**10. Phage problem frequencies and consequences depend on product portfolio**

Fermentation problems in the dairy plant can be related with: low starter activity, fermentation conditions (e.g. temperature fluctuations), milk composition (year, season, 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. Phages can also sometimes turn a dairy staff life into a 'nightmare'.

### **10.1. Fermented milk beverages**

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

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

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].

**10. Phage problem frequencies and consequences depend on product** 

Fermentation problems in the dairy plant can be related with: low starter activity, fermentation conditions (e.g. temperature fluctuations), milk composition (year, season,

**9.3. Starter cultures** 

undefined culture is very difficult.

**9.4. Equipment/air** 

**portfolio** 

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

### **10.2. Ripened cheese**

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

quality suitable only for processed cheese production and, in some extreme cases, complete loss of product.

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

**Concentration** (%)

0.1- antiseptic

Diversey Sodium hypochlorite 0.1 – 3.0 cool 10 – 20

Sodium hydride 0.25 – 1.0 20 15

Sodium hydride 0.2 – 0.5 20 – 60 15

Hydrogen peroxide 0.2 – 0.5 20 5 – 20

**Disinfectant Supplier/** 

Deptil PA 5 Hypred

special 150 Novadan

active 150 Ecolab

ZS Ecolab

CD Ecolab

Divosan Hypochlorite

VT3

Oxidan

Hypochlor

P-3 Oxonia

P3 – Oxysan

P-3 Horolith

\*exposure time

P-3

**Producer** 

Johnson

DES Novadan Sodium hypochlorite,

Hypochloran Ecolab Sodium hypochlorite,

Clarin spezial Clarin Peracetic acid,

**Main active substances** 

Hydrogen peroxide, Peracetic acid, Acetic acid

Hydrogen peroxide, Peracetic acid, Acetic acid

Hydrogen peroxide, Peracetic acid, Acetic acid

Hydrogen peroxide, Peracetic acid, Acetic acid,

Peroxyoctanoic acid

Nitric acid, Phosphoric acid, Polyhexamethylene biguanide hydrochloride

**Table 2.** Characteristics of CIP disinfectants used in the dairy industry.

Desinfect CL Novadan Sodium hypochlorite 0.20 – 1.0 5 – 40 10 – 15 P-3 Oxonia Ecolab Hydrogen peroxide 0.5 – 1.0 ca. 10 5 – 30

Disinfection is becoming more and more important in the current strategies used by the dairy industry to limit bacteriophage infections. The virucidal efficacy of disinfectants against bacteria, yeasts, moulds, including pathogens, is well-documented in supplier specifications, but very seldom the information on the efficacy against phages is available. It is wrong to consider that disinfectants active against bacteria will also inactivate bacteriophages [123]. The virucidal activity of commercially available disinfectants is unknown or known only against lab reference phages proposed by the established in 1989

to Lower Phage Contamination in Dairy Fermentation 51

**Conditions recommended by supplier** 

2.5-fungicidal 30 20

0.1 – 0.35 5 – 40 5 – 60

0.1 – 0.2 ca. 10 5 – 30

0.5 – 1.5 50 – 70 10

max. 40 5 – 30

0.10 ca. 10

**Temp.**  (°C)

**Time\***  (min.)
