Authentication of leguminous-based products by targeted biomarkers using high resolution time of flight mass spectrometry

Highlights

• New biomarkers for food authentication by high-resolution mass spectrometry (HRMS).

• Simultaneous identification/quantification of legumes, meat and honey by LC-MS/MS.

• The method is independent from the kind of processed food matrix.

• The method could be used to prove food labeling of vegetarian/vegan food products.

• Reproducible recovery rates of 70–113% and LOQ of 10–50 ppm were achieved.

Abstract

A growing number of health-conscious individuals supplements their diet with protein-rich plant-based products to reduce their meat consumption. Analytical methods are needed to authenticate these new vegetarian products not only for the correct labelling of ingredients according to European legislation but also to discourage food fraud. This paper presents new biomarkers for a targeted proteomics LC-MS/MS work-flow that can simultaneously prove the presence/absence of garden pea, a protein-rich legume, meat and honey and quantify their content in processed vegan food. We show a novel rapid strategy to identify biomarkers for species authentication and the steps for the multi-parameter LC-MS/MS method validation and quantification. A high resolution triple time of flight mass spectrometer (HRMS) with SWATH Acquisition was used for the rapid discovery of all measurable trypsin-digested proteins in the individual ingredients. From these proteins, species-selective biomarkers were identified with BLAST and Skyline. Vicilin and convicilin (UniProt: D3VND9, Q9M3X6) allow pea authentication with regard to other legume species. Myostatin (UniProt: O18831) is a single biomarker for all meat types. For honey, we identified three selective proteins (UniProt: C6K481, C6K482, Q3L6329). The final LC-MS/MS method can identity and quantify these markers simultaneously. Quantification occurs via external matrix calibration.

Introduction

The world population is expected to grow from 6.9 billion in 2010 to 9.5 billion by 2050 (United Nations Department of Economic and Social Affairs, 2012). To provide enough food in the form of the essential amino acids only found in proteins, protein-rich plant sources, such as soy and other leguminous seeds but also algae or insect proteins, offer new opportunities and are already a growing supplement option in the human diet (Petrusán et al., 2016, van der Spiegel et al., 2013). For instance, in 2015, there was a 26% growth for vegetarian and vegan products in German retail (IFH, 2016). The estimated annual growth rate for the global market value of these protein-rich sources is 6% in the next five years, reaching a value of $58.49 billion by 2022 (Markets, 2017).

The strong interest in products based on plant proteins is ascribable to consumers who reduce meat consumption for sustainability and health reasons. The reported health benefits of such a diet for metabolic health, blood pressure and reduced risk of type 2 diabetes (Derbyshire, 2016, Kim and Bae, 2015, Rodenas et al., 2011, Turner-McGrievy et al., 2015) will likely increase the number of consumers for plant-based products. The so-called “flexitarians”, including vegetarian and vegan consumers, represent currently 29% and 37% of consumers in Britain and Germany respectively (Derbyshire, 2016, GfK, 2016). Flexitarians primarily focus their buying decisions on nutritional and health relevance and the sustainability of the food supply but also on the transparency of labelling (authenticity of ingredients, regional origin) and on allergen, GMO and synthetic additive free products (Cavaliere et al., 2015, Comission Regulation (EU) No 432/2012, 2012, Regulation (EC) No 1924/2006, 2006).

In April 2016, a first unified legal definition of the terms “vegan” and “vegetarian” for the labelling of food was introduced in Germany in reaction to the demand of clear labelling practices from consumer interest groups and the food industry (European Vegetarian Union, 2016). This definition will be effective until the European Commission issues standardized regulations as stipulated in Article 36(3) (b) of (Regulation (EU) No 1169/2011, 2011). The new definition has two main objectives: (1) absence of meat and (2) description of improved quality attributes as compared to animal-based products.

The product labelling needs to be combined with an authentication analysis of plant ingredients and additives to increase consumer confidence and to prevent false labelling and adulteration (Primrose et al., 2010, Regulation (EU) 2017/625, 2017). The Grocery Manufacturers Association of America reported that up to 10% of the food in the developed world and 20% in the developing world is affected by food fraud (Johnson, 2014). Therefore, food manufactures are required to provide and confirm the authenticity of vegan/vegetarian products with respect to the regulation (EU) No. 1169/2011, (EU) No. 178/2002 and the USA FDA Food Safety Modernization Act signed on January 4, 2011. Reliable analytical methods are thus needed to check the suitability of vegetarian/vegan products as well as the authenticity and quality of ingredients.

Different analytical techniques can test authenticity and detect adulterations of food ingredients, f.i. measurement ratios of stable isotopes (mostly ¹³C/¹²C), spectroscopic, chromatographic, molecular biological, and electrochemical methods. Molecular biological methods such as real-time PCR have been used to identify and quantify food, for example meat species (Laube et al., 2007, Montowska et al., 2015). A broad variety of analytical methods tried to authenticate honey (for a review see (Trifkovic, Andric, Ristivojevic, Guzelmeric, & Yesilada, 2017)). However, some methods are not suitable for processed food, because processing especially thermal treatment leads to a change in the structure and properties of the targeted analytes. For instance, DNA can undergo heat denaturation and therefore a standard PCR analysis that produces copies of DNA fragments is likely to over- or underestimate the targeted molecular species. Processing also introduces new structures into food, a problem for metabolomics analytical strategies.

The recent developments in proteomics and mass spectrometry (MS) offer new opportunities for the authentication of food ingredients due to the fast and easy sample preparation, high throughput processing, the incorporation of post-translational and processing-dependent modifications and quantification of analytes. Processing also effects the protein structure and properties, but a well-defined proteomics strategy can analyze processed products, when it utilizes thermal stable parts/fragments of the targeted protein during the analysis. MS-based protein biomarkers have successfully been used to differentiate and authenticate ingredients and food additives (Garcia-Canas et al., 2012, Huschek et al., 2016, Orduna et al., 2015, Primrose et al., 2010). For example, for meat species and meat products, an authentication based on a peptide biomarker analysis protocol has recently been published (Montowska et al., 2015). Similarly, for cereal-based baked products, an analytical method for the differentiation of cereals species has been developed (Bönick, Huschek, & Rawel, 2017).

We add to this field of authentication in proteomics by focusing on the market for plant-based protein–rich processed foods that should be free of meat and/or honey (for vegan products). We developed a targeted-proteomics LC/MS/MS multi-parameter method that can simultaneously detect the presence/absence of garden pea, meat and honey in processed plant-based products and quantify these ingredients. Another novelty aspect addressed is the new biomarkers for these food ingredients that were derived by employing an innovative rapid identification of potential species-selective biomarkers from trypsin-digested proteins using high-resolution mass spectrometry (HRMS) in combination with bioinformatics tools.