (0.025 % TFA in UHQ water), filtered through a 0.20-µm syringe filter (Sartorius, Goettingen, Germany), and applied to the chromatographic system. For certified reference material, 3 g of powder were dissolved in 30 mL UHQ water, which was then used as a sample, following the described protocol.

2.4. UHPLC conditions Chromatographic analysis of B12 vitamers was performed on a Waters AcquityTM Ultra Performance LC (UPLCTM) H-class system (Waters, Milford, MA) equipped with an AcquityTM photodiode array detector (PDA), and an Acquity UPLCTM column (BEH C18, 50  2.1 mm i.d., 1.7 µm) was used for separation. Mobile phases consisted of UHQ water, modified with 0.025% trifluoroacetic acid (TFA) (solvent A) and HPLC-grade methanol (solvent B). Gradient elution was conducted at 40 °C with a flow rate of 0.6 mL min−1 at a composition of 0 min, 0% B; 4 min, 100% B; 6 min, 100% B, 6.5 min, 0% B and 8.5 min, 0% B. The detection was performed at different wavelengths, namely 348 nm for OHCbl, 361 nm for CNCbl, 262 nm for AdoCbl, and 254 nm for CH3Cbl, at a sampling rate of 20 points/s and a resolution of 1.2 nm. Injection volume was set to 50 µL. Chromatographic data were collected and processed using Waters Empower 3TM software and Microsoft Excel 2007. The value of

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t0 for the determination of the retention factor (k´) was evaluated by injecting 50 µL of a 20 mg/L thiourea solution at isocratic conditions of 75% methanol and 25% water. Detection was at 254 nm.

2.5. Validation parameters The developed method was validated following international guidelines (ICH, 2005) and regarding the following parameters: linearity, specificity, method accuracy, recovery, precision and detection limits. Precision was assessed as repeatability (n = 6, three different concentration levels (low, medium, high), intermediate precision (n = 3, three different concentration levels, over a period of four days) and precision of sample preparation, by preparing the same sample six times over a period of two days (n = 3 per day, 2 days). Detection limits were assessed as limit of detection (LOD, signal-to-noise ratio S/N = 3) and quantification (LOQ, S/N = 10) using Empower 3 with baseline drift correction. Method accuracy was assessed using certified reference material (ERM®-BD600, dried milk powder, n = 6, over 3 non-consecutive days, n = 2 per day, 2 operators and changed solvents). Retention factor (k´) was calculated according to k´=(tr − t0)/t0. Recovery experiments were conducted by adding required amounts of OHCbl standard to the weight in insects, prior to dispersing (Level 1: + 50% and Level 2: + 100%).

3. Results and Discussion 3.1. 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, Kitchens, & Prough, 1979; Kelly, Gruner, Furlong, & Sykes, 2006; Szterk, Roszko, Małek, Czerwonka, & Waszkiewicz-Robak, 2012; Viñas, López-Erroz, Balsalobre, & Hernández-Córdoba, 2003), but separation-time was drastically reduced by employing UHPLC technology, which allowed the separation of all four targeted vitamers within 2.6 min. So far only one study has achieved a

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similar separation speed (Owen, Lee, & Grissom, 2011), but did not achieve baseline separation of all four studied B12 vitamers. Determination of t0 was as suggested by the vendor (thiourea), and was found to be 0.25 min, resulting in k´ for chosen B12 vitamers of 5.55 to 9.12 (Table 1). All were significantly greater than 2.0, hence illustrating sufficient retention behaviour. Thus, the proposed UHPLC separation in conjunction with preparative immunoaffinity chromatography was subject to method performance evaluation.

