**11. Phage control strategy**

As previously stated, phages represent a constant threat of serious economic losses in the dairy industry. Dairy microbiologists have attempted for almost 80 years to eliminate or, at least, bring under better control, bacteriophages that interfere with the manufacture of fermented milk products. Phages rapidly disseminate in dairy environment and are difficult to eliminate. The important procedures for phage control are: adapted factory design, design of starters, cleaning and disinfection, and air control [102].

### **11.1. Cleaning and disinfection**

The classical operations of cleaning and disinfection are an essential part of milk processing. Cleaning-in-place (CIP) procedures are usually applied in milk processing lines. The basic procedure consists of the following sequence operations: i) pre-rinse with cold water to remove gross residues; ii) circulation of alkali detergent to remove the remaining minor residues (from time to time acidic detergent is incorporated to remove precipitated minerals and milkstone deposits in the following sequence: alkali detergent, water rinse, acidic detergent); iii) rinse with cold water to flush out the detergent; iv) circulation of disinfectant to inactivate residual microorganisms and phages (still in many dairies this stage is not performed in each cleaning cycle); v) final rinse with cold water to flush out the detergent and cooling line [122]. The cleaning process can remove 90% or more of microorganisms associated with the surface, but cannot kill all of them. One of the drawbacks of the cleaning process is that residual live bacteria can redeposit and, in longer periods of time, can form a biofilm. The presence of LAB among the residual microorganisms increases phage risk contamination. The main role of disinfection is to kill microorganisms that survive the cleaning procedures.


\*exposure time

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

**10.3. Fresh cheese (cottage cheese, quark, tvarog)** 

loss of product.

**11. Phage control strategy** 

**11.1. Cleaning and disinfection** 

cleaning procedures.

quality suitable only for processed cheese production and, in some extreme cases, complete

Cottage cheese and traditional tvarog productions are the most sensitive processes to phages infection. Fermentation delays in production of cottage cheese lead often to complete loss of the final product. However, symptoms of phage contamination are most visible in production of traditional tvarog, where curd quality depends on the activity of lactic acid bacteria alone (rennet is not used). It is estimated that more than 70% of technological disruptions during tvarog manufacture is related to phage contaminations, which usually lead to the following consequences: delay or halt in milk acidification, curd lamination or its drop to the bottom of the tank or vat (which, in effect, causes problem with curd handling), prolonged process of whey separation due to the loss of the curd syneresis, low tvarog yield, contamination with foreign microbiota, including pathogens, intensive growth of post-

As previously stated, phages represent a constant threat of serious economic losses in the dairy industry. Dairy microbiologists have attempted for almost 80 years to eliminate or, at least, bring under better control, bacteriophages that interfere with the manufacture of fermented milk products. Phages rapidly disseminate in dairy environment and are difficult to eliminate. The important procedures for phage control are: adapted factory design,

The classical operations of cleaning and disinfection are an essential part of milk processing. Cleaning-in-place (CIP) procedures are usually applied in milk processing lines. The basic procedure consists of the following sequence operations: i) pre-rinse with cold water to remove gross residues; ii) circulation of alkali detergent to remove the remaining minor residues (from time to time acidic detergent is incorporated to remove precipitated minerals and milkstone deposits in the following sequence: alkali detergent, water rinse, acidic detergent); iii) rinse with cold water to flush out the detergent; iv) circulation of disinfectant to inactivate residual microorganisms and phages (still in many dairies this stage is not performed in each cleaning cycle); v) final rinse with cold water to flush out the detergent and cooling line [122]. The cleaning process can remove 90% or more of microorganisms associated with the surface, but cannot kill all of them. One of the drawbacks of the cleaning process is that residual live bacteria can redeposit and, in longer periods of time, can form a biofilm. The presence of LAB among the residual microorganisms increases phage risk contamination. The main role of disinfection is to kill microorganisms that survive the

pasteurization microbiota, off-flavor and texture alterations of the tvarog [121].

design of starters, cleaning and disinfection, and air control [102].

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

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


Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

Sodium hydride 0.5 – 1.5 RT 15

(CAS No 8001-54-5) 0.50 - 1 RT 10 – 20

Activator – citric acid 0.01 – 0.05 RT 10

**Table 3.** Characteristics of the disinfectants for surfaces, equipment, shoe baths and hands used in dairy

CEN committee for harmonizing the method of evaluating the efficacy of disinfectants [124]. Factors influencing the efficiency of a given disinfectant are: concentration, temperature and exposure time. Among them, the most important is the concentration of active substances. Most of disinfectants are less effective against phages in the presence of interfering proteins (milk or whey) or hard water. The virucidal activity of most disinfectants is improved by increasing the temperature and is usually the lowest in cold water. Therefore, at low temperatures and/or in the presence of proteins, disinfectant concentration and/or contact time should be increased. It is always advisable to combine biocides and heat rather than use them separately at extreme conditions [125]. However, no disinfectant will be fully effective when sanitized surfaces are not cleaned and proteins or biofilm-living cells are present [126]. Under certain conditions phage particles may exist as aggregates, which may also impair complete inactivation. Peracetic acid and sodium hypochlorite are the most efficient biocides of the CIP system in the dairy industry; however, literature data indicate that some LAB phages may be resistant to sodium hypochlorite [125,127-130]. Nonetheless, the most recently available disinfectants are a combination of several biocides. Table 2 presents the chemical content of CIP disinfectants and conditions of their use in the dairy

