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Antifungal Activity of Lactic Acid Bacteria, Isolated from (Mytilus Galloprovincialis Lam.) in the Bulgarian Black Sea Aquatory

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Antifungal Activity of Lactic Acid Bacteria, Isolated from (Mytilus Galloprovincialis Lam.) in the Bulgarian Black Sea Aquatory https://doi.org/10.5272/jimab.2020261.2875 Journal of IMAB Journal of IMAB - Annual Proceeding (Scientific Papers). 2020 Jan-Mar;26(1) ISSN: 1312-773X https://www.journal-imab-bg.org Original article ANTIFUNGAL ACTIVITY OF LACTIC ACID BACTERIA, ISOLATED FROM (MYTILUS GALLOPROVINCIALIS LAM.) IN THE BULGARIAN BLACK SEA AQUATORY Sevginar Ibryamova1, Nina Arhangelova2, Teodora Koynova1, Dimitar Dimitrov1, Zheni Dimitrova1, Radoslav Ivanov1, Karamphil Kalchev1, Nescho Chipev1, Nikolay Natchev1, Tsveteslava Ignatova-Ivanova1* 1) Department of Biology, Shumen University, Shumen, Bulgaria 2) Department of Physics and Astronomy, Shumen University, Shumen, Bulgaria.. ABSTRACT: Currently, aquaculture has become an important economic In the present study we present the results from an- activity in many countries. In large-scale production fa- tifungal activity of Lactic Acid Bacteria, isolated from M. cilities, where aquatic animals are exposed to stressful con- galloprovincialis Lam. from the Bulgarian Black Sea ditions, problems related to diseases and deterioration of aquatory. The Black Sea mussel (M. galloprovincialis environmental conditions often result in serious economic Lam.) is in fact the only marine species grown as losses. During recent decades, prevention and control of aquaculture in the Bulgarian section of the Black Sea. diseases have led to a substantial increase in the use of The mussels have an exceptional nutritional value, mak- veterinary medicines. However, the utility of antimicro- ing them highly suitable for the human diet. The cultiva- bial agents as apreventive measure has been questioned, tion of sea products, in particular the Black Sea clam, is given extensive documentation of the evolution of anti- becoming an important part of the Western Black Sea in- microbial resistance among pathogenic bacteria [1]. From dustry. With increasing of the tourist pressure and the con- a social point of view, safe food forms a significant prob- struction activities on the Black Sea coast, maritime pol- lem for modern civilization. The cultivation of sea prod- lution increased in the last decades. The pollutants in- ucts, in particular the Black Sea clam, had becom an im- clude various pathogenic microorganisms - Escherichia portant part of the Western Black Sea industry. The mus- coli, Staphylococcus aureus, Salmonella thyphimurium, sels are flter-feeding organisms and can concentrate bacte- Hymeniacidon perlevis, incl. the cholera cause (Vibrioo ria in high number [2]. The microbiota found in shellfishes cholera). Such pathogenic microorganisms are often can be divided into autochtonous and allochtonous mi- present in the mussels and can cause the spread of vari- croorganisms, and reflects the microbial population of the ous diseases. In this study 2 isolates Lactic acid bacteria, water in which they grow [3]. On the other hand, it has from M. galloprovincialis Lam have been identified as been found that seafood is a source of lactic acid bacteria Lactobacillus sp. and characterized as cultures with prom- (LAB), which synthesize substances with antimicrobial ising antifungal activity. The antifungal activity of our activity against many pathogenic microorganisms [2, 4]. new isolates Lactobacillus sp. lineages, seems to be a Consuming foods containing high quantities of chemical promising advantage, suggesting their potential applica- conservants provoked the interest in the use of natural and tions in different food technologies. minimally processed foods, with a focus on the use of natu- rally produced antimicrobial agents - bioconversants such Keywords: Bivalvia, Mytilus galloprovincialis Lam, as bacteriocins [5]. Bacteriocin producing LABs can im- probiotic bacteria, pathogens, Black Sea, prove the aquatic environment of the shrimp and fish aqua- cultures. The development of bacteriocinogenic strains and INTRODUCTION the isolation of bacteriocins from seafood and products Worldwide, people obtain about 25% of their ani- may result in valuable strains that are used both as mal protein from fish and shellfish and consumer’s demand bioconversants and as food (and feed) additives in aqua- for fish continues to climb. The Black Sea mussel (M. culture animals [6]. galloprovincialis Lam.) is the only species of mussels Currently, the food industry depends on chemical grown as aquaculture in the Bulgarian Black Sea aquatory. preservatives to extend the shelf life and control the growth Mussels have an exceptional nutritional value making them of spoilage fungi. The consumer’s awareness about the suitable for the human diet. Mussel flesh is rich in sele- health