**9.1. Raw milk**

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

given sample volume.

**level of academic or innovation labs** 

analyses in the dairy industry [104].

fermentations [105-108].

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

**8.3. Sensitive methods (including ELISA and molecular DNA techniques) at the** 

Plaque assays and acidification tests are microbiological methods that are economically accessible and sensitive enough for detection of phages in the dairy industry. These techniques are time consuming, but provide many practical data for both dairy plants and starter producers. The polymerase chain reaction (PCR), ELISA and flow cytometry-based methods have been designed for detecting phages and are often used to complement microbiology tests. However, they have still many drawbacks to be applied for routine

PCR-based methods detect virulent and non-virulent phages; thus, microbial methods should be used in parallel to precisely distinguish the virulent phages. PCR-based methods can also be too expensive and too specific (only phages targeted by specifically-designed primers are detected) for routine experiments. However, PCR is a fast method able to confirm the presence of bacteriophages within 30 minutes and can be applied to determine the potential utility and quality of big batches of milk. At the same time, the method could be handy in finding niches of phage accumulation, in order to reduce their impact in dairy

ELISA techniques use for phage detection antibodies which are highly specific against structural proteins of phage capsids. Due to the wide phage diversity in the dairy environment, development of several antibodies detecting various groups of phages was required. ELISA is regarded as a highly useful method for monitoring specific phages in the dairy environment, but a single assay cannot be used to detect phages with different 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.

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

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.

### **9.3. Starter cultures**

The starter culture itself can be a source of phages, when strains contain temperate phages. Temperate phages are incorporated into the bacterial chromosome and their genome replicates in synchrony with the bacterial genome. Prophages are carried in many LAB strains. The analysis of bacterial genomes revealed that prophages are more widespread than previously considered [113-114]. Phages may be induced from lysogenic to lytic form by the manufacturing conditions. Serial subculturing of temperate phages in milk may result in their replacement by a virulent mutant. Prophage induction from multiple lysogenic starter culture strains has the potential to influence fermentation. Induction can occur under stress conditions, such as heat, salts, acidity, bacteriocins, starvation or UV [115- 116], and can also occur naturally with a frequency of even up to 9% [117]. Starter culture producers make huge efforts to eliminate strains containing prophages using a screening assay for strain lysogeny. Usually, easily lysogenized strains are difficult to find in defined strain cultures. The main source of lysogenic strains are undefined cultures, which are still commonly used (for example, kefir grains). This is due to two main reasons: i) the exact strain composition of these starters is unknown; ii) elimination of lysogenic strains from undefined culture is very difficult.

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

occurrence of mastitis, mineral levels, lactation period, microbial and enzymatic composition), presence of inhibitors in milk (antibiotics, detergents) and phage infections.

However, phages are the primary source of fermentation problems in the dairy industry. Bacteriophages can cause great economic losses due to fermentation failure in dairy plants. About one third of the annual world production of around 500 million tons is converted into fermented products. Two thirds of all processed milk is fermented by *Lactococcus lactis* and *Leuconostoc* spp. Thermophilic *Lactobacillus* and *Streptococcus thermophilus* spp. account for fermentation of the remaining major part of the milk. According to estimations, from 0.1% to 10% of all milk fermentations are negatively affected by virulent phages [102]. Phage contaminations can slow down or even halt the milk fermentation process. Consequences of the phage presence include: alteration of the product quality, such as taste, flavor, texture, and its microbiota composition. Phage contaminations due to the delay in lactic acid production can also lead to development of undesired microbiota during the fermentation process. In the worst cases, the inoculated milk must be discarded. The frequencies of phage contaminations and their consequences depend on the type of milk product produced.

Among dairy products, the least phage affected are fermented milk beverages (yoghurt, kefir, butter-milk, Actimel®-like products, etc.). There are many reasons behind this phenomenon. Milk for beverage production usually undergoes treatment at temperatures much higher than in cheese manufacturing. Moreover, some drinking yoghurts are produced from UHT milk. Beverages are made in relatively aseptic conditions, including more and more aseptic inoculation systems, where the fermented product is minimally exposed to the factory environment. In spite of that, phage contamination is sufficiently frequent and has become the primary source of fermentation problems in milk beverage production. Phage contaminations in this particular case lead to fermentation delays or

In cheese production the risk of phage infection is very high. A large cheese plant can process more than 500 tons of milk per day, very often in many vats, lasting more than one shift. Pasteurized milk (very often low temperature-treated milk or even raw milk) is used in cheese fermentation and many phages as well as microorganisms remain viable after pasteurization. Contamination, also by phages, increases during curd handling and whey separation in open vats. The consequences of phage infection in cheese production can be: delay or halt in milk acidification, cheese contamination with foreign microbiota, including pathogens, preferential growth of post-pasteurization microbiota, problems in whey separation (syneresis), higher water and lactose content in the final cheese product, abnormal or irregular holes (eyes), or no eyes, and alterations of flavor and texture [5]. To conclude, phage contamination may result in lower quality of cheese or cheese

inhibition, product alterations in taste and flavor as well as texture properties.

Phages can also sometimes turn a dairy staff life into a 'nightmare'.

**10.1. Fermented milk beverages** 

**10.2. Ripened cheese** 

to Lower Phage Contamination in Dairy Fermentation 49

### **9.4. Equipment/air**

The one of the most probable sources of virulent phages is the dairy plant environment. Phages are commonly present on working surfaces. For propagation, phages need the presence of their bacterial hosts, in this case lactic acid bacteria. Due to this fact, they are usually found in places where conditions for LAB development are favorable. The most common sources of phage contaminations are valves, crevices and "dead ends" (difficult cleaning and disinfection places) of production lines. Also, the formation of biofilms on dairy equipment can lead to serious phage problems. Moreover, phages were detected at high levels on various equipments and objects found in cheese plants, such as walls, pipes, door handles, floors, office tables and even on cleaning materials [118]. Raw milk handling, cheese milk processed in open vats and whey handling can lead to spreading of phages in the air. Phage aerolization can occur during air displacements around contaminated places (fluids or surfaces) or by liquid splashes. Virulent phages can circulate through the air far away from their aerosolization source due to the ability to bind to small particles (< 2.1 µm) [118]. Taking into account high levels of phages detected in the air, it is hard to precisely determine whether phage propagation already took place or if it is likely to occur. Concentrations of up to 108 PFU per m3 of air have been detected in a cheese manufacturing plant in Germany; however, mainly in specific areas of the fermentation line [119-120].
