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Bio-Products against Dangerous Infections

#A-1156


Study of the Mechanism of Mycocins and Bacteriocins Action on Especially Dangerous Infections (Cholera, Plaque, etc.) and Elaboration of a New Biopreparation Production Technology

Tech Area / Field

  • BIO-MIB/Microbiology/Biotechnology
  • MED-DRG/Drug Discovery/Medicine

Status
3 Approved without Funding

Registration date
18.05.2004

Leading Institute
Scientific and Production Center "Armbiotechnology" NAS RA, Armenia, Yerevan

Supporting institutes

  • Center of Prophylaxis of Especially Dangerous Infections, Armenia, Yerevan

Collaborators

  • CNRS / Universite Henri Poincare, France, Vandœuvre lés Nancy\nLawrence Livermore National Laboratory, USA, CA, Livermore\nPacific Northwest National Laboratory, USA, WA, Richland

Project summary

Study of microorganisms’ metabolic products, their biological activity opens new opportunities in their application spectra.

Antagonistic properties of a wide variety of bacteria and yeasts as probiotics are known for a long time, however practically there are no researches on their usage against several pathogens of especially dangerous infections (EDI), cholera, plague, anthrax, etc. These microorganisms are the bioterrorism weapons; hence today’s actual problem is combating against them Strong disinfectant substances, practically poisons, such as sublimate, lysol, chlorine-containing substances, phenol, various acids are used as means against EDI pathogens [1,2]. However, the chemical agents affect an environment, ecology and people working with pathogens [3]. Therefore new biological methods of inhibition are of significant interest nowadays. Researches were carried out in the Institute of Biotechnology in cooperation with the Center of Prophylaxis of Especially Dangerous Infections (CPEDI) to study the effect of metabolic products of the Saccharomycopsis fibuliger SP 13 and Lactobacillus acidophilus 1991 bacteria on several EDI pathogens growth: Yersinia pestis 3344, Vibrio cholerae eltor 2590, S. enterocolitica 2840, Brucella abortis 544, Bacillus anthrocoides, etc., as well as Pseudomonas, Shigella, Salmonella, Staphylococcus.

Antagonistic properties of the strain Lactobacillus acidophilus against the pathogenic microorganisms causing digestive tract (DT) diseases have been known for a long time and the treatment and prophylactic products derived on their basis had a wide circulation [4,5].

Our team has been using the yeast strain Saccharomycopsis fibuliger SP 13 as a producer of fodder yeasts within 5 years. The effect of this strain was observed in decrease of DT diseases in birds and pigs and as consequence increase in animals’ weight. Certificates (protocols) and publications testify these facts. Therefore it was natural to assume the existence of antagonistic properties in fodder yeast strain against several pathogens causing DT diseases in birds.

The cultivation of the studied strains Saccharomycopsis fibuliger SP 13 and Lactobacillus acidophilus 1991, study of their metabolic products has shown that in cultural liquors there were the substances (bacteriocins and mycocins) with antagonistic properties against a number of microorganisms [6].

The bioinhibitors, extracellular biologically active substances, obtained after biomass separation, inhibited the microorganisms growth in 90% in case of Lactobacillus acidophilus 1991, and in 45% in case of Saccharomycopsis fibuliger SP 13. Their metabolic products were of wide spectrum of action on microbes of various toxicological groups, in particular, they inhibited the growth of some microorganisms, causing foodstuff spoiling, a few representatives of spore Bacillus cultures, a number of phytopathogenic fungi [7].

Preliminary data (fractioning method) have shown, that inhibiting substances extracted from Lactobacillus acidophilus 1991 and Saccharomycopsis fibuliger SP 13 cultural liquors, could appear the low-molecular weight peptides (mycocins and bacteriocines), which as separate fractions inhibit the growth of the studied microorganisms [6]. The isolated three fractions from the Lactobacillus acidophilus 1991 were thermostable up to 120˚C for 45 min. and their maximum effect against spore form was observed at pH=3.5-4.5. The isolated two fractions from the Saccharomycopsis fibuliger SP 13 were thermostable up to 70˚C for an hour and their maximum effect against spore culture was observed at pH=7.8. The preliminary experiments demonstrated that the secondary metabolism products of the studied Saccharomycopsis fibuliger SP 13 and Lactobacillus acidophilus 1991 in CL inhibited the growth of a number of EDI pathogens, particularly, Yersinia 3344, Vibrio cholerae eltor 2590 and microorganisms causing infections in vitro, whereas the antagonistic effect of the cultures themselves wasn’t significant. The fact that both of the studied cultures and their fractions inhibited the growth of the various taxonomic groups not similarly, had different thermostability and pH range but successfully cultivated in the nutrient medium of the similar composition suggested an idea of joint cultivation technology for producing the cultural liquor of both inhibitors of wide spectrum. There are numerous literary data on joint cultivation of yeasts and lactic acid bacteria. However elaborations on aimed cultivation followed by extraction of the biologically active substances with antagonistic action on the EDI lack.

The present project by our opinion is of scientific and practical interest, hence the further profound basic research of the nature and the bioinhibitors-microorganisms interaction mechanism for elaboration of the commercial preparation is of utmost importance.

Three specific tasks are an object of research of the proposed project:

1. Elaboration of the technological process including joint cultivation of the obtained yeast strains and bacteria; product isolation and purification; economical and technological expediency characteristics of its application.


2. Comparison of the active substances nature; evaluation of spectral characteristics and their physical and chemical properties, mechanisms of interaction with cellular membrane and DNA, ribosome (in vitro).
3. Biopreparation tests in lab. conditions for evaluation of its potential application against EDI pathogens and various infections (in vivo).

The nature of the bioinhibitor will be determined by gel filtration method, electrophoresis in polyacrylamide gradient density, UV- and VIS-spectroscopy, circular dichroism method. The following parameters as molecular weight, amino acid structure, isoelectric point, spectral characteristics, specific rotation and enantiomeric analysis of amino acids will be measured.

Comparison of the obtained data will allow revealing:

1. General mechanism of the antagonistic effect of the metabolism products (bacteriocines and mycocins) of the studied yeasts and bacteria on the microorganisms growth; bioinhibitor nature.


2. Their wide application spectra against EDI pathogens and various infections.

The principle executors of the project are former weapon researches experienced in elaboration of biologically active substances (amino acids, proteins, enzymes) production technologies which were successfully introduced at the facilities in Russia, Ukraine, Byelorussia, China, Germany and France. At the certain stages the experts will be involved from other institutions of RA. Participation of young specialists will allow preparing the highly skilled personnel in biotechnology and medical microbiology.


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