**11.2. Design of starter cultures rotation system based on phage contamination control**

Starter cultures are a key factor influencing the diversity of phage population in a dairy plant. Application of undefined multispecies and multistrain cultures was the main strategy to overcome production problems related to phages in many factories (Flora Danica - Chr. Hansen, Probat 505 - Danisco) in the past. One complex culture (e.g. Flora Danica) allowed producing many products: maturated cheese, fresh cheese (tvarog and quark), butter, butter-milk and other mesophilic fermented beverages. Complex multispecies and multistrain cultures are relatively phage tolerant and even upon high phage contamination give products with small deviations that are accepted for marketing. In the past, when dairy plants produced a wide range of products, mainly for the local market, complex undefined cultures fulfilled the expectations of the dairy business.

**Figure 2.** Example of well-designed culture rotation and disinfection frequency strategy for phage control in dairy plant.

Modern industrial fermentations increasingly rely on well-defined, direct vat inoculated (DVI), high concentrated (> 1010 cfu g-1) and product-optimized starters, containing from two to five phage-unrelated strains [131-132]. Market share of bulk starters (semi-direct inoculation) diminished very fast in the last two decades and does not exceeded 20% for dairy beverages and 60% for cheese of the total global processed milk. The defined cultures have been widely adapted in large-scale production facilities due to the significant degree of control over fermentation processes and complementary fermentation properties, such as rapid acidification, gas formation, texturization, and development of flavor and aroma compounds. Each defined culture is designed in two or three phage-unrelated options, which can consistently enable producers to obtain high quality standard products. Rotation of defined phage-unrelated cultures is an efficient phage control method. Usually the rotation strategy in big dairy plants is elaborated in tight collaboration with culture suppliers based on individual phage monitoring programs. Ideally, sterilized products or whey samples are delivered on a routine basis at agreed intervals to the phage lab of the culture supplier. In longer perspective, successful cooperation of culture suppliers and users in monitoring different culture rotation strategies allows designing sequences of culture rotation and safe intervals between rotations as well as elaborate the cleaning and disinfection strategy adapted to specific dairy environments (Fig.2).

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

cultures fulfilled the expectations of the dairy business.

**control** 

control in dairy plant.

Disinfectants recommended mainly for surfaces, equipment, hands and shoe sanitization are listed in Table 3. Disinfectants are in liquid, foam or aerosol form, depending on their application. The efficacy of such disinfectants for phage inactivation, especially those based on alcohols, are lower in comparison to CIP disinfectants. Among biocides, particularly ineffective in phage inactivation is isopropanol [125]. However, taking into account a lower

**11.2. Design of starter cultures rotation system based on phage contamination** 

**Figure 2.** Example of well-designed culture rotation and disinfection frequency strategy for phage

Modern industrial fermentations increasingly rely on well-defined, direct vat inoculated (DVI), high concentrated (> 1010 cfu g-1) and product-optimized starters, containing from two to five phage-unrelated strains [131-132]. Market share of bulk starters (semi-direct inoculation) diminished very fast in the last two decades and does not exceeded 20% for dairy beverages and 60% for cheese of the total global processed milk. The defined cultures have been widely adapted in large-scale production facilities due to the significant degree of control over fermentation processes and complementary fermentation properties, such as rapid acidification, gas formation, texturization, and development of flavor and aroma compounds. Each defined culture is designed in two or

Starter cultures are a key factor influencing the diversity of phage population in a dairy plant. Application of undefined multispecies and multistrain cultures was the main strategy to overcome production problems related to phages in many factories (Flora Danica - Chr. Hansen, Probat 505 - Danisco) in the past. One complex culture (e.g. Flora Danica) allowed producing many products: maturated cheese, fresh cheese (tvarog and quark), butter, butter-milk and other mesophilic fermented beverages. Complex multispecies and multistrain cultures are relatively phage tolerant and even upon high phage contamination give products with small deviations that are accepted for marketing. In the past, when dairy plants produced a wide range of products, mainly for the local market, complex undefined

number of phages in an environment, it can be sufficient for their elimination.

