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OPTIMAL PHYSIC-CHEMICAL FACTORS AND CULTURAL CONDITIONS FOR THE PRODUCTION OF ANTIMICROBIAL METABOLITES BY SOME STRAINS LACTIC
ACID BACTERIA
Kuliyev A.A.1, Katerina Demnerova2, Abdullayeva N.F.3, Abdullayeva U.C.4 ®
1 2 3
Baku State University; University of Chemistry and Technology; PhD;
4PhD student
Summary
Lactic acid bacteria widely distributed in the nature and play an important role in much food and feed fermentations with increased shelf life. In the present investigation the influence of different chemical and physical factors on the production of antimicrobial metabolites were studied. Like a physic-chemical factors temperature, pH, several enzymes were used and antimicrobial activity was tested against the indicator pathogenic strains of bacteria.
Keywords: lactic acid bacteria, enzymes, MRS broth, bacteriocins, well diffusion method.
Анотация
Молочнокислые бактерии широко распространены в природе и играют важную роль во многих процессах ферментации пищевых продуктов продлевая их срок хранения. В данной исследовательской работе было изучено влияние различных физико-химических факторов на образование антимикробных метаболитов. В качестве данных факторов выступают влияние температуры, рН и различных ферментов, была изучена антимикробная активность против ряда патогенных бактерий.
1. INTRODUCTION
Lactobacillus's are an order of gram-positive, acid-tolerant, generally nonsporulating, nonrespiring, either rod- or coccus-shaped bacteria that share common metabolic and physiological characteristics. Lactic acid bacteria widely distributed in the nature and occurring indigenous microflora in raw milk that play an important role in much food and feed fermentations with increased shelf life. Within the LAB group, the genus Lactobacillus is the most widely encountered for probiotics because they display numerous antimicrobial activities. This is mainly due to the production of antimicrobial metabolites including organic acids, hydrogen peroxide and bacteriocins. Among these, bacteriocins have gained increasing interest. Bacteriocins, as defined by Ogunbanwo et al, (2003) are proteinaceous compounds produced by bacteria that exhibit a bactericidal mode of action against related as well as unrelated organisms. Bacteriocin generally exert their antimicrobial action by interfering with the cell wall or the membrane of target organisms, either by inhibiting cell wall biosynthesis or causing pore formation, subsequently resulting in death [1, 2-4].
Among these are bacteriocins (e.g. nisin), antibiotics (e.g. reutericyclin) and small antibiotic-like molecules such as reuterin. The current need for biopreservation has renewed the interest in the search for food compatible antimicrobials produced by microorganisms [1, 2-4].
The important contribution of probiotic LAB in food preservation has been attracting much attention because of the nutritional qualities of the raw material through an extended shelf life of food and their ability to inhibit spoilage and foodborne pathogens, which is interesting for the food industry [1, 2-4].
2. MATERIALS AND METHODS
2.1 Collection and identification of microorganisms
The part ofexperiment was carried out at the University of Chemistry and Technology in Prague (Czech Republic) and the other part was done at Baku State University, Baku Azerbaijan.
® Kuliyev A.A., Katerina Demnerova, Abdullayeva N.F., Abdullayeva U.C., 2017 г.
Some strains for the research were conferred, from the collection of lactic acid bacteria of the Department of Biochemistry and microbiology and faculty of Food and biochemical technology of the University of Chemistry and Technology and other part was isolated from six sorts of cheese prepared in different regions of Azerbaijan. Antimicrobial metabolites and their production were studied by analysis of antagonistic relations between lactic acid bacteria strains and test-cultures of pathogenic microorganisms.
List of microorganisms used as a test- cultures in the experiment.
