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This study (purchase required) used, high-performance liquid chromatography, combined with chemometric analysis, to classify buffalo vs cows ricotta based on the profile of water soluble peptides.  The authors then identified specific peptides that could be used as species markers . Both mid-infrared spectroscopy and electrophoresis were also investigated as peptide measurement methods by were found to give insufficient discrimination, with IR overly affected by storage time of the extracts.

The authors created  11 experimental cheese formulations by increasing the proportions of cow whey mixed with buffalo whey. Water-soluble peptides were analysed using mid-infrared spectroscopy, high-performance liquid chromatography and electrophoresis. The data obtained from mid-infrared spectroscopy and high-performance liquid chromatography were statistically processed using principal component analysis, analysis of covariance and multiple linear regression..

High-performance liquid chromatography identified 14 peptide peaks, with three recognized as specific markers for cow whey in adulterated samples. PCA explained 77% of the variance, distinguishing pure and adulterated ricotta. Multiple linear regression modelling of high-performance liquid chromatography data predicted cow whey concentration with a correlation of R = 0.87. High-performance liquid chromatography with chemometrics was effective for detecting buffalo ricotta adulteration.

When applied to 14 commercial samples, the model suggested that nine contained adulteration ranging from 10% to 100% cow whey.

Photo by Conor Brown on Unsplash

This study (open access) proposes a strategy to verify the authenticity of Mozzarella di Bufala Campana (MdBC).  MdBC is, a Protected Designation of Origin (PDO) cheese, Buffalo breeds are highly similar genetically, so detecting foreign buffalo milk in commercial cheese is more complicated than identifying cow, goat, or sheep milk. Fraud involving cow milk is particularly concerning because it is cheaper and more widely available, especially during peak MdBC production seasons

The researchers used a reference set of sixty-four anonymized PDO MdBC and foreign mozzarella samples provided by the Italian Central Inspectorate for Fraud Repression and Quality Protection of the Agrifood Products and Food, Ministry of Agricultural and Forestry Policies (Rome, Italy).  They used a sequential approach to verifying foreign milk species in buffalo mozzarella.  As a first screen, the casein was separated on a polyacrylamide gel.  This was generally sufficient to identify extraneous cows’ milk proteins.  In a second stage, the isolate casein was then digested with trypsin and the peptides analysed by MALDI-ToF-MS.

In cases requiring confirmation, nano-liquid chromatography coupled to electrospray tandem mass spectrometry (nano-LC-ESI-MS/MS) is used in central state laboratories for the highly sensitive detection of extraneous milk proteins in PDO buffalo MdBC cheese. The researchers report that analysis of the pH 4.6 soluble fraction from buffalo blue cheese identified 2828 buffalo-derived peptides and several bovine specific peptides, confirming milk adulteration.

They conclude that, despite a lower detection extent in the pH 4.6 insoluble fraction following tryptic hydrolysis, the presence of bovine peptides was still sufficient to verify fraud. This integrated proteomic approach, which combines electrophoresis and mass spectrometry technologies, significantly improves milk adulteration detection.

Photo by Audric Wonkam on Unsplash