Microbial Dynamics of a Specialty Italian Raw Ewe's Milk Cheese Curdled with Extracts from Spontaneous and Cultivated Onopordum tauricum Willd

Abstract

Milk coagulants prepared by maceration of flowers harvested from both spontaneous and cultivated Onopordum tauricum Willd. and a commercially available coagulant obtained from Cynara cardunculus L. (control) were assayed for small-scale manufacturing of Caciofiore, an Italian specialty raw ewe's milk cheese produced in a family run dairy farm located in the Marche region (Central Italy). The microbiota of the three thistle-based milk coagulants and their effect on the microbial dynamics of raw milk cheeses during fermentation and maturation (from day 0 up until day 60) were investigated through a combined approach based on viable counting and Illumina DNA sequencing. In both the control and experimental cheeses, despite the slight differences emerged depending on the coagulant used, Lactococcus lactis and Debaryomyces hansenii were the prevalent species among bacteria and fungi, respectively. Moreover, raw ewe's milk was the main factor affecting the evolution of both the bacterial and fungal microbiota in all cheeses. The overall similarities between control and experimental cheeses herein analyzed supports the exploitation of Onopordum tauricum Willd. as an alternative milk coagulating agent for production of Caciofiore and, more in general, raw ewe's milk cheeses.

Keywords: Caciofiore cheese; Illumina DNA sequencing; Onopordum tauricum Willd.; microbial dynamics; thistle rennet; vegetable rennet.

Figure 1

Flow chart illustrating the followed steps for the preparation of crude extracts (CE) from Onopordum tauricum Willd. flowers heads.

Figure 2

Experimental plan of the cheesemaking trials. Two production rounds were run, the first (R1) between April and July 2021, and the second (R2) between September and November 2021; for each Round, trials were repeated on two different days (Batch 1, B1, and Batch 2, B2, respectively). On the same day, both control (CF_C) and experimental (CF_Ot_st in R1 and CF_Ot_ct in R2) Caciofiore cheeses were produced.

Figure 3

Results of pH and TTA in round 1 (a) and round 2 (b). pH values are indicated in the primary axis and the results represented with a continuous line; in the secondary axis, TTA values are indicated, and results represented with a dashed line.

Figure 4

Relative frequency of the bacterial ASVs detected by Illumina DNA sequencing in thistle-based coagulants (Cc, CE_st, CE_ct), raw milk (R1_M, R2_M), control (CF_C), and experimental (CF_Ot_st and CF_Ot_ct) cheeses sampled during ripening. Only ASVs with an incidence above 1% in at least two samples are shown.

Figure 5

Principal component analysis (PCA) based on amplicon sequence variant (ASV) relative frequencies of thistle-based coagulants, raw milk, and Caciofiore cheeses for bacterial biota (panel (a)) and mycobiota (panel (b)). Samples are coded as follows: 1-green, coagulants; 2-black, milk used in round 1 (R1_M); 3-gray, milk used in round 2 (R2_M); 4-cyan, cheese manufactured with commercial coagulant (Cc) in R1; 5-blue, cheese manufactured with coagulant obtained from spontaneous O. tauricum flowers (CE_st) in R1; 6-pink, cheese manufactured with commercial coagulant (Cc) in round 2; 7-red, cheese manufactured with coagulant obtained from O. tauricum cultivated flowers (CE_st) in R2.

Figure 6

Boxplots showing relative frequencies of bacterial differential ASVs evaluated through the Kruskal–Wallis test (p-value < 0.05) in experimental Caciofiore cheeses.

Figure 7

Relative frequency of the fungal ASVs detected by Illumina DNA sequencing in thistle-based coagulants (Cc, CE_st, CE_ct), milk (R1_M, R2_M), control (CF_C), and experimental (CF_Ot_st and CF_Ot_ct) cheeses sampled during ripening. Only ASVs with an incidence above 1% in at least two samples are shown.

Figure 8

Boxplots showing relative frequencies of mycobiota differential ASVs evaluated through the Kruskal–Wallis test (p-value < 0.05) in experimental Caciofiore cheeses.