Game meat and high-resolution magic angle spinning nuclear magnetic resonance spectroscopy: a traditional foodstuff versus a novel analysis technology

- The inclusion of game meat could diversify the meat market; hence, carcass merit and meat quality parameters must be standardized to ensure consumers’ requirements. - Enhancing both consistency and accuracy on game meat quality parameters implies the implementation of new technologies. - The metabolic profile of game meat obtained by High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR) Spectroscopy together with multivariate analysis is a powerful tool to characterize game meats according to the species of origin. Bats, lizards, snakes, alligators, rats, turtles, etc. are a variety of wildlife species that remain a cheap source of protein, particularly for the population in the developing world, with the correspondent food security challenges (Hoffman and Cawthorn, 2012). It has been estimated that, in the developed world, only 5%–10% of the population consumes game meat. In Europe, consumers consider game meat as innovative food, natural and sustainable, and positively respond to an increase in market availability (Soriano and Sánchez-García, 2021). In North America, although the higher nutritional value of game meat is highly valued by consumers, its market availability is still defined by direct purchases from ranches and/or farms (Hedman et al., 2020). More information regarding the relationship between game meat and consumers’ perceptions and attitudes and the correlation with age, gender, socioeconomic factors, etc. was described by Corradini et al. (2022). Consequently, the implementation of standardized procedures in the field and cutting plant to ensure safety, quality, and traceability should be considered. In addition, authentication is necessary to avoid fraudulent practices derived from the substitution of the most valued species for those of lesser commercial and organoleptic value. Likewise, species identification is important to verify compliance with the prohibitions and bans established in hunting laws. Since game animal carcass and game meat quality parameters are yet to be standardized, the development of rapid and noninvasive technologies for carcass merit and meat quality evaluation should be considered a pending work in progress. Metabolomics is a branch of the omics sciences that investigates a huge range of small molecules in an organism, such as organic acids, amino acids, carbohydrates, lipids, etc. Nuclear Magnetic Resonance (NMR) has recently grown in popularity as one of the most used analytical nondestructive techniques, since it enables the detection and measurement of metabolic components in biological matrices with concentrations equal to or greater than 1 µM (Tilgner et al., 2019). It must be mentioned that any biological process is defined not by one but by several biomarkers. Therefore, statistical methods are typically used in conjunction with NMR analyses. The traditional classification of game/hunting species differentiates between “large and small” and “fur and feathered” game animals. For example, wild boar and roe deer would be large-furred game animals, while hare and rabbit would be small-furred ones. In addition, considering feather game, animals could be classified depending on their habitat: land (partridge, pigeon, quail, and woodcock), mountain, and water (wild duck). In addition, a classification based on the type of animal husbandry is also possible (Soriano and Sánchez-García, 2021). On one hand, “wild game” considers the animals that live in the wild, including mammals that live in enclosed areas in conditions of freedom similar to those of wild game animals, including ungulates (wild boar, deer), wild lagomorphs (rabbits, hares), rodents, and wild birds (pheasant, duck, partridge, quail). On the other hand, “farmed game” considers animals raised in controlled conditions including farmed ratites (ostriches, emu) and farmed land mammals (cattle, goats, sheep, horses) (Regulation (EU) 2021/1374). Soriano and Sánchez-García (2021) compared livestock to the most consumed species of wild game meat (red and fallow deer, wild boar, hare, and wild rabbit) from Central and Mediterranean Europe and found that, in general, the latter showed a trend to a higher protein percentage and a beneficial composition of essential amino acids, a lower fat content (<3%–4%), and a limited saturated fat content and a higher proportion of polyunsaturated fatty acids, especially omega 3. Wild game meat also showed high concentrations of Zn and the bioavailable form of Fe, together with optimal amounts of vitamin E and vitamins of the B group. NMR spectroscopy is one of the main technologies used in metabolomics and has been utilized for decades in food science and nutritional investigations. NMR is relatively less sensitive than mass spectrometry (MS) but an alternative for the analysis of metabolites that are unstable and/or sensitive to aggressive conditions such as high voltage in MS. Limited spectral resolution, difficulties in measuring lipids and high-field instrument cost are drawbacks that have to be still addressed. However, numerous characteristics of NMR including its high reproducibility and quantitative abilities (one single internal reference is enough for quantitation of all detected metabolites), its nonselective and noninvasive nature, and the ability to identify unknown metabolites in complex mixtures, among others, offer numerous benefits to the metabolomics field (Nagana Gowda & Raftery, 2021; Sobolev et al., 2022). Molecular mobility is needed in NMR, thus most of the analyses are