Species identification of culinary spices with two-locus DNA barcoding

Highlights

• We evaluated five DNA barcodes for efficient identification of 19 culinary spices and three common crop-based adulterants.

• On their own, barcodes were insufficient to definitively identify spice types.

• Combining ITS2 with psbA-trnH considerably improved spice identification.

• All spices, except the Cambodian cardamom, were identified using this combination.

• We detected adulteration (6.7%) in 30 commercial products.

Abstract

Culinary spices are widely used as both condiments and food. However, some cheaper species with similar morphological characteristics are utilized as substitutes or additives, causing issues with quality control and product safety. This study aimed to develop a DNA barcoding method for the species identification of culinary spices. We evaluated the ability of five barcodes (ITS2, rbcL, trnL (UAA), trnL (P6 Loop), and psbA-trnH) to detect 19 culinary spices. The reliability of barcodes for distinguishing species was assessed using PCR amplification and sequencing, genetic distance, barcoding gap, neighbor-joining phylogenetic tree, and BLAST analyses. When the five barcodes were used independently, the 19 types of culinary spices and three common crop-based adulterants could not be definitively identified simultaneously. The ITS2 region, followed by the psbA-trnH, trnL (UAA), and rbcL regions, had intra- (0.0099) and inter- (0.8955) specific divergences, a barcoding gap, and an 86.7% species resolution rate. The combination of ITS2 and psbA-trnH barcodes improved the species identification ability (95.5%). Except for the Cambodian cardamom, all the tested culinary spices were successfully identified using the ITS2 + psbA-trnH barcode group. Utilizing the ITS2 + psbA-trnH barcode, we tested the authenticity of 30 commercial products. The recovery rate of DNA barcodes from culinary spice products was 100.0%, with 93.3% of the tested products being authentic, and 6.7% indicating adulteration with Oryza sativa. Our findings provide an effective tool for culinary spice identification that is easy, accurate, and quick. This method can be used to standardize labels on commercial culinary spices.

Introduction

Culinary spices are derived from dried plants that contain aromatic flavors or volatile oils and include flowers and buds (cloves), bark (cinnamon), rhizomes (ginger), fruits (pepper, cumin), and seeds (cardamom, coriander seeds) (Shelef, 1984; Siruguri & Bhat, 2015). They not only enhance food flavor, aroma, and color but may also protect consumers from acute, chronic, and non-communicable diseases according to traditional medicine approaches (Jiang, 2019). Culinary spices are extensively consumed worldwide, with the global spice and seasoning market projected to reach $21.3 billion in 2021 (Ford, Berger, & Jackoway, 2022). Generally, culinary spices are sold in broken or ground form, which alters their original appearance and increases the risk of adulteration of natural culinary spices and their derivatives (Lakner, Szabó, Szűcs, & Székács, 2018). Moreover, long and complex supply chains present many opportunities for economically motivated adulteration (EMA) (Kaavya et al., 2020). Some unscrupulous traders intentionally substitute or add cheaper substances with similar physical properties for high economic incomes (Barbosa, Nogueira, Gadanho, & Chaves, 2019; Ford & Embuscado, 2019; Zhang et al., 2019).

A survey showed that from 2015 to 2020, herbs and spices adulteration accounted for 2.0% of 4375 food fraud cases, including food allergen-related cases, as recorded by MedISys-FF (Marvin et al., 2022). As a common food allergen, the presence of peanuts in cumin powder poses a significant threat to the health of consumers allergic to peanut (Lima et al., 2020). Food anaphylaxis directories in the US reported that peanuts adulteration caused 59% of 63 anaphylaxis-related deaths between 2001 and 2007 (Uncu & Uncu, 2020). The US Food and Drug Administration (FDA) issued 20 recall notifications for “Cumin Powder” in 2014 and 2015 due to the detection of undeclared peanuts in 100 different products, (Moyer, DeVries, & Spink, 2017; Silvis, van Ruth, van der Fels-Klerx, & Luning, 2017). The same situation exists in China. Li, Cheng, Luo, Li, and Wu (2022) investigated food fraud news reported from 2001 to 2019, and found that condiments and culinary spices accounted for 3.5% of these cases in China. The adulteration of culinary spice violates the legitimate rights and interests of consumers. Further, it poses a serious threat to public health (Negi, Pare, & Meenatchi, 2021). Therefore, the development of detection methods for culinary spice testing is of major relevance for regulatory authorities, enterprises, and consumers.