3.2. Method performance The proposed method performed well regarding linearity, specificity, method accuracy, recovery, instrument precision, precision of sample preparation, LOD and LOQ (Table 1 and Table 2). Specificity of the proposed method was ensured by using highly selective immunoaffinity columns for sample preparation, which is a well-established approach (Chamlagain et al., 2015; Nakos et al., 2017). Furthermore, all naturally occurring B12 vitamers (OHCbl, AdoCbl, CH3Cbl) as well as the targeted artificial vitamer CNCbl were satisfactory separated by UHPLC. Finally, specificity was also illustrated by using OHCbl for recovery experiments, which was entirely converted to CNCbl, as intended. Linearity of the calibration was confirmed by a value for R2 > 0.99 for HOCbl and CNCbl, and > 0.94 for AdoCbl and CH3Cbl. With the exception of AdoCbl and CH3Cbl, derived regression (R2) of the calibration of the standard vitamers was greater than 0.99, which can be attributed to their extreme light sensitivity (Szterk et al., 2012; Viñas et al., 2003), and was found to be satisfactory. Range of calibration levels comprised 10 levels for HOCbl and CNCbl ranging from 2.5 µM to 600 µM, or 6 levels for AdoCbl ranging from 10 µM to 600 µM, and from 10 to 400 µM for CH3Cbl over 5 levels. The determination of vitamin B12 in insects is based on an unpublished validated in-house method for the quantification of vitamin B12 in milk, which has been validated regarding method accuracy by determining vitamin B12 in CRM and resulted in good agreement of determined and certified values for vitamin B12 (Fig. 2). Since insects represent an entirely different matrix to milk, consequently accuracy was assessed by adding known amounts of analyte at two different levels (+50% and +100%). In order to assess the effectiveness of the conversion of possible vitamers into

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the targeted CNCbl, recovery was conducted with HOCbl standards. Results were satisfactory, ranging from 89% to 104% (Table 2), and fell well within the range of recovery experiments reported for other food matrices (Chamlagain et al., 2015; Nakos et al., 2017; Zironi et al., 2013). Furthermore, recovery experiments were of utmost importance for confirming peak identity, when interfering compounds were observed. Precision of the chromatographic system was assessed by evaluating repeatability and intermediate precision. Repeatability was assessed by consecutive injections of the four targeted vitamer standards (OHCbl, CNCbl, AdoCbl, CH3Cbl) over one day, and was found to be ≤4.5%. Intermediate precision was assessed over a longer period, including changes of operators and chromatographic solvents, and was found to be below ≤5.6% (Table 1). Precision of sample preparation was also evaluated over several days and resulted in variations below 7.6% (Table 2). Values for precision are superior (Zironi et al., 2013) or in good agreement (Nakos et al., 2017) compared to other studies. Determination of limit of detection (LOD) and limit of quantification (LOQ) using PDA detection at chosen wavelengths ranged from 0.12 to 1.61 nM for LOD, and 0.40 to 5.34 nM for LOQ, and were found to be satisfactory for studying B12 vitamers in insects. LOD and LOQ were superior to other methods employing the same methodology (Chamlagain et al., 2015; Nakos et al., 2017; Owen et al., 2011), but were inferior when compared to methods employing detection based on mass spectrometry (Szterk et al., 2012). Hence, the proposed method was found to be suitable for the determination of vitamin B12 in insects using the proposed approach utilizing immunoaffinity chromatography and UHPLC.

3.3. Vitamin B12 content in edible insects As the results for method validation were satisfactory, the proposed method was subsequently employed to study the content of vitamin B12, based on assessing CNCbl, in edible insects as a novel nutritional source. Values for vitamin B12 in studied species (mealworm, grasshopper, cricket and cockroach) ranged from 0.84 to 13.21 µg/100 g dry weight (Table 2), which falls well within the range of other food of animal origin, such as pork meat (1.0 µg/100 g) or fish like mackerel (9.0 µg/100 g) (Souci et al., 2008). This confirms that insects are an excellent source of vitamin B12 and