Strains and microorganisms Media
1. Candidapseudotropicalis ENITIAA YPD (Yeast extract/Peptone/Dextroza)
2. Echerichiacoli ATCC 23355 LB (Luria-Bertani) and BHI broth
3. Enterococcusdurans ENITIAA MRS(De Man, Rogosa and Sharpe)
4. Lactobacillusbulgaricus 340 MRSbroth
5. Lactobacilluscasei DSM 20011 MRSbroth
6. Lactococcuslactis spp. cremoris 5345 MRS broth
7. Listeriainnocua CIP 80.11 BH (Brain-Heart)
8. Sacchromycescerevisiae ENITIAA YPD
9. Staphylococcusaureus ENITIAA MRS broth
10. Lactobacillus reuteri 291 MRS broth
11. Lactobacillus fermentum G1 MRS broth
Lactobacillus crispatus G2, 313 MRS broth
Bacillus cereus BHI broth
Listeria ivanovii BHI broth
2.2 Preparation of strains of LAB and pathogenic microorganisms used as a test cultures.
Pathogenic cultures of bacteria Bacilluscereus, Listeriaivanovii, Staphylococcus aureusuEcherichia coli ATCC 23355, Candida pseudotropicalis ENITIAA for the research were conferred from the collection of lactic acid bacteria of the Department of Biochemistry and microbiology and faculty of Food and biochemical technology of the University of Chemistry and Technology. They were cultivated on BHI broth and YPD (Yeast extract/Peptone/Dextrose) for 18 -24 hours at the temperature 37 °C and used as test-pathogenic microorganisms.
Strains of Lactobacillus and Enterococcus were cultivated on MRS broth for 24 hours at the temperature 37 °C in anaerobic conditions with the present of 5% CO2 [2, 3-6; 3, 3-4].
2.3 Evaluation of antimicrobial activity of Lactobacillus and Enterococcus strains by well diffusion method
The antimicrobial spectrum of Lactobacillus and Enterococcus cultures were screened for their antagonistic activity against bacteriocin sensitive strains. An overnight culture of each isolate were grown in MRS broth at 37°C and standardized to an optical density of 0.5 at a wavelength of 600 nm (spectrophotometer). One percent of standardized culture was used to inoculate MRS broth. After incubation at 37°C for 24 hrs, cells were removed by centrifugation at 10,000 rpm for 15 min. The pH of one portion of supernatant was adjusted to 7.0 and filtered through 0.22 mm membranes. The filtrates were used to evaluate antimicrobial activity method by agar well diffusion method. Positive results were recorded when the zone of inhibition of at least 1 mm around the wells was observed [3, 4-5].
2.3.1 Well diffusion assay
The antimicrobial activity of the isolated LAB (cell free filtrate) against (Escherichia coli, Staphylococcus aureus, Listeria ivanovii, Bacillus cereus, Bacillus cereus), was performed by the well diffusion assay. The pathogenic test bacteria were incubated in BHI broth at appropriate temperature for 24 hrs. petridishes containing 20 ml of MRS agar were prepared previously and inoculated with 0.1 ml of 24 hrs. broth culture of pathogenic bacteria. Once solidified the dishes were stored for 2 hrs. in a refrigerator. Four wells were made and filled using different
concentration like 25 pi, 50 pl, 75 pl, 100 pi of cell-free filtrate and the petridishes were incubated at 37°C for 24 hrs. Then the diameter of the inhibition zone was measured with calipers in mm. The antimicrobial activity was determined by measuring the clear zone around the wells [2, 3-5].
2.4 Effect of various cultural conditions on the metabolite production and activity of crude supernatant
The selected lactic acid strains were subjected to different culture conditions to derive optimum conditions for crude supernatant production. To study the effect of varying culture conditions, growth and crude supernatant production and thus bacteriocin activity was estimated at varied temperatures (15, 30, 37, and 45°C) pH (5.0, 6.0, 7.0 and 8.0), sodium chloride (NaCl) concentrations (2.0, 4.0, 6.0 and 8.0% w/v) and duration of incubation (24, 48) in MRS broth. All samples were collected after 48hrs, except for those measuring incubation time effects before inhibitory activity was determined by agar well diffusion assay as described above [1, 4-6].
2.4.1 Kinetics of the production of bacteriocins by selected strains LAB
Thekinetics of the production of bacteriocins was tested at optimal growth temperature of isolated bacteria. For this reason the 10 ml of bacterial culture was inoculated into 200 ml of MRS -broth at define meaning of ph. The dynamics of the growth were controlled horal by determination of intensity at wavelength 620 nm. Parallel the cultural suspension was measured as well.