hazards associated with chemicals has recently in- nium, calcium, iron, magnesium, phosphorous and vita- creased, and they are demanding for processed foods that mins (A, B1, B2, B6, B12 and C). Mussel farming has an are free of preservatives. There are many natural alterna- important economic impact in many European countries. tive preservatives produced by certain microorganisms, J of IMAB. 2020 Jan-Mar;26(1) https://www.journal-imab-bg.org 2875 however these microorganisms have to be non-toxic, easy 18 h) on MRS at 370C and in limitation of oxygen (tubes to grow and require simple media for cultivation [7]. Lac- or Petri dishes with the strains were incubated in plastic tic acid bacteria (LABs) represent a known potential source bags, which limited the oxygen content). for generating a variety of secondary metabolits such as X-Gal-based medium bacteriocines, organic acids and peptides [8]. In the past The modified MRS agar was used as the medium for decade the interest for antifungal LABs has increased and the incorporation of 5-bromo-4-chloro-3-indolyl β-D- different studies have showed that many LAB strains have galactopyranoside X-Gal (Boehringer Mannheim, the potential to impact the proliferation of fungi in vari- Montréal, Canada) [14]. ous food and feed materials [9]. LABs, due to their long API 50CHL System Assay Initial identiûcation of history of safe use in food and feed fermentations, [10] all the strains was performed by API 50CHL system have received both GRAS (Generally Recognised as Safe) (BioMerieux, Craponne, France), according to the manu- and QPS (Qualified Presumption of Safety) status in the facturer’s instructions. The fermentation proûles were read EU. Therefore, LABs have high potential as antifungal after incubation at 37C in anaerobic conditions, for 3 days. biocontrol agents in the future. The antifungal effect by Test Microrganisms Aspergillus niger, Penicillium the culture supernatant of various LAB strains against few claviforme, Saccharomyces cerevisae, Candida albicans fungi including food-deteriogens has been studied by vari- 8673 and Candida glabrata 72 were obtained from the ous authors [7]. Several compounds have been proposed National Bank for Industrial Microorganisms and Cell Cul- as responsible for the antifungal activity of LAB, like or- tures, Sofia, Bulgaria. All the isolates were checked for ganic acids, low molecular weight compounds, phenylace- purity and maintained in slants of Nutrient agar. tic and fatty acids, cyclic dipeptides, proteinaceous com- Assay for Antifungal Activity Antifungal assay was pounds and other miscellaneous compounds e.g. lactones performed by the well disc diffusion method using soft [11]. The fungistatic activity of pentocin TV35b against 0.8% agar. Agar medium was added to sterile Petri dishes Candida albicans and of two LAB strains against Fusarium seeded with 100 µL of each test yeast and fungi strains. (isolated from cereals) has been reported [12]. Due to their Wells of equal distance were dug on the seeded plates. inhibitory spectrum, these peptides are considered to be Before the assays, the strains LAB were twice pre-cultured powerful antimicrobials to target bacteria, fungi and para- in MRS broth, for 24h at 37°C. Exponential Lactobacillus sites [13]. The use of probiotics or beneficial bacteria, cultures in MRS broth were used as inoculum for the anti- which control pathogens through a variety of mechanisms, fungal tests. The plates were incubated at 37°C for 48 h. is increasingly viewed as an alternative to antibiotic treat- The antifungal activity was assayed by measuring the di- ment. ameter of the inhibition zone formed around the well [15]. In this study 2 isolates Lactic acid bacteria, from M. All Antifungal Activity of Lactic Acid Bacteria, Isolated galloprovincialis Lam have been identified as Lactoba- from M. galloprovincialis Lam 3 experiments were per- cillus sp. and characterized as cultures with promising an- formed in triplicates. tifungal activity. The antifungal activity of the new iso- lates Lactobacillus sp., seems to be possess potential for RESULTS AND DISCUSSION applications in different food technologies. The microscopic pictures were performed using stereomicroscope OPTIKA (Italy) with a camera Canon MATERIALS AND METHODS EOS 60D. Place and duration of the study: The study was con- The microorganisms were isolated from M. gallo- ducted at the Department of Biology, University of Shumen, provincialis Lam. (figure 1) in the Bulgarian Black Sea Bulgaria, from September 2018 to December 2018. aquatory. The microscopic pictures were performed using Collection of samples stereomicroscope OPTIKA (Italy) with a camera Canon The mussels were harvested from northern part of EOS 60D. the Bulgarian Black Sea aquatory. After collection, the samples (about 10 kg) were immediately refrigerated (4°C) Fig. 1. General view of a dissected M. galloprovin- and transported to
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