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can significantly contribute to nutritional requirements. The number of original research data on the content of vitamin B12 in insects is extremely limited (Table 3). Although the subject has already been addressed in reviews (Nowak, Persijn, Rittenschober, & Charrondiere, 2016; Rumpold & Schlüter, 2013; van Huis, 2013), to the best of the authors’ knowledge, only six studies have reported on vitamin B12 in mealworm, cricket, cockroach or grasshopper (Table 3). Most information is available on mealworm (Tenebrio molitor larvae), as it is the best studied insect species in terms of a novel food source and has long been a focus for protein (Lenaerts et al., 2018; Purschke, Mendez Sanchez, & Jäger, 2018; Yi, Lakemond, Sagis, Eisner-Schadler, van Huis, & van Boekel, 2013) and fat production (Lenaerts et al., 2018; Purschke, Stegmann, Schreiner, & Jäger, 2017). In this study, mealworm was found to contain 1.08 µg/100 g dry mass. Reported values regarding vitamin B12 in mealworm differ greatly, ranging from 0.13 to 6.16 µg/100 g dry mass (Table 3). Jones et al. (1972) reported the highest values, which might be attributed to the presence of pseudovitamin B12, as an MBA was employed. The found value of this study agrees well with the work of Finke ( 2008). However, other studies report values that are significantly lower (0.13–0.56 µg/100 g) than the present study (Finke, 2002, 2013, 2015). A possible explanation for this is that in those studies, mealworms were starved for a certain period (12–24 h), in order to empty their gastrointestinal system prior to vitamin analysis. As insects themselves are not capable of vitamin B12 synthesis, and were exclusively reared on plant material, the only source of vitamin B12 can be their gastrointestinal microbiota, which would explain higher B12 levels in this study. Finally, it is worthy to note that levels of vitamin B12 found in freeze dried mealworms are marginally exceeded compared to recent studies (Lenaerts et al., 2018). Besides the mealworms, crickets (Gryllus assimilis) are the second most studied insect for novel applications as food and feed. The found value for vitamin B12 in crickets was 2.88 µg/100 g dry mass. Reports on the content of vitamin B12 are significantly fewer in number, and range from 5.37 up to 38.1 µg/100 g dry mass of vitamin B12 (Table 3). These values significantly exceed the found content. This might be explained by the fact that despite highly selective sample preparation using immunoaffinity columns, interfering compounds were observed (Fig 3). The possible presence of

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pseudovitamin B12 in the sample is likely, when obtained chromatograms are compared with other studies on pseudovitamin B12 (Chamlagain et al.,

2015). However, final proof would require

identification using mass spectrometry, which unfortunately is poorly compatible with the used solvent modifier (TFA). Nevertheless, it is obvious that false-positive results for vitamin B12 in crickets, even when chromatographic approaches are used, are likely to occur. Finally, it has to be mentioned that reported values for vitamin B12 in crickets exhibit limited comparability, as two of the three studies report on Acheta domesticus (adult) or on nymphs, and not adults of Gryllus assimilis as in the present study. For cockroach (Shelfordella lateralis), the found amount of vitamin B12 was 13.2 µg/100 g dry mass. Only one other study reports on the content of vitamin B12, which clearly exceeds the found value (Table 3). The mismatch of the two values can not only be explained by analytical interferences (Fig. 3), but also by the fact that Finke (2012) studied nymphs of Shelfordella lateralis, which is not in accordance with the present study. With only 0.84 µg/100 g dry mass, grasshoppers showed the lowest content of vitamin B12 among the studied insects. No values for vitamin B12 in grasshopper could be found in the existing literature, making this study the first of its kind. This can be explained by the fact that whole grasshoppers were used as sample material. With characteristic legs and rather large wings, they have a different ratio of extremities to thorax, compared to other studied insect species, which may result in a lower overall content of vitamin B12.