After centrifugation, neutralization and filtration the antimicrobial activity of cultural suspension was measured as mentioned before [1, 4-6].
2.4.2 Effect of the temperature and pH on the production of antimicrobial metabolites
The effect of pH and temperature on the metabolite production was studied by inoculating
24 h-48 h old culture in MRS broth. An overnight culture of each isolate were grown in MRS broth at 37°C and standardized to an optical density of 0.5 at a wavelength of 600 nm (spectrophotometer). One percent of standardized culture was used to inoculate MRS broth. After incubation at 37°C for 24 hrs.,cells were removed by centrifugation at 10,000 rpm for 15 min. The pH of one portion of supernatant was adjusted to 7.0 and filtered through 0.22 mm membranes. Obtained supernatant was treated by catalase with the final concentration 1 mg/ml, later the pH of supernatant was adjusted to 6.5 with the help of NaOH and HCl acid. For the temperature study strains, were tested at the temperature range from 15 °С -45°Cand at extreme conditions as well, at boiling temperature. The experiments were repeated with each producer strain of Lactobacillus and Enterococcus respectively a few times. The production of antimicrobial metabolites was tested for antimicrobial activity after 120 h of incubation by well diffusion assay against B. subtilis, L.ivanovii, E.coli, S.aureus[1,4-6].
2.4.3 Influence of different enzymes on the activity of antimicrobial agents and their production
Sensitivity of each supernatant with antimicrobial activity to the influence of proteolytic enzymes were tested by treatment of cultural liquids by the solutions of proteinase K, lipase 7, amylase - 2-A during 90 minutes at the temperature 37 °С.
The solutions of used enzymes with the final concentration of 1mg/ml were prepared in sterile 20 mM K-phosphate buffer (pH=7). Buffer solution without enzyme was used as a control one. After incubation, the activity was determined by well diffusion method.
Sensitivity to high temperature was determined by thermal treatment of supernatant during 20 - 60 minutes at the temperatures 20 °С, 60 °С, 80 V and 100 °С and autoclaved for 15 minutes (1 amp.121°C)
3. RESULTS AND DISCUSSION
Total seven strains of lactic acid bacteria were used for the determination of optimal physical, chemical and cultural conditions for cultivation of strains of lactic acid bacteria. The results of antimicrobial activity checking, have shown, that all the strains of Lactobacillus show the antimicrobial activity against the pathogenic test microorganisms (Bacilluscereus, Listeriaivanovii, Staphylococcus aureusи Escherichia coli) with the different spectra of activity. The inhibition zones of between 6 -18 mm were observed.
Based on the influence of the temperature on the production of antimicrobial metabolites were obtained that at 15°C the production of metabolites is observed after 10 hours in a small amount (10 per/ml). Future cultivation of bacteria is results again in a small increase of the amount of produced metabolite.
At the temperature 45 °C, the high antimicrobial activity of bacteriocin is observed in comparison with the temperature 15 °C and make 80 per/ml of cultural liquid, which is 8 times higher than previous one. In this case, the activity is observed earlier than in first case, after 6 hours of cultivation.
The influence of pH on the production of bacteriocinsof L. paracasei spp. ParacaseiBN ATS5wis also important point in the production of antimicrobial metabolites. The approximate meaning of pH was maintained my 5M of NaCl. Based on this we chose the pH range between 4.56.5.
At the diagram 2 we can see the production of metabolite at pH 4.5 by strains ofL.paracasei spp. ParacaseiBN ATS 5w.
Рис. 3.6. Growth dinamics (•) and synthesis of (■) stmmL.paracasei spp. ParacaseiBN ATS 5w
(test-culture L.bulgaricus340, рН4.5)
Based on the composition of media 2 typesof media were used (MRS agar orBHI agar) were used with different concentration of agar inside (15% and 8%). The high production and more antimicrobial activity against pathogen strains were observed in the types of media with the less concentration of agar inside.
Therefore, the optimal pH for the production and collection of antimicrobial metabolites is 5.5. At boiling temperature, which was considered as an extreme condition the production of antimicrobial metabolite, was not observed because of protein nature of the metabolites.
Literature
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