**Concentration** (%)

> 1.0 - static method 2.0 - foam method

**Disinfectant Supplier/** 

Ekoserwis

P-3 Topax 91 Ecolab Benzalkonium chloride

P-3 Topax 99 Ecolab Alkyl ammonium acetate

Eko Javel PUT

P-3 topactive DES Ecolab

Anthium Dioxide 5% active chlorine

industry.

P-3 Monodes Ecolab

**Producer Main active substances** 

Acetic acid

Acetic acid Amino-oxide

GSG Chlorine dioxide

\* RT – room temperature, \*\* exposure time

industry as recommended by the suppliers.

Benzyl alcohol Propanol-2-ol Ethanol

Hydrogen peroxide

Sodium hypochlorite

to Lower Phage Contamination in Dairy Fermentation 53

**Conditions recommended by supplier** 

> **Temp\*.**  (°C)

1.0-3.0 RT 10-30

undiluted RT 0.5

**Time\*\***  (min.)

RT 10 – 20


\* RT – room temperature, \*\* exposure time

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

**Producer Main active substances** 

products

Propan-1-ol

Amphoteric surfactants (amines, N-C10-C16- alkyl trimethylenedi, reaction

with chloroacetic acid)

Benzalkonium chloride

aminopropyl)-N-

CAS: 2372-82-9 Sodium carbonate Disodium tetraborate

decahydrate

diamine

propanol

m chloride Glutaraldehyde Propan-2-ol

(CAS:5064-31-3) N-Dodecylpropane-1,3-

(CAS: 5538-95-4)

(CAS: 34590-94-8) reaction product of

alkyl diazapentane (CAS: 139734-65-9)

Benzalkonium chloride Dimetylodidecyloammoniu

Cationic surfactants (N-(3-

Trisodium nitrilotriacetate

2-methoxymethylethoxy

alkylamino acetic acid and

dodecylpropane-1,3-diamine

**Conditions recommended by supplier** 

0.5 – 1.0 TR

0.5 – 2.0 RT .

1.0 – 2.0 RT

0.5 – 1.0 RT 60

**Temp\*.**  (°C)

max 50 15 - 60

max 50 5 - 30

max 50 15- 60

**Time\*\***  (min.)

**Concentration** (%)

Didecyldimonium chloride 0.3 – 2.5 RT 5

Sorbic acid undiluted RT 5

dipropylenediamine 1.0 20 - 90 5 - 15

Propan-2-ol 50 – 100 RT 5 - 15

(CAS No 8001-54-5) 0.4 – 0.8 RT 60 - 240

**Disinfectant Supplier/** 

Mycocide S Hypred Propan-2-ol

Deptil BFC Hypred Laurylamine

Johnson Diversey

Johnson Diversey

Johnson Diversey

Diversey

Johnson Diversey

Virocid CID Lines

Deptil HDS Hypred Ethanol

Deptil

Tego 2000 VT 25

Divodes FG VT 29

Suredis VT1 Johnson

Divosan Extra VT 55

Tego Hygiene 2001

**Table 3.** Characteristics of the disinfectants for surfaces, equipment, shoe baths and hands used in dairy industry.

CEN committee for harmonizing the method of evaluating the efficacy of disinfectants [124]. Factors influencing the efficiency of a given disinfectant are: concentration, temperature and exposure time. Among them, the most important is the concentration of active substances. Most of disinfectants are less effective against phages in the presence of interfering proteins (milk or whey) or hard water. The virucidal activity of most disinfectants is improved by increasing the temperature and is usually the lowest in cold water. Therefore, at low temperatures and/or in the presence of proteins, disinfectant concentration and/or contact time should be increased. It is always advisable to combine biocides and heat rather than use them separately at extreme conditions [125]. However, no disinfectant will be fully effective when sanitized surfaces are not cleaned and proteins or biofilm-living cells are present [126]. Under certain conditions phage particles may exist as aggregates, which may also impair complete inactivation. Peracetic acid and sodium hypochlorite are the most efficient biocides of the CIP system in the dairy industry; however, literature data indicate that some LAB phages may be resistant to sodium hypochlorite [125,127-130]. Nonetheless, the most recently available disinfectants are a combination of several biocides. Table 2 presents the chemical content of CIP disinfectants and conditions of their use in the dairy industry as recommended by the suppliers.

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

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

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

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

dairy environments (Fig.2).

to Lower Phage Contamination in Dairy Fermentation 55
