###### Abstract  
The addition of frozen curd (FC) during the production process of \"Mozzaarella di Buffalo Campana\", an Italian cheese with Protected Designation of Origin (PDO), is a common fraud not involving modifications of the chemical composition in the final product. Its detection cannot thus be easily obtained by common analytical methods, which are targeted at changes in concentrations of diagnostic chemical species. In this work, the possibility of spotting this fraud by focusing on the modifications of the supramolecular structure of the food matrix, detected by time domain nuclear magnetic resonance (TD-NMR) experiments, was investigated. Cheese samples were manufactured in triplicate, according to the PDO disciplinary of production, except for using variable amounts of FC (i.e., 0, 15, 30, and 50% \(w/w\)). Relaxation data were analysed through different approaches: (i) Discrete multi-exponential fitting, (ii) continuous Laplace inverse fitting, and (iii) chemometrics approach. The strategy that lead to best detection results was the chemometrics analysis of raw Carr-Purcell-Meiboom-Gill (CPMG) decays, allowing to discriminate between compliant and adulterated samples, with as low as 15% of FC addition. The strategy is based on the use of machine learning for projection on latent structures of raw CPMG data and classification tasks for fraud detection, using quadratic discriminant analysis. By coupling TD-NMR raw decays with machine learning, this work opens the way to set up a system for detecting common food frauds modifying the matrix structure, for which no official authentication methods are yet available. food structure; chemometrics; relaxometry; machine learning +
Footnote †: journal: Medicalcurd (FC), as no effective molecular markers of this kind of adulteration have been detected so far, at a difference with other frauds such as geographical or milk species origin [3; 4]. The fragment 69-209 of beta-casein, deriving from the hydrolysis of \(\beta\) casein by plasmin, also called \(\gamma\)4-CN, was reported by some authors [5] as marker of frozen milk and curd in buffalo mozzarella cheese with Protected Designation of Origin (PDO). However, further studies did not confirm these findings [6]. A different approach based on supramolecular structure characterisation could therefore prove to be efficient where traditional methods fail. Foods are indeed structurally complex materials, due to the compresence of different physical states as well as wide lengths and time scales of their components [7]. As such, the food structure could be used as a fingerprint of a product with a specific formulation and transformation process. This approach to authentication has been applied to the case study of \"Mozzarella di Bufala Campana\" PDO cheese. Mozzarella cheese is a complex system and its water distribution differs from most other cheeses because of a particular step of the cheese-making process that corresponds to curd stretching. This step is responsible for changes in its microstructure: During the stretching, an elastic network of oriented parallel protein fibres is created [8; 9]. As certified within the European quality label \"Protect Denomination of Origin\", the product must be compliant to the regulations regarding geographical origin, raw materials, and production procedures [10] (D.P.C.M. 10/05/1993). However, due to the scarcity of buffalo milk supply during certain years, some producers freeze part of the fresh curd produced during periods of abundance of raw material, to be used when needed in mixtures of fresh and frozen curd [6]. This is a fraudulent practice, deceptive for the consumer and representing a form of unfair competition and a damage to the product brand as well. Food control authorities require analytical or spectroscopic methods to discriminate between fresh water-buffalo Mozzarella PDO cheese and Mozzarella produced from frozen intermediates, which are not compliant to the PDO manufacturing process.