Culinary spices can be distinguished based on morphological, microscopic, physical, and chemical indicators (Zhang et al., 2019). Once culinary spice products have been processed, the morphological and microscopic identification cannot be performed owing to the destruction of external and internal appearance (Sunil Kumar, 2013). Physical and chemical methods are mainly based on the chemometric characteristics of polysaccharides, fats, metal elements, and other components present in culinary spices, which are easily affected by external conditions such as geography, climate, and processing (Reinholds, Bartkevics, Silvis, van Ruth, & Esslinger, 2015). DNA-based methods, such as polymerase chain reaction (PCR) (Sousa, Ferreira, & Faria, 2019), random amplified polymorphic DNA (RAPD) (Corcolon, Laurena, & Dionisio-Sese, 2015), and sequence characterized amplified region (SCAR) (Bansal, Thakur, Mangal, Mangal, & Gupta, 2019), provide cheap, accurate, and reproducible means of fraud detection (Galvin-King, Haughey, & Elliott, 2018), having been rapidly developed and widely adopted for the identification of species present in culinary spices. However, most methods typically detect a single species of interest. Hebert (Pennisi, 2019) was the first to propose DNA barcodes, which do not require prior knowledge of the sample composition. This approach utilizes one standard and relatively short segment of DNA as a genetic marker to identify the species present by comparing genetic relationships through phylogenetic analysis (Hebert, Cywinska, Ball, & DeWaard, 2003). The selection of a barcode locus is a significant challenge in DNA barcoding (Kress, Wurdack, Zimmer, Weigt, & Janzen, 2005). The Plant Working Group of the Consortium for the Barcode of Life (CBOL) suggests using ribulose 1,5-bisphosphate carboxylase (rbcL) and maturase K (matK) as core plant barcodes, with tRNA His-photosystem II protein D1 (trnH-psbA) and internal transcribed spacer (ITS) as supplementary barcodes (Hollingsworth et al., 2009). However, the universal plant barcode remains debatable because of the widespread occurrence of plant hybridization among terrestrial plants (Gogoi, Wann, & Saikia, 2020; Gu, Yang, Chen, & Chen, 2020).

DNA barcoding has been successfully used as an efficient and inexpensive method for identifying culinary spices. Parvathy et al. (2014) compared 3 loci, trnH-psbA, rbcL and ribonucleicacid polymerise C1 subunit (rpoC1), to authenticate chili in traded black pepper powder and found that the trnH-psbA spacer could be a preferred barcode, which was still valid at a 0.5% adulteration ratio. Subsequently, they discriminated turmeric, nutmeg, and cinnamon from their adulterants using DNA barcoding (Parvathy, Swetha, Sheeja, & Sasikumar, 2015; Swetha, Parvathy, Sheeja & Sasikumar, 2014, 2017). As a potential DNA barcode, the ITS gene, particularly ITS2 gene, has been employed as part of multi-locus DNA barcodes (Fu et al., 2016). Zhang et al. (2019) demonstrated that ITS2 and psbA-trnH could be used to effectively identify 16 kinds of spices and their adulterants, revealing a certain degree of adulteration in commercial culinary spices, whereas lower PCR amplification of villosum and nutmeg resulted in unsuccessful identification.

This study examined the authenticity of nineteen different culinary spices. By comparing the identification abilities of five different barcodes (ITS2, rbcL, trnL (UAA), trnL (P6 Loop), and psbA-trnH), we selected the optimal barcodes, and established a DNA barcoding method for culinary spice identification and used it to identify market samples based on the selected DNA markers.