carried out on liquid samples. High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR) spectroscopy allows the application of NMR spectroscopy on semisolid and solid materials, a much-needed approach, since a lot of foods are solid or semisolid, with a similar resolution. This technique consists of a rapid spinning (4–6 kHz) of the sample at an angle of 54.7° relative to the applied magnetic field. This angle called the magic angle cancels the term (3 cos2 θ–1), present in the first-order term of the Hamiltonian of the chemical shift anisotropy associated with semisolid samples and the dipolar interaction, removes the susceptibility broadening effects, and leaves spectra with linewidths comparable to the ones obtained with liquid samples (Jensen & Bertram, 2019). One of the first applications of HR-MAS to meat was developed in 2002. The authors described the meat molecular profile from fresh minced beef, including various amino acids, glucose, fatty acids, lactate, creatinine, and carnosine. Both water-soluble and fat-soluble components were detected, with a better performance of HR-MAS in terms of sensitivity than traditional NMR technology (Brescia et al., 2002). Over the next decade, the application of HR-MAS was described to recognize and differentiate lamb and beef meat according to the breed, geographical origin, feeding regime, etc. together with the metabolic profile of meat of young bulls of buffalo Chianina, Maremmana, and Holstein-Friesian breeding varieties and the meat of the purebred Garganica goat (Mazzei et al., 2018). Recently, Castejón et al. (2015) demonstrated that meat exudates may advantageously represent the molecular composition of the corresponding meat. 1H spectra of both tenderloin beef meat and its exudates, as by HR-MAS and traditional liquid-state NMR, respectively, appeared strongly correlated to each other, except for the liposoluble components, which, as expected, were detected only by HR-MAS. Because an increase in muscle dystrophy in chickens was observed in the last decade, Sundekilde et al. (2017) used HR-MAS to evaluate the pectoralis major meat samples deriving from either healthy or dystrophic chickens and identified that carnosine and anserine were downregulated in the dystrophic muscles. In 2018, García-García et al. developed the first complete metabolic profile of dry-cured fermented sausages and studied variations in the concentration of metabolites involved in ripening. The authors demonstrated that HR-MAS can be very useful in evaluating the metabolome variations as a function of processing conditions. Recent research of our research group has been focused on the fact that game meat is often a target for fraudulent labelling as a result of the high commercial value associated with its products. In a recent study carried out by our research group, the species Roe deer (Capreolus capreolus), European deer (Cervus elephus), Wood pigeon (Columba palumbus), Iberian hare (Lepus granatensis), European rabbit (Oryctolagus cuniculus), and Red-legged partridge (Alectoris rufa) were considered. HR-MAS NMR spectroscopy was performed at 500.13 MHz using a Bruker AVIII500 HD spectrometer 11.7 T. The samples were placed within a 50 μL zirconium oxide rotor with a cylindrical insert and spun at 5 kHz. In the first approach, all the game meats studied presented the same types or families of metabolites, including different amino acids, dipeptides, carbohydrates, fatty acids, organic acids and nucleotides, as already reported in meat from slaughter animals (Ritota et al., 2012; Castejón et al., 2015). Despite this similarity, substantial quantitative differences were observed, resulting in characteristic metabolomics profiles for each game meat. For example, Figure 1 shows 1H-NMR spectra from Roe deer and Red-legged partridge and differences in both the number of signals and the signal intensity are shown. Examples of 1H-NMR spectra corresponding to Cervus elephus (upper) and Alectoris rufa (lower). To carry out a precise elucidation of the differences associated with the meat of each of the different species, a Principal Component (PC) Analysis was used, an unsupervised multivariate statistical analysis that constitutes a transformation to a new coordinate system in which the maximum variance of the data matrix is sought. The largest variance is explained on the first axis corresponding to the first principal component, PC1, the second largest on the second axis, PC2, and so on. As shown in Figure 2 (PC1 vs. PC2, explain 75% and 10% of the total variance, respectively), there was a grouping of the samples analyzed in different clusters depending on the species. Clustering of samples according to species by PC analysis of the metabolic profile obtained by HR-MAS. As feature knowledge and summarizing, quantitative differences in the concentrations of the dipeptides anserine and carnosine, the amino acids histidine, taurine and glycine, the amino acid derivative carnitine, and together with glycerol and glucose defined the species differentiation. It is concluded that the HR-MAS NMR spectroscopy together with multivariate analysis is a powerful tool to be used for the characterization of game meats according to the species of origin. Nevertheless, a variety of specific applications in the game meat field may be waiting to be implemented. For example, the discrimination of cuts of different commercial value to protect high-quality products and the development of a quality control system. Furthermore, information regarding the application of HR-MAS to provide knowledge about directly related metabolites to taste, sensory, and texture properties is scarce. José Segura