3.4. Occurrence of possible pseudovitamin B12 in edible insects Non-bioactive forms of vitamin B12, so-called pseudovitamin B12, have been reported multiple times over the years (Bito et al., 2016; Chamlagain et al., 2015; Watanabe et al., 1999). The change in the lower ligand from 5,6-dimethylbenzimidazole to adenine makes them biologically inactive for humans and mammals (Taga & Walker, 2008). It is noteworthy that this is not the case for most microorganisms, as they can utilize pseudovitamin B12 as an enzymatic cofactor (Taga & Walker, 2008). Moreover, if the global amount of B12 derivatives is considered, pseudovitamin B12 is most

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likely to represent the bulk component. B12 vitamers accumulate along the food chain in mammals, which have sophisticated mechanisms for their selective uptake and storage (Nielsen et al., 2012). For foodstuffs, this explains why the highest found values are in liver from pig and cow (Souci et al., 2008). Regarding the formation of pseudovitamin B12, it has to be considered that microorganisms are capable of vitamin B12 synthesis only under aerobic conditions. When confronted with limited oxygen supply, such as in the gastrointestinal tract of insects, mostly pseudovitamin B12 is formed (Taga & Walker, 2008). Although no structural confirmation based on mass spectrometry was available at the time of this research, the presence of presumed pseudovitamin B12 in the four studied insect species is likely due to the extreme selectivity of employed immunoaffinity sample preparation. This has several implications: Firstly, MBAs are not suitable assays for the determination of vitamin B12 in insects or insect-derived foods, as they lack sensitivity and may report higher values. Secondly, the impact of further processing on the content of vitamin B12 and possible derivatives has yet to be determined. At least for Europe and the US, it is likely that insects will not be consumed as a whole in the near future, due to consumer acceptance issues (House, 2016; Tan, Fischer, Tinchan, Stieger, Steenbekkers, & van Trijp, 2015). A more realistic scenario is that insects, such as mealworms, will be used in production systems for certain components, such as protein and fat, or may be utilized as feed for animal husbandry. It has yet to be determined where vitamin B12 is located within the individual insect (muscle, gastrointestinal tract) and how processing and extraction technologies can focus on minor nutrients such as vitamin B12. Thirdly, A deeper understanding of the intestinal microbiota and how it is affected by the rearing conditions (e.g., insect feed) is necessary in order to understand and maybe influence the synthesis of B12 vitamers within insects. Thus, further studies are needed, not only to increase analytical certainty but also on processing technologies and rearing strategies, in order to meet the demand for major and minor nutrients of the world population by 2050 and beyond.

4. Conclusion

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In this work, an ultra-high-performance liquid chromatography method, with prior preparative immunoaffinity chromatography, was proposed for the determination of vitamin B 12 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 nutritional sources for vitamin B12. Further studies should employ mass spectrometry in order to confirm presumed pseudovitamin B12. Finally, the composition of the gut microbiota as well as feeding practices and their possible influences on the content of vitamin and pseudovitamin B12 would be of great importance in the utilisation of insects as a novel source of vitamin B12.

Acknowledgement 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.

References AOAC. (2006). Official method 986.23: Cobalamin in milk-based infant formula. AOAC International. Bito, T., Bito, M., Asai, Y., Takenaka, S., Yabuta, Y., Tago, K., Ohnishi, M., Mizoguchi, T., & Watanabe, F. (2016). Characterization and quantitation of vitamin B12 compounds in various Chlorella supplements. Journal of Agricultural and Food Chemistry, 64(45), 8516-8524. Burk, D., & Winzler, R. J. (1943). Heat-labile, avidin-uncombinable, species-specific and other vitamers of biotin. Science, 97(2507), 57-60. Chamlagain, B., Edelmann, M., Kariluoto, S., Ollilainen, V., & Piironen, V. (2015). Ultra-high performance liquid chromatographic and mass spectrometric analysis of active vitamin B12 in cells of Propionibacterium and fermented cereal matrices. Food Chemistry, 166, 630-638. Cohen, J. E. (2003). Human population: The next half century. Science, 302(5648), 1172-1175.

