Among the 68 isolates, selected from 13 raw-milk samples in Finland (that originate from farm, truck or silo tanks), 60 (88%) were psychrotrophs. All the isolates were characterized by the determination of their spoilage and phenotypic features: proteolytic and lipolytic activities, the production of lecithinases and hemolytic factors were considered. Phenotypic characterization of the isolates was mainly performed with API 20NE and BIOLOG GN2 identification systems; the results were system-dependent, although the presence of representatives of the Pseudomonas genus (for the majority of the isolates) was suggested by both systems. The results of the numerical profile analyses by API 20NE proposed that some strains might be members of Stenotrophomonas, Burkholderia and Acinetobacter genera; however, the identity of many isolates remained doubtful or controversial.

Pure N(2) gas was introduced in the headspace of test bottles containing raw milk that were then stored at either 6.0, 7.0, or 12.0 degrees C. Treatment with N(2) significantly reduced the growth of total bacteria, and the growth of bacterial subgroups such as psychrotrophs, enterobacteria, protease- and lipase-producing bacteria, and Listeria spp, and completely excluded Bacillus cereus growth on MYP plates. The inhibitory effect was maximal at 6.0 degrees C, and bacterial growth could be halted at this temperature for 11 days. At 12.0 degrees C, N(2) was able to inhibit growth during the first 48 h. No alarming or undesirable effects, such as the excessive growth of anaerobes or lactobacilli, were observed during the course of the study. The same treatments also halted the growth of one bacterial isolate in pure culture that expressed multiresistance to antibiotics. The continuous flushing of raw milk with pure N(2) gas in a so-called open system that allows gas exchanges with the environment positively impacted the microbiological quality of the raw milk at a temperature range of 6.0-12.0 degrees C. This procedure should therefore be considered as a possible complementary method to refrigeration in controlling bacterial growth and the spoilage potential of both psychrotrophs and mesophiles in raw milk.

The present study proposed to evaluate the breadth of antibiotic resistance among psychrotrophic bacteria spoiling raw milks in Finland. A core of 60 isolates, retrieved from farms, trucks, and silos, phenotypically characterized, were analysed with ATB PSE strips, composed of a panel of 17 beta-lactams and non-beta-lactams antibiotics, representatives of five classes. Surprisingly, 60% of the psychrotrophs harboured multiresistant traits. We observed an increase of this feature along the cold chain of raw milk transportation.

The quality and safety of raw milk still remains a worldwide challenge. Culture-dependent methods indicated that the continuous N2 gas-flushing of raw milk reduced the bacterial growth during cold storage by up to four orders of magnitude, compared to cold storage alone. This study investigated the influence of N2 gas-flushing on bacterial diversity in bovine raw-milk samples, that were either cold stored at 6°C or additionally flushed with pure N2 for up to one week. Next-generation sequencing (NGS) of the V1-V2 hypervariable regions of 16S rRNA genes, derived from amplified cDNA, which was obtained from RNA directly isolated from raw-milk samples, was performed. The reads, which were clustered into 2448 operational taxonomic units (OTUs), were phylogenetically classified. Our data revealed a drastic reduction in the diversity of OTUs in raw milk during cold storage at 6°C at 97% similarity level; but, the N2-flushing treatment alleviated this reduction and substantially limited the loss of bacterial diversity during the same cold-storage period. Compared to cold-stored milk, the initial raw-milk samples contained less Proteobacteria (mainly Pseudomonadaceae, Moraxellaceae and Enterobacteriaceae) but more Firmicutes (mainly Ruminococcaceaea, Lachnospiraceae and Oscillospiraceaea) and Bacteroidetes (mainly Bacteroidales). Significant differences between cold-stored and additionally N2-flushed milk were mainly related to higher levels of Pseudomononadaceae (including the genera Pseudomonas and Acinetobacter) in cold-stored milk samples; furthermore, rare taxa were better preserved by the N2 gas flushing compared to the cold storage alone. No major changes in bacterial composition with time were found regarding the distribution of the major 9 OTUs, that dominated the Pseudomonas genus in N2-flushed or non-flushed milk samples, other than an intriguing predominance of bacteria related to P. veronii. Overall, this study established that neither bacteria causing milk spoilage nor any well-known human pathogen or anaerobe benefited from the N2 gas flushing even though the N2-flushed and non-flushed cold-stored milk differed in bacterial counts by up to 104-fold.

Psychrotrophic bacteria in raw milk are most well known for their spoilage potential and cause significant economic losses in the dairy industry. Despite their ability to produce several exoenzyme types at low temperatures, psychrotrophs that dominate the microflora at the time of spoilage are generally considered benign bacteria. It was recently reported that raw milk-spoiling Gram-negative-psychrotrophs frequently carried antibiotic resistance (AR) features. The present study evaluated AR to four antibiotics (ABs) (gentamicin, ceftazidime, levofloxacin, and trimethoprim-sulfamethoxazole) in mesophilic and psychrotrophic bacterial populations recovered from 18 raw milk samples, after four days storage at C or C. Robust analysis of variance and non parametric statistics (e.g., REGW and NPS) revealed that AR prevalence among psychrotrophs, for milk samples stored at C, often equalled the initial levels and equalled or increased during the cold storage at C, depending on the AB. The study performed at C with an intermediate sampling point at day 2 suggested that (1) different psychrotrophic communities with varying AR levels dominate over time and (2) that AR (determined from relative amounts) was most prevalent, transiently, after 2-day storage in psychrotrophic or mesophilic populations, most importantly at a stage where total counts were below or around  CFU/mL, at levels at which the milk is acceptable for industrial dairy industrial processes.

