Effect of the use of chlorine usage in milking equipment cleaning procedures on raw milk microbiota

Aim Proper milking machine cleaning and disinfection procedures are essential to ensure milk hygienic quality, and sodium hypochlorite is generally present in commercially available products (Gleeson et al. 2012), but it has been previously demonstrated that nonchlorine detergent products give satisfactory results when used with sufficiently hot water (Teagasc et al. 2012). In addition, some studies highlighted the effect of chlorine on milk bacterial count, encouraging a deeper comprehension of the relationship between the use of chlorine products and milk microbiota, which represents a key point for raw milk cheese production (Gleeson et al. 2013). The aim of the study was to evaluate the influence of chlorine products usage for cleaning and sanitizing the milking equipment on raw milk and the deriving whey-starter microbiota in Trentingrana production. Trentingrana is a Protected Designation of Origin (PDO) hard cheese manufactured in the valleys of Trento province (eastern Italian Alps) by several small cooperative dairies linked in a consortium (Bittante et al. 2011). Materials and Methods Three farms (here named as F1, F2 and F3) located in Trentino and associated to a factory producing Trentingrana cheese, were involved in the study. The herds were composed by 55, 48 and 91 lactating cows (Italian Holstein-Friesian, Brown and Italian Simmental in different ratios). The farms were free stall barns with cubicles with mattress covered with sawdust and lactating cows were fed hay associated with concentrate. The herds were milked twice a day in DeLaval herringbone milking plants (F1 (5+5), F2 (4+4), F3 (6+6). The cleaning routine practices and products usage rates were those recommended by the producers and the water temperatures appropriate. Milk was collected thrice weekly during the sodium hypoclorite detergent usage (six-weeks period, C) and in a subsequent, analogous, nonchlorine detergent period (period NC); a 4-weeks interval was established between the two experimental periods in order to allow bacterial population adaptation to the new detergent. In the last three days of each experimental week, bulk milk was sampled from tank. The whey-starter used that day for cheese-making was sampled too. Within 12 h from collection, samples were submitted to microbiological analyses (Standard Plate Count SPC, coliforms, staphylococci coagulase positive, lactic acid bacteria LAB). For microbiome analysis, bacterial DNA was extracted from 5 ml of milk or whey-starter samples using a protocol previously described (Cremonesi et al. 2006) with some modifications. 16S rRNA gene (V3-V4 region) was amplified following standard 16S Metagenomic Sequencing Library Preparation and sequenced on a paired 2x250 bp run on Illumina MiSeq platform. Data analyses (alpha and beta diversity, taxonomical abundances) were performed using the QIIME pipeline (release 1.8.0; (Kuczynski et al., 2011). Results and Discussion Even if no microbiological significant differences were observed, higher levels of SPC and LAB in bulk milk were recorded going from period C to period NC (4,06 ±0,16 vs 4,10 ±0,20 log cfu/mL), while coliforms and staphylococci tended to be lower in NC period (1,97±1,02 vs 1,60±0,68 log ufc/mL and 2,17±0,48 vs 2,07±0,57 log cfu/mL respectively). An increase in LAB count, although not significant, was observed in LAB content in whey-starter (8,11±0,43 log ufc/mL in period N vs 8,66 ±0,45 log cfu/mL in period NC). The microbiota composition was quite peculiar in the three farms analysed, and between C and NC periods. Alpha- and beta-diversity analyses revealed significant differences among farms (p values: 0.003 and 0.001, respectively): in fact, farm F1 and F2 microbial taxonomic composition was dominated by Firmicutes (rel. ab. respectively 60.5% and 42.8% in period C; 57.5% and 52.1% in period NC), while farm F3 showed a microbial composition dominated by Bacteriodetes (47.4% period C; 52.1% period NC). Differences in the microbiota profile (beta-diversity) between C and NC periods within each farm were found to be significant (p < 0.05). Relative abundances analysis revealed that farm F1 and F2 had a significant change in Oscillospira genus and, respectively, in Chryseobacterium and Clostridium genera; farm F3 showed Acinetobacter and Trichococcus genera significantly decreased. Though raw milk microbiota varied among farms, whey-starter bacterial composition consisted mainly of Lactobacillus genus. At a species-level, Lb. helveticus was predominant during period C (62.9% rel. ab.) and significantly reduced during period NC (31.8%), whereas Lb. delbrueckii had the exact opposite trend (28.1% period C; 58.6% period NC). Conclusion Our preliminary results report that is reasonable to further study chlorine influence on milk microbiota, in order to better understand the changes in its composition and, consequently, the possible effect on raw milk cheese production performances and overall quality.