Many types of probiotic products with desired traits are produced throughout the world and delivered to the target recipients. With the classical view in mind, the bacteria need to be live to exert their probiotic effects. Indeed, it is the case in most of the situations. Also, the regulatory guidelines of WHO state that the probiotics need to be administered at some minimum CFU. Wouldn’t it be fantastic then if the probiotics could be quantified? With the advancement in scientific technology it is now quite a reality to quantify the probiotic strains. The dairy products besides containing the useful microflora might contain many undesired fungi, bacteria and viruses. The quantification methods thus employed should be specific enough to distinguish between the useful and undesired bacterial cells and their strains. Refinements in the traditional methods of bacterial cell quantification has brought about a revolution in this field and one can understand what they are consuming as also their relative amounts to maintain the proper probiotic usage guidelines.
Classical Counting Method And The Disadvantages:
The counting of cells from time immemorial has been performed routinely by the use of plate count methods. This is a very tedious job of cell enumeration done manually and at times with the help of counting gauge like haemocytometer, etc. So, there is always the chance of manual errors. The method would also require relatively longer times as the colonies need time to grow. A little modification of the system brings about the viable plate counting. But according to the reports of Breeuwer et al., the method is severely compromised due to clump formation, growth media contents and encroachment by the neighboring colony forming cells. The probiotic effects not only depends on the culturable cells but also largely on their activity. Therefore, it is essential to get all the relevant informations on the probiotic product. Bacteria may exist in dormant, active and dead forms. There is also a sub-population that is non-culturable but active. Such dormant strains of Bifidobacterium are quite common in dairy products after the long exposure to stress during storage. All these factors should be kept in mind before venturing for the newer technologies that can help in the quantification of the cells.
PCR-Based Methods for Quantification:
As a replacement of the classical cell counting techniques, DNA and RNA based enumeration methods are being contemplated. Among them Polymerase chain reaction (PCR) methods have found useful applications for food products. PCR methods can with good effect be used in the identification, detection and quantification of both beneficial as well as pathogenic bacterial cells. In addition, they can generate the useful fingerprints of the active but non-culturable probiotic strains. They can also be employed to detect or quantify the genes specific for a desired beneficial property. Therefore, there has been a great expansion in the use of such PCR based methods in recent times. The traditional PCR coupled with DNA gel electrophoresis system and the use of sensitive DNA labeling dye like Ethidium bromide has made possible the successful identification of specific species or strains present within the probiotic products and even in the fecal samples. The viability of the probiotics is an essential feature for many of its functionalities. However, the PCR methods cannot distinguish between the DNA from live or dead cells and will provide similar amplification. A deviation from the existing PCR technique was performed by the group of Nocker with the application of dyes (e.g., Ethidium or Propidium monoazide) that can intercalate within the DNA bases of the membrane ruptured dead cells and thereby prevent their DNA amplification through the formation of strong photo-activable covalent linkages with DNA.
Among the available techniques, quantitative or semi-quantitative real time PCR (qRT-PCR) can be effective in understanding the physiological properties and activities of the probiotics. Most of the organisms have a relative abundance of mRNA transcripts as compared to DNA and protein. The qRT-PCR is more effective in the sense that they need only a small amount of mRNA and reverse transcribed to identify signature genes of the species or even strains. The slightest variation in the quantity of such genes can be easily detected through highly sensitive dye like SYBR green or probes like TaqMan or the molecular beacons. The 16s rRNA has been routinely used to identify the variations among the different strains of probiotics.
Inspite of all its added advantages in the identification and enumeration of probiotic strains, the PCR based techniques need to overcome the deficiency in the ability to distinguish between the DNA and RNA from dead or live cells. Therefore, even the DNA or RNA from the inactive probiotic strains contaminates the data available through the PCR-based methods. This can be overcome with the use of techniques like FISH alongwith FACS that will enable efficient sorting and separation of the live and dead cells. The best way to distinguish between live and dead cells is to exploit the membrane integrity of the cells. While the live cells have an intact membrane but that of the dead cells are irreversibly damaged and ruptured. That is why the live cells can efficiently exclude the DNA intercalating dyes but the dead cells cannot and the dyes penetrate through the compromised membranes of such cells. This principle has been used in the differential staining of live and dead cells. Even dual staining techniques where one of the dyes will stain the live cells while the other stains the dead cells have also been based on the membrane integrity properties of the cells. In such a work by Alakomi, the usefulness of the viability kit, BacLight LIVE/DEAD was used together with FACS. The kit consisted of Propidium iodide (stains the dead cells red) and SYTO9 dyes (stains the live cells green). However, the technique cannot differentiate between species or strains.
The proper identification of the probiotic strains is an important aspect which needs utmost attention and care. The activities and functions of the probiotics vary considerably among strains with similar phenotypes. Therefore, the development of techniques for the better identification and quantification of probiotic strains as also between the live and dead cells can provide probiotic products with greater beneficial traits.