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Increased phage control in dairies through highly specific molecular phage detection methods and an orthogonal process strategy to reduce the phage count in whey
Project
Project code: MRI-MBT-08-KI-320-1040 Phagen, AiF 19353 N
Contract period: 01.03.2017
- 29.02.2020
Purpose of research: Experimental development
Keywords: food security, food production
According to literature, raw milk may contain bacteriophages of lactic acid bacteria (Lactococcus lactis). As previously shown in two FEI projects (AiF 14339 N, 2007; AiF 15886 N, 2010), the majority of dairy phages survive short-term pasteurization. Thus, phages might contaminate the cheese manufacturing process. In the presence of phage-sensitive host bacteria, these phages may multiply to high titers up to 109 plaque-forming units (pfu) / ml of whey, resulting in severe fermentation problems when whey or whey products are recycled in processes within the dairy. Until now, phage free whey cannot be guaranteed; moreover, rapid phage detection systems are not yet available. In a previous AiF project (AiF 16714 N, 2015), a specific molecular detection system based on the LAMP method was developed. Within 1.5 hours, four particularly heat resistant Lactococcus lactis phages of the 936 phage group could be specifically detected in whey with a detection limit of 103 pfu / ml. In the previous AiF project (16714 N, 2015) we developed a non-thermal alternative for phage reduction in whey products ensuring a high recovery of native whey proteins. A 4-log reduction of phages in native whey was achieved by microfiltration. At low phage contamination levels, filtered whey could be reused in dairies or processed further. However, in the case of high initial phage contamination, the filtration process alone would not guarantee a phage secure product. In those incidences, a so-called orthogonal process control strategy (i.e., UV-C treatment of filtered whey) combined with a fast phage detection tool would be applicable. A UV-C treatment of food is already approved in the US, but in Germany it is only permitted for disinfection of drinking water, hard cheese surfaces and fruit preparations, respectively. Other products, e.g. wine (AiF 18688 N, ongoing project) might be added based on the availability of data on efficiency and health safety. Thus, approval of UV-C treatment of whey is foreseeable, when sufficient scientific data will be available. Therefore, the research objective is (i) to detect lactococcal phages in whey via rapid molecular methods and (ii) to reduce phages up to 9-log units by means of an orthogonal process design with sequential filtration and UV-C treatment steps without negative product changes such as whey protein denaturation.
For optimization of a detection system for Lactococcus lactis phages (MRI), a DNA polymerase with higher amplification rate and yield was tested: The reaction time of the LAMP method for molecular phage detection was reduced by 30 min, allowing the application of a LAMP-based phage detection system specific for phages of L. lactis starter cultures within a short detection time below 1 h. For the UV-C irradiation experiments at the Univ. Hohenheim) a set of 11 L. lactis phages was selected with phages from different phage groups and with different morphologies and genome sizes. Phage preparations were purified and concentrated by CsCl density gradient ultracentrifugation. Phage samples were checked by transmission electron microscopy for selection of phage samples with high numbers of intact phages ( 90%). UV-C screening (Univ. Hohenheim) was done so far with the L. lactis phages P008 and P001 (with different morphotypes) suspended in water, and significant phage inactivation occurred above a minimal dosis of 5 mJ cm-2. Both phages revealed similar resistance phenotypes to UV-C irradiation, and a UV-C dosis of 100 mJ cm-2 was required for maximal phage reduction (8-9 log unit reduction). For both phages, UV-C irradiation of various doses did not cause morphological damaging of the phage particles. For characterization of phage populations in raw milk at MRI, a total of 20 L. lactis phages were isolated from a set of 52 raw milk samples from different farms. Their phage titers varied from 10 to 106 plaque-forming units (pfu) per ml. These raw milk samples showed a significant biodiversity. The majority of these phages were assigned to different subgroups of the P335 phages. One single phage isolate was identified as a member of the 949 group of phages. With the exception of the 949 type phage, all raw milk samples exhibited a narrow host range. Diversity of the P335 phages from raw milk was confirmed by restriction enzyme and genome sequence analysis. Therefore, phage populations in raw milk differed from those that are predominant in dairies. Furthermore, a few raw milk phages did also show similarity with novel and atypical Streptococcus thermophilus phages. Complete inactivation of the raw milk phages was achieved by 1-min thermal treatments at 75°C. Hence these phages are not expected to survive the thermal hurdle of raw milk pasteurization.
Section overview
Subjects
- Food Processing
- Toxicology
Framework programme
Funding programme
Excutive institution
MRI - Department of Microbiology and Biotechnology (MRI-MBT)