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Council Regulation (2015). (EC) No 258/97. Council Regulation (2018). (EC) No 2015/2283. Fang, H., Kang, J., & Zhang, D. (2017). Microbial production of vitamin B12: a review and future perspectives. Microbial Cell Factories, 16(1), 15. Finke, M. D. (2008). Nutrient Content of Insects. In J. L. Capinera (Ed.), Encyclopedia of Entomology 2nd ed., vol. 3 (pp. 2623-2646): Springer. Finke, M. D. (2002). Complete nutrient composition of commercially raised invertebrates used as food for insectivores. Zoo Biology, 21(3), 269-285. Finke, M. D. (2013). Complete nutrient content of four species of feeder insects. Zoo Biology, 32(1), 27-36. Finke, M. D. (2015). Complete nutrient content of four species of commercially available feeder insects fed enhanced diets during growth. Zoo Biology, 34(6), 554-564. Frenkel, E. P., Kitchens, R. L., & Prough, R. (1979). High-performance liquid chromatographic separation of cobalamins. Journal of Chromatography A, 174(2), 393-400. Froese, D. S., & Gravel, R. A. (2010). Genetic disorders of vitamin B12 metabolism: Eight complementation groups - Eight genes. Expert Reviews in Molecular Medicine, 12. House, J. (2016). Consumer acceptance of insect-based foods in the Netherlands: Academic and commercial implications. Appetite, 107, 47-58. ICH. (2005). Harmonised tripartite guideline: Validation of analyctical procedures: Text and Methodology (Q2)R1. Jones, L. D., Cooper, R. W., & Harding, R. S. (1972). Composition of mealworm Tenebrio molitor larvae. The Journal of Zoo Animal Medicine, 3(4), 34-41. Kelly, R. J., Gruner, T. M., Furlong, J. M., & Sykes, A. R. (2006). Analysis of corrinoids in ovine tissues. Biomedical Chromatography, 20(8), 806-814. Lenaerts, S., Van Der Borght, M., Callens, A., & Van Campenhout, L. (2018). Suitability of microwave drying for mealworms (Tenebrio molitor) as alternative to freeze drying: Impact on nutritional quality and colour. Food Chemistry, 254, 129-136. Nakos, M., Pepelanova, I., Beutel, S., Krings, U., Berger, R. G., & Scheper, T. (2017). Isolation and analysis of vitamin B12 from plant samples. Food Chemistry, 216, 301-308. Nielsen, M. J., Rasmussen, M. R., Andersen, C. B. F., Nexø, E., & Moestrup, S. K. (2012). Vitamin B12 transport from food to the body's cells - A sophisticated, multistep pathway. Nature Reviews Gastroenterology and Hepatology, 9(6), 345-354. Nowak, V., Persijn, D., Rittenschober, D., & Charrondiere, U. R. (2016). Review of food composition data for edible insects. Food Chemistry, 193, 39-46.

