Abstract
Hay farming has been a traditional agricultural practice for small-scale mountain farms in Alpine regions for centuries. Increasing recognition of haymilk highlights its contributions to environmental sustainability and biodiversity. Haymilk, produced through hay and pasture feeding, yields high-quality milk and dairy products with favorable protein quality and fatty acid profile such as higher concentrations of beneficial polyunsaturated fatty acids compared to conventional milk. Despite its official recognition under the EU’s “Traditional Specialty Guaranteed” (TSG) quality scheme in 2016, current haymilk production specification lacks a standardized method to verify compliance with its strict feeding regulations. This lack in verification system presents challenges to ensure uniformity across different producers and regions of haymilk. Cyclopropane fatty acids (CPFAs), released by lactic acid bacteria during silage fermentation, can be indicators of silage feeding in dairy products, which is prohibited in haymilk production. Nuclear magnetic resonance (NMR) spectroscopy presents a powerful tool in the assessment of haymilk authenticity due to its accurate detection of CPFAs in the upfield region of the NMR spectra, which is free of any interfering signals. This thesis aimed to develop a robust 1H NMR-based method for haymilk authentication. To the best of our knowledge, no prior study has applied an NMR approach to verify the authenticity of haymilk, which makesthis research a novel contribution in this field. Furthermore, it also aimed to overcome the limitations of NMR in CPFAs detection, especially due to their low concentrations in milk, by introducing a novel 1H-NMR technique- homonuclear decoupling sequence- to enhance detection sensitivity. The study involved the analysis of 245 milk samples from South Tyrol (Italy), categorized as haymilk, milk from cows fed either maize or grass silage. The 1H-NMR method successfully differentiated authentic haymilk samples from milk from silage feeding, detecting and quantifying the CPFAs as molecular marker for silage feeding in milk products. The authenticity of haymilk in yoghurt products were further verified by 1H NMR and gas chromatography-mass spectrometry (GC-MS). Results confirmed the absence of CPFAs in all haymilk samples, while 97 % of milk samples from maize silage and 77 % of grass silage feeding systems exhibited distinct CPFAs signals, confirming the authenticity of haymilk. Furthermore, 1H-NMR homonuclear decoupling sequence was applied to enhance the detection sensitivity of the CPFAs signal in milk. The NMR technique significantly improved the detection limits for CPFAs by threefold increasing in the sensitivity compared to previous NMR studies under similar conditions. The adoption of NMR methodology to haymilk authenticity through CPFAs analysis provides an efficient and reliable tool for assessing compliance with haymilk TSG production standards. Establishing such an official analytical method can enhance the transparency and integrity of haymilk products, supporting the premium market position of haymilk as well as geographically protected Alpine dairy products produced exclusively from haymilk in the competitive dairy market.