Plaza joined the Department of Food Technology (Complutense University of Madrid, UCM) as an Assistant professor in September 2022 and currently works on the application of NRM and computer vision systems (CVS), as non-destructive technologies, on food matrices. He has more than 12 years of experience in meat science. From 2019 to 2022, he worked at the Agriculture and Agri-Food Canada Lacombe Research and Development Centre in the Canadian Meat Science Program - Livestock Carcass Merit and Market Competitiveness. Throughout these 42 months, he published several studies on the applicability of video image analysis (VIA) and/or dual-energy x-ray absorptiometry (DEXA) for the evaluation of beef carcass merit. Corresponding author:josesegu@ucm.es Víctor Remiro Yagüe is currently working on his PhD in the Department of Food Technology (Complutense University of Madrid, UCM) on the application of non-destructive methods based on nuclear magnetic resonance (NMR) for the rapid analysis of food matrices. He has a degree in Food Science and Technology from the University of Zaragoza and holds a Master's Degree in Food Engineering applied to Health from the Polytechnic University of Madrid. Throughout this time, his work and studies have been closely related to meat science. M. Dolores Romero-de-Ávila Hidalgo works as an Associate Professor in the Department of Food Technology in the Veterinary Faculty at the Complutense University of Madrid (UCM). She is a member of the UCM research group Animal Food Technology, TECNOLALIMA. Her research is mainly related to the composition, structure, and quality of food products of animal origin. Initially, studying the composition and sensory and textural properties of meat products and in recent years, working on the application of different nuclear magnetic resonance techniques to study structural changes associated with the production process and quality of final product, in different food matrices. During her PhD, she researched about conditions and viability to use different cold binding agents for use in fresh in the preparation of boneless dry-cured ham. Palmira Villa Valverde holds a degree in Physics from the University of Cantabria and a PhD in Telecommunication Engineering from the Polytechnic University of Madrid. She has been working since 1993 at the Nuclear Magnetic Resonance (NMR) research assistance centre of the Complutense University of Madrid, which has recently been incorporated into the map of Research Infrastructures, specifically the Complutense Bioimagen Unit (BioImac). Palmira’s research focuses on Metabolomics using NMR, in both biological and food samples. This research is carried out on both liquid and semi-solid samples using the High-Resolution Magic Angle Spinning technique. In addition to Metabolomics, her research interest also includes the design of coils for Magnetic Resonance Imaging scanners, the subject on which she completed her PhD David Castejón Ferrer is Research Assistant at the Complutense University. He has a PhD “Cum Laude” in Analytical Chemistry from Complutense University (2015) and a degree in Chemical Sciences from the University of Vigo (2001). He has performed research stages at the University College London and the Friedrich Schiller University Jena. He has worked in the Analytical Department at GlaxoSmithKline performing NMR structural elucidation of new antimalarial drugs. Since 2004 he has been a member of the Bio-NMR Facility of Complutense University working with both MRI and Spectroscopy. He focuses his research work on the development of new advances and applications of NMR. He is currently part of the research team of the research project Strategies and methodologies for the development, characterization, and control of challenges currently faced by the meat sector and the teaching innovation project Application of MRI in the neurological diagnosis of exotic animals. Carlos Santos Arnaiz is an Assistant Professor at the Food Technology Department of the Complutense University of Madrid (Spain). His research is focused on the analysis of food using nuclear magnetic resonance (NMR), specifically its relationship with the different stages of the production process of fish and pork products. In addition, Carlos has ongoing collaborations with the industry related to eggshell rheological properties and the profile of fatty acids and organic volatile compounds of meat from poultry fed different diets, dry-cured ham, cooked ham and other meat products. M. Isabel Cambero Rodríguez is a Full Professor of Food Technology at the Complutense University (UCM). Degree in veterinary medicine (1982) and PhD from the UCM (1987). She has made several both research and teaching stays in several countries. She teaches in the Degrees of Food Science and Technology (CYTA) and Veterinary Science, in PhD programs and has participated in Master's Degrees from different universities, being the author of eight books oriented to teaching. Her research work is related to composition, application of non-thermal preservation methods, new meat products and application of different NMR techniques for food control. She has supervised ten doctoral theses and tutored several Degree and Master´s Thesis. She has been the director of the Institute of Meat Science and Technology of the UCM (2000-2009) and Deputy to the Area of Food Science and Technology (2005-2009) of the ANEP. For the altruistic sample supply, to the farm “La Garganta” (Ciudad Real, Spain). State property of the Duchy of Westminster. Project PID2019-107542RB-C22 from the Spanish Ministry of Science and Innovation.

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