The research was conducted at Kiev Institute of Microbiology and Virology of Ukrainian Academy of Science.
I. MRET activated water inhibitory effect on the growth of conditionally pathogenic bacteria Escherichia coli K-12 in an aerobic environment
The investigation revealed the significant effect of MRET activated nutrient medium in an aerobic environment on the process of growth and reproduction of E.coli microorganisms, their division, the size of colonies and the modification of forms of culture cells. It was observed that at a low initial concentration of cells of investigated culture Escherichia coli K-12 MRET nutrient medium activated during 30 minutes and 60 minutes inhibited the growth of culture 27 and 303 times respectively during the 25 hours of the experiment.
Initial view of Petri dishes with different fractions of nutrient medium at the beginning of experiment is presented on Fig 1.
Fig 1: Petri dishes at the beginning of experiment. Identical very small amount of Escherichia coli K-12 cells was introduced on a surface of non-activated nutrient medium of control dishes and on a surface of dishes with nutrient medium MRET activated for 30 and 60 minutes(tact=0.5h and tact=1.0h) in aerobic environment. There are no colonies of microorganisms in Petri dishes in the beginning of experiment.
Petri dishes with grown colonies and statistical parameters of the colonies after 23 hours of experiments are presented on Fig 2: (a) non-activated nutrient medium (control); (b) nutrient medium activated for 30 minutes; (c) nutrient medium activated for 60 minutes. Petri dishes after 29 hours of experiment are shown on Fig 3. The significant inhibition of growth of E.coli in activated samples was revealed and it confirmed the strong bacteriostatic effect of MRET activated medium.
(a) Control:
Number of colonies NC = 1.7x108 CFU/ml.
Average diameter of grown colonies d = 1.1 mm.
(b) MRET activated, tact = 30 minutes:
Number of colonies N0.5 = 6.4x106 CFU/ml.
Average diameter of grown colonies d = 1.8 mm.
(c) MRET activated, tact = 60 minutes:
Number of colonies N1.0 = 5.2x105 CFU/ml.
Average diameter of grown colonies is d = 1.5 mm.
Fig 2: Petri dishes after 23 hours of the experiment.
This experiment shows that MRET-activation process has very strong bacteriostatic effect on conditionally pathogenic E.coli microorganisms and that the inhibition of E.coli growth is more effective when activation time is increased. It was observed that at low initial concentration of cells of E.coli in nutrient medium MRET-activation during 30 minutes and 60 minutes period of time inhibited the culture growth NC/N0.5 = 27 and NC/N1.0 = 303 times respectively after 25 hours of experiment (Fig 4). Consequently, the level of bacteriostatic activity was 96% in 30 minutes activated nutrient medium and 99.7% in 60 minutes activated medium. Thus, the direct correlation between bacteriostatic activity of MRET activated nutrient medium and the time of activation was confirmed.
Inhibition of E.coli bacteria growth in aerobic environment:
This experiment also revealed the strong effect of MRET activated water on the process of division of E.coli microorganisms, the modification of forms of culture cells and the size of colonies. It was observed that one of the reasons of abnormally low growth of E.coli bacteria was related to the modification of the process of cell division in MRET activated nutrient medium. In the process of growth and reproduction a large number of cells did not separate from each other and the linear line-ups consisting of 2-3 sequentially paired cells were formed. The culture cells grown in non-activated and MRET activated medium are shown on Fig 5 and Fig 6 respectively.
Fig 5: Cells of Escherichia coli K-12 grown in non-activated medium.
Fig 6: Cells of Escherichia coli K-12 grown in MRET activated medium (tact =1 hour).
K – Control;
0.5 – 0.5 hour MRET activated water;
1.0 – 1 hour MRET activated water
RC = 0.2, R0.5 = 0.1, R1.0 = 0.4
K0.5 = 0.5, K1.0 = 2.0
DC = D0.5 = D1.0
RC = 25, R0.5 = 9, R1.0 = 19
K0.5 = 0.36, K1.0 = 0.76
DC = D0.5 = D1.0
II. The effect of MRET activated water on metabolic activity of E.coli bacteria in aerobic and anaerobic environment
Reductant activity is an integral characteristic of metabolic activity of microorganisms and it is measured with the help of Sodium Resasurine color indicator in the percentage degree of discoloration (purple = 0%, red = 50%, transparent = 100%). The reductant activity of E.coli bacteria reduced up to 3 times in 30 minutes MRET activated water and up to 1.6 times in 60 minutes activated water during the first 6 hours of an experiment in an aerobic environment (Fig 7 and 8).
