Starter Cultures and Fermented Milk Products

Module 6. Causes of slowness of starters

Lesson 10
BACTERIOPHAGE ACTION ULTRA STRUCTURES, CLASSIFICATION, DETECTION AND CONTROL

10.1 Introduction

Bacteriophages are the viruses that infect bacteria. Viruses are not plants, animals, or bacteria, but they are the quintessential parasites of the living kingdoms. ‘Phage’ literally means devouring just like phagocyte (to swallow or eat up greedily). Bacteriophages are widely distributed in nature & most abundant in intestinal contents of animals and Bacteriophages were first invented by Twort in 1915.

All viruses contain nucleic acid, either DNA or RNA (but not both), and a protein coat, which encases the nucleic acid. Some viruses are also enclosed by an envelope of fat and protein molecules. Without a host cell, viruses cannot carry out their life-sustaining functions or reproduce. They cannot synthesize proteins, because they lack ribosomes and must use the ribosomes of their host cells to translate viral messenger RNA into viral proteins. Viruses cannot generate or store energy in the form of adenosine triphosphate (ATP), but have to derive their energy, and all other metabolic functions, from the host cell. They also parasitize the cell for basic building materials, such as amino acids, nucleotides, and lipids (fats).

10.2 Bacteriophage

They are significant from processing point of view. Phages attack Lactic acid bacteria & lyses them by multiplying inside the host cell leading to the release of many phages which can re-infect the fresh cells in the culture. This results in the failure of starters to act & bring about the changes during preparation of fermented products.

• Bacteriophages are highly host specific, a rotation of starter cultures help to control the problem to some extent.
• Ca ion deficient medium for maintaining starter cultures prevent phage attack as the ion helps in phage adsorption to host cell.
• Genetic manipulations to construct phage resistant strains.

Phages attach themselves to living cells & as they divide phages also multiply. The infective material is injected (nucleic acid) through hollow tail of the phage particle into the bacterial cell. The rate of phage proliferation is greater than that of bacterium. Consequently, a point is reached at which lysis begins. Lysis is preceded by swelling of the bacteria to 6-16 times their normal size. Phage action may cause a number of variations in the properties of bacteria. Irreversible adsorption of phage to the cell triggers a no. of steps which lead to the transport of viral nucleic acids into the cytoplasm & nucleic acid gets metabolically active transforming cellular function to one of synthesizing viral components. As the phage assembly completed the lysis of the host cell occur. All this occur approx. in one generation time of the host phage multiplying to hundred in one generation & in two generations to 10000& so on.

Ultra structures : Typical bacteriophagehas a structure like tadpole.

Head – enclosing nucleic acids in protein sheath.

Tail – a hollow tube of proteins & bearing tail plate & tail fibers.

10.2.1 Structure of Bac

Fig. 10.1 Common bacteriophase structure
(Courtesy: Southwest Biotechnology and Informatics Center)

1. Head
2. Tail
3. Nucleic acid
4. Capsid
5. Collar
6. Sheath
7. Tail fiber
8. Spikes
9. Base plate

Phages of lactic-acid bacteria are having either prolate or isometric heads with contractile tails in some cases and a variety of terminal fibres or plates in addition to collars and other structures along their length.

Phages of Mesophilic Streptococci i.e all Lactococcus lactis ssp lactis and Lactococcus lactis ssp cremoris haveisomeric heads of 45-65 nm diameter with non-contractile tails 100-250 nm longor, less frequently, prolate heads(55-65 nm X 40-48 nm) with shorter non-contractile tails (80-110 nm long)

Phages of Thermophilic Streptococci i.e Str.thermophilus contains isomeric heads of 50-70 nm diameter with non-contractile tails 200-300 nm long

The bacteriophages of Lactobacillushelveticus show isometric heads(49-56 nm diameter) and contractile tails(150-230 nm long) with sheaths. Lb.bulgaricus phages have isometric heads (44-55nm diameter), non-contractile tails 120-215 nm in length with cross-bars andcollars and base plates sometimes present

Since temperate phages released from lysogens may show virulence against other starter strains or mutate to virulence against their own lysogenic host,meaningful distinction between temperate and virulent phages is not always possible.

10.2.2 Classification of bacteriophages

On the basis of presence of single or double strands of genetic material, the bacteriophages are categorized as under:

1. The ssDNA Bacteriophages
2. The dsDNA Phages
3. The ssRNA phages
4. The dsRNA phages

The dsDNA tailed phages, or Caudovirales,account for 95% of all the phages reported in the scientific literature, and possibly make up the majority of phages on the planet. However, there are other phages that occur abundantly in the biosphere, phages with different virions,genomes and lifestyles. Phages are classified by the International Committee on Taxonomy of Viruses (ICTV) according to morphology and nucleic acid.

Table 10.1 ICTV classification of phages

10.3 Detection

A method for detection,identification and/or quantification of bacteriophage of bacterial host specificity for bacterial genus, speciesor serotype, based upon the occurrence of release of cell contents,particularly nucleotides e.g. ATP, on lysis of bacterial cell walls on incubation with bacterial host cells. When new phage particles are released at the end of the phage replication cycle nucleotide levels are measured and compared with controls. The method provides for the detection of specific phages which is faster and more sensitive than known techniques. The method is only limited by the availability of host bacteria/target phage pairings.

Viruses are ubiquitous in natural environments where they can exist as natural inhabitants or as contaminants from the disposal of human and animal wastes. A modified hybridization assay is available for detection of bacteriophage, which employs DNase protection and slot blot methods to measure quantitatively the concentration of soluble and bacteriophage-encapsulated DNA in fluid samples. The potential use of this assay for estimating virus viability was tested with a model system consisting of inactivating bacteriophage lambda particles. These experiments show that the new hybridization assay provides upper-limit estimates of bacteriophage viability when inactivation results in the release of DNA.

10.4 Precautionary Measures to Control Phage Attack

1) Use aseptic techniques for propagation and production of starter cultures

2) Ensure effective sterilization of equipment and utensils

3) Ensure proper heat treatment of the milk

4) Restrict movement of plant personnel in starter handling room, and locate starter room far away from production area

5) Check filtration of air into the starter room and production area

6) Fog the atmosphere in the starter room with hypochlorite solution, or the use of laminar flow cabinets for small scale culture transfers

7) Grow starter cultures in phage inhibitory/resistant medium(PIM/PRM)

8) Use a daily rotation of phageun related strains(or phage resistant strains)

9) Produce the bulk starter culture or even the retail product using a direct-to-vat system