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Owen, S. C., Lee, M., & Grissom, C. B. (2011). Ultra-performance liquid chromatography separation and mass spectrometric quantitiation of physiologica cobalamins. Journal of Chromatographic Science, 49, 228-233 Purschke, B., Mendez Sanchez, Y. D., & Jäger, H. (2018). Centrifugal fractionation of mealworm larvae (Tenebrio molitor, L.) for protein recovery and concentration. LWT - Food Science and Technology, 89, 224-228. Purschke, B., Stegmann, T., Schreiner, M., & Jäger, H. (2017). Pilot-scale supercritical CO2 extraction of edible insect oil from Tenebrio molitor larvae – Influence of extraction conditions on kinetics, defatting performance and compositional properties. European Journal of Lipid Science and Technology, 119(2). Rumpold, B. A., & Schlüter, O. K. (2013). Nutritional composition and safety aspects of edible insects. Molecular Nutrition and Food Research, 57(5), 802-823. Santos, F., Vera, J. L., Lamosa, P., de Valdez, G. F., de Vos, W. M., Santos, H., Sesma, F., & Hugenholtz, J. (2007). Pseudovitamin B12 is the corrinoid produced by Lactobacillus reuteri CRL1098 under anaerobic conditions. FEBS Letters, 581(25), 4865-4870. Schimpf, K., Spiegel, R., Thompson, L., & Dowell, D. (2012). Determination of vitamin B12 in infant formula and adult nutritionals by HPLC: First action 2011.10. Journal of AOAC International, 95(2), 313-318. Souci, S. W., Fachmann, W., & Kraut, H. (2008). Food composition and nutrition tables (7th ed.). Boca Raton: Taylor & Francis Group. Szterk, A., Roszko, M., Małek, K., Czerwonka, M., & Waszkiewicz-Robak, B. (2012). Application of the SPE reversed phase HPLC/MS technique to determine vitamin B12 bio-active forms in beef. Meat Science, 91(4), 408-413. Taga, M. E., & Walker, G. C. (2008). Pseudo-B12 joins the cofactor family. Journal of Bacteriology, 190(4), 1157-1159. Tan, H. S. G., Fischer, A. R. H., Tinchan, P., Stieger, M., Steenbekkers, L. P. A., & van Trijp, H. C. M. (2015). Insects as food: Exploring cultural exposure and individual experience as determinants of acceptance. Food Quality and Preference, 42, 78-89. van Huis, A. (2013). Edible insects : future prospects for food and feed security. Rome: Food and Agriculture Organization of the United Nations. van Huis, A., & Oonincx, D. G. A. B. (2017). The environmental sustainability of insects as food and feed. A review. Agronomy for Sustainable Development, 37(5). Viñas, P., López-Erroz, C., Balsalobre, N., & Hernández-Córdoba, M. (2003). Speciation of cobalamins in biological samples using liquid chromatography with diode-array detection. Chromatographia, 58(1-2), 5-10.

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Watanabe, F., Katsura, H., Takenaka, S., Fujita, T., Abe, K., Tamura, Y., Nakatsuka, T., & Nakano, Y. (1999). Pseudovitamin B12 is the predominant cobamide of an algal health food, Spirulina tablets. Journal of Agricultural and Food Chemistry, 47(11), 4736-4741. Wilkinson, K., Muhlhausler, B., Motley, C., Crump, A., Bray, H., & Ankeny, R. (2018). Australian consumers’ awareness and acceptance of insects as food. Insects, 9(2). Yi, L., Lakemond, C. M. M., Sagis, L. M. C., Eisner-Schadler, V., van Huis, A., & van Boekel, M. A. J. S. (2013). Extraction and characterisation of protein fractions from five insect species. Food Chemistry, 141(4), 3341-3348. Zironi, E., Gazzotti, T., Barbarossa, A., Devicienti, C., Scardilli, M., & Pagliuca, G. (2013). Technical note: Development and validation of a method using ultra performance liquid chromatography coupled with tandem mass spectrometry for determination of vitamin B12 concentrations in milk and dairy products. Journal of Dairy Science, 96(5), 2832-2836.

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Fig. 1. UHPLC separation of four B12 vitamers, hydroxocobalamin (HOCbl, 50nM, 348nm), cyanocobalamin (CNCbl, 200 nM, 361 nm), 5´-adenosylcobalamin (AdoCbl, 200 nM, 262 nm) and methylcobalamin (CH3Cbl, 400 nM, 254 nm)

Fig. 2. A: Comparison of the certified value and result for the determination of vitamin B12 (n = 6) using the proposed method on certified reference material (dried milk powder, ERM®-BD600); B: Corresponding chromatogram of cyanocobalamin (CNCbl) in certified reference material.

Fig. 3. Chromatograms of CNCbl and Pseudovitamin B12 at 361 nm: 1 mealworm (Tenebrio molitor larvae), 2 cockroach (Shelfordella lateralis), 3 cricket (Gryllus assimilis), and 4 grasshopper (Locusta migratoria).

