# Exploring New Fruit- and Vegetable-Derived Rennet for Cheese Making
## 3 Results
### FFA Determination  
Based on a UHPLC-MS analysis in negative ion mode, thirteen free fatty acids (FFAs) were identified and quantified. They were medium- and long-chain FFAs, whose qualitative profiles are reported in Figure 4. The total content of PUFAs in the control cheese, obtained using the calf rennet, was very similar to that calculated for the other samples, in which plant and fruit extracts were employed as milk-clotting agents. Linoleic acid (18:2, \(\omega\)-6), whose identity was confirmed by injection of the pure standard compound, was the most abundant one in all the samples, followed by \(\alpha\)-linolenic acid (18:3, \(\omega\)-3). Their ratio ranged from 1.4 in the cartoon-based clotted cheese to 2.7 in the mushroom-based sample. A second octadecadienoic acid was tentatively identified as rumen acid, whereas the less common octadecenoic acid was putatively vaccine acid. \begin{table}
\begin{tabular}{c c c c c c c c} \hline  & **Calf** & **Internal** & & & & & \\
**Rennet** & **Bracts of** & **Cardoon** & **Pineapple** & **Papaya** & **Fig Milky Sap** & **Oyster** \\  & **Arithoke** & & & & & & \\ \hline pH & 5.05 \(\pm\) 0.09 & 5.09 \(\pm\) 0.07 & 5.04 \(\pm\) 0.02 & 5.07 \(\pm\) 0.03 & 5.05 \(\pm\) 0.04 & 5.06 \(\pm\) 0.03 & 5.08 \(\pm\) 0.04 \\ \hline moisture & 38.1 \(\pm\)  
Among the samples, slight differences were observed with regard to the content in the MUFAs and SFAs. In the first FFA subgroup, oleic acid (18:1; \(m/z\) 281.2489) contributed the most to the total percentage, especially in cheeses made with calf rennet, and milky sap from _F. carica_ and _P. ostreatus_ (about 21 and 20%, respectively). Finally, among the saturated FA, palmitic acid was the main constituent, with an estimated content equal to 22% in the calf rennet sample and variable percentages in the plant-based clotted cheese, ranging from a minimum of 12% in the case of cartoon to a maximum of 24% in the case of mushroom. The relative quantitation of free fatty acids in the produced cheeses is reported in Figure 5A. The quantitative profile was obtained from the UHPLC-HRMS analyses, and each FFA content was expressed as mg/g of cheese. Figure 5B shows the plot of the data using a principal component analysis. The graph highlights that while a large part of the saturated and monounsaturated fatty acids do not discriminate the cheeses produced, the richness in palmitic, oleic, and linoleic acid differentiates the chees produced with fig rennet, oyrnet mushroom, and artichoke. The relatively higher content of \(\alpha\)-linolenic acid places chees made from thistle, pineapple, and papaya rennet in a common quadrant. Indeed, calculating the \(\omega 6/\omega 3\) ratio, it was observed that cheeses from pineapple and papaya shared a value equal to 1.6, and cartoon rennet-based cheese showed a linoleic acid/\(\alpha\)-linolenic acid ratio equal to 1.4. Cheeses from artichoke, fig milky sap, and oyrnet mushroom exhibited \(\omega 6/\omega 3\) ratios equal to 2.1, 2.3, and 2.7, respectively. This finding highlights that the free fatty acid profile was positively affected by using vegetable rennet and allowed \(\alpha\)-linolenic acid, the essential \(\omega 3\) fatty acid, to be increased for a healthier diet. To support this statement, based on the FFA quantitative profile, lipid quality indices were also determined. It was found that the use of pineapple, cardoon, and papaya extracts as clotting agents guaranteed their improvement when compared to the control cheese. Indeed, hypocholesterolemic fatty acids (DFAs) were slightly enhanced, whereas hypercholesterolemia fatty acids (OFAs) decreased, giving rise to 1.01-, 1.16-, and 1.24-fold overall increases in their ratio, respectively (Figure 6A). Moreover, lower values of the atheogenicity (AI) and thrombogenicity (TI) indices were found for the same samples, with a more pronounced effect observed for cardoon (1.6 and 1.8-fold reductions, respectively) (Figure 6B).