Rotation strategy of defined multiple strain cultures demands selection of strains resistant to a wide range of phages, which could replace infected strains. This aspect can be a drawback when considering continuous and effective use of this method. Moreover, continual rotation of multiple strains during fermentation processes has an effect on phage co-evolution and was shown to increase phage diversity and their abundance in the dairy environment [133]. It also requires constant selection of starter strains with specific fermentative properties. An alternative is the use of a single, highly specialized phage-resistant strain and its variants carrying phage resistance plasmids obtained from naturally resistant strains. This strategy was termed by Sing and Klaenhammer as the phage defense rotation strategy (PDRS) [134]. The success of designed rotations systems of phage-resistant single strain derivatives is assessed by the Heap-Lawrence starter culture activity test (SAT) performed usually in phage-contaminated milk or whey from earlier cycles [135]. Continuous rotation in repeated cycles of single starter lactococcal strain derivatives, where each carries a different type or a combination of various phage defense systems (e.g. R/M or Abi), has been recognized as an effective method of limiting phages during industrial processes [134,136]. Sing and Klaenhammer have shown that the rotation system of three *Lactococcus lactis* derivative strains encoding different phage defense mechanisms provided resistance to the culture during nine rotation cycles against 106 PFU ml-1 of whey composition containing as many as 160 phage isolates [134]. The strategy was then shown to demand precise determination of the type of defense systems to be used as well as the rotation order of the strains. Expression of several phage defense systems relying on different mechanisms conferred complementary defense against phage infection of single strain-derived cultures. Even if one defense system has been overcome, the phage can be inactivated by another. In the study of Durmaz and Klaenhammer (1995) three single starter *Lactococcus lactis* subsp. *lactis*  derivatives, containing different plasmid-encoded phage defense mechanisms, were subjected to a 9-day rotation process challenged by two isometric phages (ul36 and Ф31) or a combination of 10 industrial phages at high titer [136]. Moreover, in most cases examined, an additive effect of different phage R/M and Abi defense systems was observed [136]. As assessed by SAT, the culture persisted incoming infections and only one Ф31-derived mutant phage was detected, but did not disturb culture growth during 17 rotation rounds. Based on these observations, it seems that continuous rotation of at least three derivatives of a single starter strain, where each carries a different phage defense system, is an attractive method to overcome phages as well as all types of resulting phage mutants. Moreover, the use of a limited number of strains, in this case one strain and its variants, limits the phage number as well as the occurrence of novel phages in fermentation plants [135,137]. A great advantage for the industry is also the use of only one indicator strain to monitor phage occurrence. Application of PDRS by construction of novel strains carrying newly identified phage-resistance mechanisms makes this strategy broad range with unlimited variants.

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

**12.1. Classical methods (isolation and selection of phage tolerant strains against** 

In order to isolate phage-resistant mutants, a secondary culture method can be used [138], in which sensitive strains undergo selective pressure of their specific phages. Sensitive strains are inoculated in liquid medium and subsequently infected with suspensions of a selected lytic phage at specific titer. Liquid cultures exhibiting complete lysis are incubated for 24-48 hours (secondary growth). After incubation, bacteria are streaked on adequate solid medium. The grown colonies are consecutively cultured in liquid medium with the same selected phage during at least three rounds. Resultant isolates that are able to grow normally in the

Another means of natural selection of phage-resistant strains was developed by Viscardi and colleagues [140]. The approach is based on flow-cytometry technique that senses and selects bacterial cells to which phage particles that have been added to the medium did not adsorb. Two detection methods have been designed, which rely on recognition of either specifically labeled anti-phage antibodies or fluorochrome-stained phages. The presented method is an attractive alternative to other means of isolating phage-resistant strains (described earlier). In the study, several different *Streptococcus thermophilus* strains were analyzed for their potential to develop spontaneous phage resistance that could be detected by flow-cytometry technique. The designed selection methods proved quite sensitive, as phage-resistant cells could be detected after only one selection round. Nonetheless, a tworound selection based on selection with anti-phage antibodies or labeled phages and then with unlabeled phage alone was more efficient in obtaining stable and proper phageresistant mutants. Phage adsorption assays determined that majority of the isolated mutants resisted phage infection at the level of phage adsorption. Moreover, several selection rounds

The great advantage of the method is its high sensitivity (detection of 2 out 107 cells) and high analysis rates (103 cells per second). As the occurrence of spontaneous phage-resistant cells is rather low in nature, the method allows increasing the level of detection of such mutants. Furthermore, the selected *S. thermophilus* mutants were resistant to phage attack throughout multiple generations, indicating the stability of this property. The novelty of the method is the short amount (several days) of time necessary for obtaining phage-resistant mutants. This creates a possibility of fast selection of new resistant starter strains in the presence of novel phages, which constantly break away from the current defense systems.

**12.2. BIM system - exposure of sensitive strains to lytic phages (spontaneous** 

strain culture and select for mutants which have sustained phage attack.

Selection of BIMs (bacteriophage insensitive mutants) is a way to obtain phage-resistant strains without genetic manipulations. The idea of obtaining such cells is to infect a starter

**mutation in chromosomal or plasmidic genes)** 

presence of the specific phage are considered as true phage-resistant mutants [139].

using different labeled phages lead to isolating multi-phage resistant cells.

**12. Selection of phage tolerant strains** 

**the most aggressive phages from the dairy environment)** 

to Lower Phage Contamination in Dairy Fermentation 57