18

Fig. 1

19

Fig 2 B12

0,01672

A

B

0,01584 0,01496 0,01408

CNCbl

0,01320 0,01232 0,01144 0,01056 0,00968 0,00880

AU

0,00792 0,00704 0,00616 0,00528 0,00440 0,00352 0,00264

found

certified

0,00176 0,00088 0,00000 -0,00088 -0,00176 -0,00264 1,00

1,20

1,40

1,60

1,80

2,00 Minutes

2,20

2,40

2,60

2,80

3,00

ERM-Referenz BD600 0386 II; Date Acquired: 14.06.2017 16:41:21 CEST; Vial: 1:B,2; Inj #: 1; Channel: **** SampleName ERM-Referenz BD600 0386 II Date Acquired 14.06.2017 16:41:21 CEST

Reported by User: System Report Method: B12 Report Method ID:11521 11521

Project Name:

Vitamin B12 Date Printed: 26.07.2017 16:38:24 Europe/Vienna

20

presumed Pseudovitamin B12

CNCbl CNCbl CNCbl CNCbl

21

Table 1: Data for calibration and chromatographic method validation Vitamer

k´

Range Levels

R²

(µM)

LOD LOQ

Precision (%)

(nM) (nM)

Repeatability

Intermediate precision

Low Medium High Low Medium High HOCbl

5.55

2.5–

10

0.990

0.12

0.40

2.0

0.6

0.9

3.0

1.3

0.7

10

0.999

0.26

0.85

1.0

1.9

4.5

2.7

2.2

5.5

6

0.947

1.61

5.34

1.3

1.3

2.1

1.9

1.0

5.0

5

0.967

1.45

4.82

0.5

1.7

3.9

7.5

2.1

5.6

600 CNCbl

6.74

2.5– 600

AdoCbl

8.13

10– 600

CH3Cbl

9.12

10– 400

Retention factor (k´) based on t0 = 0.25 min, repeatability over one day (n = 6), intermediate precision over four days (n = 3 per day) at low = 10 nM, medium = 50 nM, high = 400 nM for hydroxocobalamin (OHCbl); low = 5 nM, medium = 50 nM, high = 500 nM for cyanocobalamin (CNCbl); low = 5 nM, medium = 50 nM, high = 500 nM for 5´adenosylcobalamin (AdoCbl); and low = 5 nM, medium = 50 nM, high = 500 nM for methylcobalamin (CH3Cbl).

22

Table 2: Results of the determination of vitamin B12 in studied insect species and recovery experiment (based on hydroxocobalamin) Insect

found

CV

Recovery [%]

µg/100g

%

Level 1

Level 2

Mealworm

1.08 ± 0.03

2.5

97.6 ± 4.9

92.1 ± 6.1

Grashopper

0.84 ± 0.06

7.6

93.6 ± 7.1

89.1 ± 3.6

Cricket

2.88 ± 0.17

5.7

92.2 ± 8.4

91.2 ± 8.9

13.21 ± 0.68

5.1

101.4 ± 2.8

Cockroach

104.0 ± 12.6

Coefficient of variation (CV) n = 6 (2 days, n = 3 each day); Level 1: +50%, Level 2: +100%

23

Table 3: Results of vitamin B12 determination in comparison to other studies Vitamin B12 [µg/100 g dry weight] Reference

Mealworm

Cricket (adult)

Cockroach (adult)

Grasshopper

(larvae)

Gryllus assimilis

Shelfordella

(adult)

lateralis

Locusta

Tenebrio molitor Present study

1.08

migratoria 2.88

13.2

0.84

0.311–0.852

-

-

-

Finke, 2015

0.13

19.3

-

-

Finke, 2013

-

-

23.77

-

Finke, 2008

1.23–1.543

17.44–38.15

-

-

Finke, 2002

0.47

5.374

-

-

0.136

-

-

-

0.567

-

-

-

6.16

-

-

-

Lenaerts

et

al.,

2018

Jones et al., 1972 1

lowest reported value, depending on employed drying technology, 2freeze dried, 3 adult, 4house cricket (adult)

Acheta domesticus, 5house cricket (nymph) Acheta domesticus, 6giant mealworm (larvae), 7nymph

24

25

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

26