**8. Phage detection in dairy industry**

### **8.1. Simple tools for phage detection at the dairy plant level**

A simple test assessing acidification activity of currently used starter cultures on a daily basis can be used successfully to monitor phage contaminations in dairy plants. Briefly, a cheese whey sample from the last production vat of the shift is collected and, before use, sterilized by filtration (0.45 µm filter-pore size). In the case of dairy beverages, a sample of the final product, before its filtration, is clarified with addition of lactic acid and centrifuged. Processed pasteurized milk or sterilized milk reconstituted from powder is inoculated in duplicate with starter cultures (including a phage alternative culture) at a standard dosage. One sample of each culture is inoculated with a whey filtrate (usually 1-2%) and the second one - with a temperature sterilized whey filtrate. After incubation (the temperature and time depend on the culture and process), the pH of the milk is measured. When the pH of the milk containing the filtrate is 0.2 units higher in comparison to the sample containing the sterilized filtrate, it indicates that phage contamination is rather high and phage-unrelated culture rotation as well as disinfection with higher concentrations of active substances should be recommended.

To avoid direct measurements of pH, bromocresol purple (100 µg ml-1) as a pH indicator may also be used. The test lasts around 6 h, for mesophilic starters, and 4 h, for thermophilic cultures. When pH of the milk drops below 5.4, the indicator turns from purple to yellow. If, at the same time, the color of the sample containing the non-sterilized filtrate becomes green or purple, it means, with high probability, that phages are present and may adversely influence the fermentation process [102].

Another approach of phage detection is continuous monitoring of pH during fermentation processes conducted in vats or tanks with short time intervals and plotting the data on a graph. Even in the case when delay of the fermentation process is not observed, but the graph shows an irregular shape not related to temperature deviation, phage contamination is suspected (Fig.1). However, in this method a delay in acidification can also result from other inhibitors than phages (e.g. antibiotics, detergents) present in the sample.

**Figure 1.** Example of pH curve during milk fermentation in the presence of virulent phages incubated with the multistrain and multispecies culture.

### **8.2. Routine service at culture supplier level**

44 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

economical consequences, job resignation

**8. Phage detection in dairy industry** 

deliveries, customer losses).

consistence

should be recommended.

influence the fermentation process [102].

**7. Problem of phage contamination in dairy industry** 

resistant, phages are detected usually after a certain period of use.

**8.1. Simple tools for phage detection at the dairy plant level** 

Phage contaminations in dairy plants can cause 3 main serious drawbacks:

There are no commercial LAB cultures available which would be completely insensitive to all phages. Even when a starter culture that is launched on the market appears to be phage




A simple test assessing acidification activity of currently used starter cultures on a daily basis can be used successfully to monitor phage contaminations in dairy plants. Briefly, a cheese whey sample from the last production vat of the shift is collected and, before use, sterilized by filtration (0.45 µm filter-pore size). In the case of dairy beverages, a sample of the final product, before its filtration, is clarified with addition of lactic acid and centrifuged. Processed pasteurized milk or sterilized milk reconstituted from powder is inoculated in duplicate with starter cultures (including a phage alternative culture) at a standard dosage. One sample of each culture is inoculated with a whey filtrate (usually 1-2%) and the second one - with a temperature sterilized whey filtrate. After incubation (the temperature and time depend on the culture and process), the pH of the milk is measured. When the pH of the milk containing the filtrate is 0.2 units higher in comparison to the sample containing the sterilized filtrate, it indicates that phage contamination is rather high and phage-unrelated culture rotation as well as disinfection with higher concentrations of active substances

To avoid direct measurements of pH, bromocresol purple (100 µg ml-1) as a pH indicator may also be used. The test lasts around 6 h, for mesophilic starters, and 4 h, for thermophilic cultures. When pH of the milk drops below 5.4, the indicator turns from purple to yellow. If, at the same time, the color of the sample containing the non-sterilized filtrate becomes green or purple, it means, with high probability, that phages are present and may adversely

