**4. Effects of the storage time and temperature on bacterial growth in raw milk**

### **4.1 Importance of low temperature**

Irrespective the ecosystem, the temperature is a key determining factor of bacterial growth. Low temperature was implemented to preserve the quality of raw milk until the processing stage. When the number of dairy plants was reduced, the raw milk had to be transported over longer distances from farms to dairies. Longer distances result in increased time that elapses between milking and processing stage.

 Milk, which leaves the udder, is at a temperature of about 35°C: a study that evaluated the impact of the storage temperature on bacterial growth in raw milk showed that high temperatures promoted rapid and intense bacterial growth. Importantly, the study also illustrated the "time limited effect" of cold storage at 5°C, as the bacterial growth was only inhibited for 36 h, after which a moderate increase was noticed [28]. Some reports mention "the critical age" (the time after which bacterial growth is observed) to be slightly above 48 h [29]: these variations may reflect differences in initial bacterial levels, in bacterial diversity, or in variable levels of the natural antimicrobial systems present in raw milk.

The temperature value, even at low temperature range, is of crucial importance: an illustration can be seen with the 12 raw milk samples considered in experiments I and II, listed in Table 1 [30]; for I and II, the initial average counts in log-units were 3.9 and 4.03, respectively; it can be observed that 4 days cold storage at 4 and 6°C, respectively, yielded bacterial counts of 6.4 and 7.8 log-units, respectively; the 2° shift showed an about 1.4 log-units (equivalent to a factor of 25) higher bacterial level at 6 compared to 4°C. A 2 log-units (a factor of 100) difference in psychrotrophic *Pseudomonas* levels was also reported by another study that compared optimal (4°C) and suboptimal (6°C) storage temperatures [31].

### **4.2 Consequences of cold storage**

At bacterial population level, cold storage results in the replacement of Gram (+) by Gram (−) bacteria [11]. DGGE-based studies first highlighted that bacterial diversity in raw milk decreased during cold storage [32–34]. That cold storage-impacted bacterial diversity in raw milk was also evidenced by the determination of the amount of

Operational Taxonomic Units (OTUs) in initial and cold-stored raw milk samples: for example, after 3–4 days at 6°C, only 33% of the initial OTUs were recovered in our studies and some bacterial types did not survive the low-temperature storage condition [24].
