Chemical profiling of processed meat

Processed red meat covers various meat products such as bacon, ham, salami, and sausage produced mainly from beef and pork meat. The high consumption of processed red meat is one of the factors increasing the risk of colorectal cancer (CRC). The underlying reason behind this association remains unknown. However, it might involve the presence of carcinogenic processing contaminants. Most processed meat products contain nitrite salts added to provide characteristic desirable colour and increase shelf-life. Adding nitrite to meat products raises controversies due to the risk of the formation of carcinogenic N-nitrosamines (NAs). NAs include volatile (VNAs) and non-volatile (NVNAs) compounds. The VNAs have been widely studied. However, the occurrence, formation and toxicity of NVNAs are relatively unexplored. Apart from NAs, other potentially harmful processing contaminants can be present in meat, e.g. heterocyclic aromatic amines (HAAs). The complex chemistry of meat processing makes it quite possible that, so far, unknown carcinogenic compounds can be formed.

The main goals for this PhD project were: 1) to develop an analytical method for quantification of VNAs and NVNAs for study of the occurrence of NAs in a broad selection of market samples. 2) to develop a multiclass method for simultaneous analysis of NAs and HAAs, 3) to investigate the impact of meat type and heat treatment on NAs formation in model processed meat product and to develop a platform for a suspect and non-target screening of processed meat, 4) to investigate the endogenous formation of NAs in digestive fluids after digestion of meat sausages with/without added nitrite and spinach emulsion.

A new, robust, sensitive and precise method for simultaneous determination of VNAs and NVNAs was developed and validated using three representative processed meat products. During the successful validation study, sensitivity, precision and trueness of the method were shown (rec 77-109%, RSDr ≤ 20%). Quantification limits (LOQs) for the validated method were between 0.1 – 0.5 ng·g^-1 for VNAs and 2.3 - 4.2 ng·g^-1 for NVNAs. Results obtained with this method can be extrapolated to all processed meat types. In general low levels of VNAs (<5 ng·g^-1) were found in processed meat samples originating from the Danish market, while NVNAs were present in considerably higher amounts up to 1600 ng·g^-1. Amounts of individual compounds varied depending on the product. Generated data will aid to the evaluation of the safety consumption of processed meat regarding NAs occurrence.

The analytical method was further adjusted and allows precise determination of NAs and HAAs in fried bacon. LOQs for the implemented method were between 0.1 to 0.3 ng·g^-1 for HAAs. The method was used for studying NAs and HAAs during frying, and it was found that the formation of HAAs is dependent on meat thickness and fat content. The presented method is the first multiclass method for simultaneous determination of carcinogenic compounds in processed meat and significantly contributes to NAs and HAAs determination in processed meat.

Model beef and pork sausages were prepared to study the role of manufacturing conditions, added additives and heat treatment on NAs. Meat type was found to be a significant factor (p < 0.05) in the formation of N-nitrosodimethylamine (NDMA); N-nitrosopiperidine (NPIP), N-nitrosoproline (NPRO) and N-nitroso-2-methyl-thiazolidine-4-carboxylic acid (NMTCA). Ascorbic acid and liquid smoke flavouring had different impacts on the NAs formation in the beef and pork model. NAs were found to be formed immediately during manufacturing, and storage of ready-to-cook products did not affect NAs levels. The impact of frying, roasting and barbequing on NAs formation showed that barbequing most significantly affects NAs levels. A significant increase (p< 0.05) in NDMA, NPYR, NDBA, and NPIP with a concomitantly decrease for NPRO, NTCA and NMTCA, which could be partly explained by the decarboxylation of NVNAs to VNAs.

Furthermore, a robust workflow for the chemical characterization of difference between unprocessed and processed meat was developed. A variety of new features were detected in nitrite cure, liquid smoked and barbequed model beef and pork sausages. Among them, a probable chemical structures of 11 candidates were elucidated and 41 tentative compounds were identified. Although unknown NAs were not found, their presence still cannot be ruled out. The developed workflow can further be used for studying complex chemical changes occurring during processing of meat and other food items.

A study on the endogenous formation of NAs in the gastrointestinal tract in vitro reflected the amounts of NAs available for uptake in the body. The results further indicate that nitrite in saliva caused a significant increase in NA levels in the gastrointestinal tract. Spinach components were found to protect against the formation of VNAs both after roasting and during digestion. Thus, adding vegetable emulsion to meat sausages might prevent exogenous and endogenous NAs formation, resulting in healthier product alternatives.