Determination of Cyclopropane Fatty Acids in Food of Animal Origin by 1H NMR.

Veronica Lolli,Augusta Caligiani,Angela Marseglia,Gerardo Palla,Emanuela Zanardi

doi:10.1155/2018/8034042

Veronica Lolli, Augusta Caligiani + Show 3 more

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https://doi.org/10.1155/2018/8034042

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Abstract

Cyclopropane fatty acids (CPFAs) are unusual fatty acids of microbial origin, recently detected in milk and dairy products. CPFAs have been demonstrated to be interesting molecular markers for authentication of dairy products obtained without ensiled feeds. Moreover, they can also be recognized as a new secondary component of human diet. Information is lacking on the presence of cyclic fatty acids in other food sources. Cyclopropane fatty acids have been detected by GC-MS analysis in cheese and other animal fats in concentration ranging from 200 to 1000 mg/kg fat, but in some cases, the complex fatty acid profile and the possible presence of interfering peaks make the separation not straightforward and the quantification uneasy. Therefore, a new reliable 1H NMR method was developed to detect and measure CPFA content in different foods of animal origin, based on the detection of the characteristic signals of cyclopropane ring. The 1H NMR (600 MHz) method showed detection limits comparable with those of full scan GC-MS, and it allowed the identification and quantitation of the cyclopropane fatty acids in different foods.

Highlights

Assignments were previously made by Knothe [16] with the aid of 2D correlations: the upfield signal is assigned to the cis-proton and the downfield signal to the trans-proton. e two methine protons of the cyclopropane ring are located at 0.68 ppm. e other Cyclopropane fatty acids (CPFAs) proton signals are located at lower fields; for example, the four protons in alpha position with respect to the cyclopropane ring display a distinct shift at 1.17 ppm and the signal of the other two protons is observed at 1.40 ppm, within the broad methylene peak [16]
Because the integral of a given peak in a 1H NMR spectrum is directly proportional to a corresponding number of resonant nuclei, peak areas of CPFA cis-methylene proton can be compared with the peak area of trimethylsilyl decanol (TMSD) trimethylsilyl group close to 0.1 ppm and used for the determination of total CPFA content. is internal standard was synthetized starting from a medium chain linear alcohol as decanol because it is not volatile, soluble in apolar solvents, and its trimethylsilyl group gives a singlet close to the CPFA selected signal
Tetramethylsilane (TMS) was commercially available but it cannot be used as quantitative internal standard due to its volatility. e synthesis of TMSD was quick and simple with a high yield. e purity was determined by GC-MS and 1H NMR

Summary

Introduction

Cyclopropane fatty acids are unusual fatty acids found in microorganisms, both Gram-negative and Gram-positive, and seed oils of some tropical plants and protozoa [1, 2]. e bacterial production of cyclopropane ring is related to changes in the membrane fatty acids composition and represents one of the most important adaptive microbial responses that favours the stress tolerance of several bacteria, such as Lactobacillus helveticus, L. bulgaricus, L. acidophilus, and L. sanfranciscensis [1]. CPFAs (mainly dihydrosterculic acid) play an important role in food authentication: they were discovered in milk and dairy products from cows fed with silages, and their determination has been demonstrated to be a powerful tool for the authentication of Protected Denomination of Origin (PDO) cheeses, such as Parmigiano Reggiano, where the use of silages in cow feeding is forbidden [6]. Is was due to the advantages of this technique as the simplicity of the sample preparation (usually it only requires the fat dissolution in deuterated chloroform) and measurement procedures, the instrumental stability, the increase of sensitivity, and modern pulse sequences, with simultaneous suppression of big signals [12] For these reasons, the use of NMR spectroscopy has established a significant role in the analysis of lipids [13]. Erefore, with the aim to investigate on the presence of CPFAs in foods, we developed a new fast and reliable quantitative 1H NMR method, to be used as alternative to gas chromatographic methods and to confirm the presence of CPFAs in foods

Experimental

Results and Discussion

Application to Real Samples

Conclusion