Another approach of phage detection is continuous monitoring of pH during fermentation processes conducted in vats or tanks with short time intervals and plotting the data on a graph. Even in the case when delay of the fermentation process is not observed, but the graph shows an irregular shape not related to temperature deviation, phage contamination The most common and most useful method of phage enumeration is the plaque assay. The method is quite old and was first described by d'Herelle shortly after the discovery of bacteriophages. Currently it is used in many labs with some modifications, but its principle has not changed [103]. The most common, practical, cheap, without using large numbers of plates and sufficiently accurate method in the dairy industry is the semi-quantitative spot test method. Using this approach, results are available after 24-48 h. The method is well suitable for detection of phages of pure lactic acid bacterial strains at relatively low levels (< 100 phages ml-1). Plague assays allow detecting the presence of phages as well as determining the number of phages in dairy samples against all individual strains present in the applied defined cultures. In case of phage contamination in a dairy plant, the method is a good tool for selecting the best phage-resistant alternative cultures. The method can also be used for hygiene monitoring by enumeration of phages in samples collected from critical places if the plant. For dairy culture producers, permanent phage monitoring can identify strains which are most sensitive in defined cultures. These strains can be systematically replaced with more phage resistant strains. Semi-solid medium supporting

bacterial growth is used for multiplication of strains in form of a smooth opaque layer or lawn on the medium surface using standard Petri dishes. Serial dilutions of phage solution previously sterilized with a filter are placed (5-20 µl) on the surface of the opaque layer. When a single phage particle develops on a recipient bacterial lawn, it forms a plaque (clear spot, no bacterial lawn) visible to the naked eye. This plaque results from the destruction of bacterial cells by the phage progeny. Growth of the plaque is limited by slow diffusion of the phage in the semi-solid medium and bacterial cell growth stops, so phage growth is also inhibited due to the fact that host cells support phage growth. No visible plaques on the plate mean that the sample is not contaminated by phages. Large clear zones (no separate plaques) on the plate indicate with high probability that the level of phages is rather high and further dilutions of the sample are required to precisely determine the phage titer. The presence of a plaque means that: i) the tested sample contains phages; ii) the phage is virulent against the tested strain; iii) the strain is sensitive to the phage. Each phage particle that gives rise to a plaque is called a plaque-forming unit (PFU). One plaque corresponds to a single phage particle and phages can easily be counted. In result, the number of PFUs corresponds to the viable phage concentration in a given sample volume.

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

structural proteins. For this reason, the sensitivity of an ELISA method to detect phages in

Flow-cytometry can also be used for detection of phages in dairy samples by discriminating the phage-infected cells from non-infected based on cell morphological changes leading to lysis. Running on the flow-cytometry of samples containing phages gives a broad distribution of cell mass (wide peak), which demonstrates the presence of both lysed and live cells, while non-infected samples give narrow peaks. Flow-cytometry allows detection of phages in real time, but expensive equipment and well-trained staff needed to perform

In dairy plants phages can originate from a variety of sources. The prime importance is to identify the potential sources of phage contamination and limit their entry to the

The most probable source of virulent phages is raw milk. LAB phages occur naturally in raw milk at low titers (between 101-103 PFU ml-1) and constitute a continuous supply of bacteriophages in dairy plants [109-110]. Phage concentrations in raw milk also depend on conditions of collecting, handling and storing of milk by the supplier (farm), on transport to the plant and, finally, handling of the milk in the plant itself. For example, reverse osmosis used to concentrate raw milk at a farm can impact the level of phages detected in milk. Almost 10% of 900 milk samples examined from various geographical areas in Spain contained *Lactococcus lactis* phages [110]. Using a multiplex PCR method *Streptococcus thermophilus* phages have been detected in more than one third of milk samples used for yoghurt production in Spain [106]. Phage biodiversity is increased by combining milk collected from different farms and these numbers can be even higher in processed milk.

Reconstituted milk from powder is used in many countries for yoghurt, fresh cheese (tvarog and quark) and even maturated cheese production. Also whey proteins are used to standardize milk before the fermentation process or to improve the taste and texture as well as the nutrient value of the final product. Recently, the modern technology of milk powder and whey protein concentrate production applies often lower temperatures of treatment than during traditional technologies. Both milk powder and whey protein concentrates can be sources of high temperature-resistant phages and can influence the quality of the final product [111-112]. For separating whey proteins, ultrafiltration or/and microfiltration are more frequently employed. Applied separation processes result in higher concentrations of phages in the permeate or the retentate. Depending on which fraction is used in subsequent processes, different concentrations of phages in whey protein samples can be detected.

the assays limits application of this technique in the dairy industry [104].

dairy a sample is rather low.

fermentation process.

**9.1. Raw milk** 

**9. Sources of phage contamination** 

**9.2. Milk powder and whey protein concentrates** 

to Lower Phage Contamination in Dairy Fermentation 47
