2.9.3. Microbial Load

After pasteurization and spray-drying, the samples were analyzed; the powder (1 g) was mixed with peptone water (PW, 9 mL) and serial decimal dilutions of the initial suspension in PW were prepared until achieving 10<sup>−</sup>6. From these solutions, different counts were performed:

*Aerobic plate count (total viable count; ISO 4833-2:2013).* The dilutions were inoculated in PCA (plate count agar) by the pour plate technique, in duplicate (LOQ = 1 log CFU/g): 1 mL of suspension was pipetted onto the plate and 15 mL of melted PCA (kept at 50 ◦C in a water bath or incubator) were poured; it was homogenized and left to solidify. It was incubated at 30 ◦C for 72 h, in reversed position; when the plates had between 15 and 300 colonies, the count was performed.

*Coliforms (and E. coli; ISO 4832:2006)*. The dilutions were inoculated in VRBLA (violet, red bile lactose agar) using the pour plate technique, in duplicate (LOQ = 1 log CFU/g): 1 mL of suspension was pipetted onto the plate and 15 mL of melted VRBLA (kept at 50 ◦C in a water bath or incubator) was poured; it was homogenized and left to solidify. On top of the medium, a top layer of 4 mL of VRBLA was poured and it was left to solidify. It was incubated at 30 ◦C for 48 h, in reversed position. When the plates had between 10 and 150 colonies, the count was performed.

*Yeasts and Moulds (ISO 21527-1*/*2:2008)*. The dilutions were inoculated in DRBC (dichloran rose bengal chloramphenicol) using the spread plate technique, in duplicate (LOQ = 1.7 log CFU/g): 0.2 mL of suspension were pipetted onto a plate containing 15 mL of the medium and were spread with a disposable spreader. It was incubated at 25 ◦C for 5 days, in the upright position. When the plates had less than 150 colonies, the count of yeast and mould colonies was performed separately after 2 and 5 days of incubation.

*Bacillus cereus (ISO 7932:2004).* The dilutions were inoculated in MYP (mannitol yolk polymyxin) using the spread plate technique, in duplicate (LOQ = 1.7 log CFU/g): 0.2 mL of suspension were pipetted onto a plate containing 15 mL of the medium and were spread with disposable spreader. It was incubated at 30 ◦C for 24–48 h, in reversed position. When the plates had between 10 and 150 colonies, the count was performed.

The microbial load of the different colouring formulations was assessed after their preparation (t0) and after 12 weeks of storage at room and refrigerated temperature.