The complete genome sequence of Lactobacillus bacteriophage LL-H was determined in 1996. Accordingly, LL-H has been used as a model phage for the infection of dairy Lactobacillus, specifically for thermophilic Lactobacillus delbrueckii ssp. lactis host strains, such as ATCC 15808. One of the major goals of phage LL-H research consisted of the characterization of the first phage-host interactions at the level of phage adsorption and phage DNA injection steps to determine effective and practical methods to minimize the risks associated with the appearance and attack of phages in the manufacture of yogurt, and Swiss or Italian hard type cheeses, which typically use thermophilic lactic acid bacteria starter cultures containing L. delbrueckii strains among others. This mini review article summarizes the present data concerning (i) the special features, particle structure, and components of phage LL-H and (ii) the structure and properties of lipoteichoic acids (LTAs), which are the phage LL-H receptor components of L. delbrueckii ssp. lactis host strains. Moreover, a model of the first, extracellular, phage-host interactions for the infection of L. delbrueckii ssp. lactis ATCC 15808 by phage LL-H is presented and further discussed.

Prolonged cold storage of raw milks favors the growth of psychrotrophs, which produce heat-resistant exoenzymes of considerable spoilage potential; the bacterial proteases and lipases affect raw milk quality; among them phospholipases (PLs) may target the milk fat globule. More importantly, bacterial PLs are key virulence factors for numerous species. Two studies examined the use of nitrogen (N(2)) gas and examined its effect on psychrotrophs, proteases and lipase producers when the milk was stored in closed vessels; however, the effect on PLs producers is unknown. Here we show that by considering an open system the PLs producers were sooner or later excluded in raw milk (whereas the PLs producers in the non-treated controls culminated at 10(8)CFU/ml), by effective gas treatments that bring oxygen (O(2)) levels in milk lower than 0.1ppm. No increase of the PLs producers among the anaerobes was noticed during the course of the experiments. In the experiments performed at 6.0 degrees C, the delay after which the PLs producers were no longer detectable seemed independent of the initial level of PLs producers in raw milk (lower than 10(3)CFU/ml). We anticipate that flushing pure N(2) gas in raw milk tanks, considered as open systems, along the cold chain of raw milk storage and transportation, may be an additional technique to control psychrotrophs, and may also constitute an interesting perspective for limiting their spoilage and pathogenic potential in food materials in general.

Cold storage aims to preserve the quality and safety of raw milk from farms to dairies; unfortunately, low temperatures also promote the growth of psychrotrophic bacteria, some of which produce heat-stable enzymes that cause spoilage of milk or dairy products. Previously, N2 gas flushing of raw milk has demonstrated significant potential as a method to hinder bacterial growth at both laboratory and pilot plant scales. Using a mass spectrometry-based lipidomics approach, we examined the impact of cold storage [at 6°C for up to 7 days, the control condition (C)], on the relative amounts of major phospholipids (phosphatidylethanolamine/PE, phosphatidylcholine/PC, phosphatidylserine/PS, phosphatidylinositol/PI, and sphingomyelin/SM) in three bovine raw milk samples, and compared it to the condition that received additional N2 gas flushing (N). As expected, bacterial growth was hindered by the N2-based treatment (over 4 log-units lower at day 7) compared to the non-treated control condition. At the end of the cold storage period, the control condition (C7) revealed higher hydrolysis of PC, SM, PE, and PS (the major species reached 27.2, 26.7, 34.6, and 9.9 μM, respectively), compared to the N2-flushed samples (N7) (the major species reached 55.6, 35.9, 54.0, and 18.8 μM, respectively). C7 samples also exhibited a three-fold higher phosphatidic acid (PA) content (6.8 μM) and a five-fold higher content (17.3 μM) of lysophospholipids (LPE, LPC, LPS, and LPI) whereas both lysophospholipids and PA remained at their initial levels for 7 days in N7 samples. Taking into consideration the significant phospholipid losses in the controls, the lipid profiling results together with the microbiological data suggest a major role of phospholipase (PLase) C (PLC) in phospholipolysis during cold storage. However, the experimental data also indicate that bacterial sphingomyelinase C, together with PLases PLD and PLA contributed to the degradation of phospholipids present in raw milk as well, and potential contributions from PLB activity cannot be excluded. Altogether, this lipidomics study highlights the beneficial effects of N2 flushing treatment on the quality and safety of raw milk through its ability to effectively hinder phospholipolysis during cold storage.