This experiment showed that there was no direct correlation between the inhibition of metabolic (reductant) activity and the inhibition of growth of E.coli (bacteriostatic activity) in MRET activated water. The bacteriostatic effect is substantially higher in 60 minutes activated water and the inhibition of reductant activity during the first 3 hours is higher in 30 minutes activated water. Thus, this experiment revealed that the optimum time of activation for the maximum inhibition of metabolic activity of E.coli bacteria in aerobic environment is 30 minutes. The same optimum time of activation was found in the process of another investigation regarding the application of MRET activated water for preventive treatment and enhancement of tumor resistance in vivo on 500 mice for two types of cancer conducted at Kiev Institute of Experimental Pathology, Oncology and Radiobiology of Ukrainian Academy of Science.
Taking in consideration that a small population of pathogenic bacteria, such as E.coli, is usually presented in complex microbial associations in the intestine of the body, the test on metabolic activity of E.coli bacteria in anaerobic environment was conducted. Anaerobic environment simulates the environmental conditions similar to the conditions in the intestine of humans and animals. The investigation showed that the reductant activity of E.coli bacteria in anaerobic environment practically did not change (Fig 9 and 10).
K – Control; 0.5 – 0.5 hour MRET activated water; 1.0 – 1 hour MRET activated water; KSTER – reference to sterility
Fig 9: Comparative test on metabolic (reductant) activity of E.coli (control samples, 0.5 hour and 1.0 hour MRET activated water, KSTER - reference to sterility) in anaerobic environment: R - reductant activity (in Control, 0.5 hour and 1.0 hour MRET activated water), K - relative reductant activity, D - optical density, V - gas volume in the bottles.
Fig 10: Reductant Activity of E.coli in 30 and 60 minutes MRET activated water and in Control non-activated samples (R0.5 - red color, R1.0 - green color, RC - blue color) in anaerobic environment.
This experiment revealed that the process of MRET activation did not have any significant effect on reductant activity of E.coli bacteria in anaerobic environment.
III. The stimulating effect of MRET water on metabolic activity of Complex Microbial Associations in anaerobic environment
In order to simulate the environmental conditions similar to the conditions in the intestine of humans and animals the test on metabolic activity of microbial associations was conducted in anaerobic environment. It was found that MRET activated water substantially increased reductant activity of complex microbial associations during the first several hours of experiment (Fig 11).
Fig 11: Comparative test on metabolic (reductant) activity of microbial associations (1.0 hour and 0.5 hour MRET- activated water and Control samples) in anaerobic environment: R - reductant activity (in Control, 0.5 hour and 1.0 hour MRET activated water), K - relative reductant activity, V - gas volume in the bottles.
This experiment revealed that the optimum time of activation for the maximum increase of metabolic activity of microbial associations in anaerobic environment was 30 minutes. The same optimum time of activation was found in the process of inhibition of metabolic activity of E.coli in aerobic environment and in another investigation regarding the application of MRET activated water for preventive treatment and enhancement of tumor resistance in vivo.
Conclusions
This investigation revealed that at a low initial concentration of cells of conditionally pathogenic microbiological culture Escherichia coli K-12 in water-based nutrient medium activated for 30 minutes and 60 minutes the growth of culture was inhibited 27 and 303 times respectively after the 25 hours of the experiment in an aerobic environment. This experiment also revealed the strong effect of MRET activated water on the process of division of E.coli microorganisms, the modification of forms of culture cells and the size of colonies. It was observed that one of the reasons for abnormally low growth of E.coli population was related to the modification of the process of cell division in MRET activated nutrient medium. These results allow admitting that the process of MRET activation and the sterilization effect of MRET water can be applied in the food industry and for water purification.
The second stage of investigation revealed that the metabolic (reductant) activity of E.coli bacteria reduced up to 3 times in 30 minutes activated water and up to 1.6 times in 60 minutes activated water during the first 6 hours of the experiment in an aerobic environment. Another experiment showed that the process of MRET-activation did not affect the reductant activity of E.coli bacteria in the anaerobic environment and, consequently, should not affect a small population of conditionally pathogenic bacteria, such as E.coli, usually presented in microbial associations in the intestine of the body.
In order to simulate the environmental conditions similar to the conditions in the intestine of humans and animals, the test on metabolic activity of complex microbial associations was conducted in an anaerobic environment. It was discovered that MRET activated process substantially increased reductant activity of complex microbial associations during the first hours of the experiment. The same 30 minutes optimum time of activation was observed in the process of inhibition of the metabolic activity of conditionally pathogenic E.coli bacteria in the aerobic environment and for the maximum increase of metabolic activity of complex microbial associations in an anaerobic environment (presented in the intestine). The previous investigation regarding the application of MRET activated water for preventive treatment and enhancement of tumor resistance in oncology in vivo on 500 mice also showed the best results on 30 minutes MRET activated water. Thus, this investigation shows that the ingestion of MRET water is beneficial for the process of digestion and can enhance the metabolism of the body.