INHIBITORY EFFECT OF ESSENTIAL OILS ON THE GROWTH OF GEOTRICHUM CANDIDUM
Denisa Foltinová1, Dana Tančinová*1, Miroslava Císarová2
Address(es): prof. Ing. Dana Tančinová, PhD., 1 Slovak University of Agriculture, Faculty of Biotechnology and Food Sciences, Department of Microbiology, Tr. A. Hlinku 2, 949 76 Nitra, Slovak republic. 2 University of SS. Cyril and Methodius, Department of Biology, Faculty of Natural Sciences, Nám. J. Herdu 2, SK-91701 Trnava, Slovak republic.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2019.9.special.380-384
ARTICLE INFO ABSTRACT
Received 27. 8. 2019 The aim of this study was focused on the determination of the inhibitory effect of selected essential oils on the growth of five isolates of Revised 9. 10. 2019 Geotrichum candidum. The isolates were obtained from the dairy products of domestic origin (sour cream, raw cow's milk, base for Accepted 10. 10. 2019 production of the brand, unsalted cheese, and cheese made from cows' milk). We studied the impact of five essential oils [pure essential -3 Published 8. 11. 2019 oil EOs (100 %)] - clove, basil, rosemary, fennel and thyme at concentration of 0.625 μL.cm of air. Gas chromatography-mass spectrometry (GC-MS) analysis was used for the identification of the main components of EOs. We tested the effect of the essential oils by the gaseous diffusion method. The isolates were cultivated on PDA (Potato dextrose agar), in the dark, at 25 ±1 °C, 14 days. The Regular article diameter growing of colonies we continuously measured on the 3rd, 7th, 11th, and 14th day of cultivation. The results of the paper suggest that clove and thyme essential oil had 100 % inhibitory effect on the growth of all tested isolates. Fennel, basil and rosemary essential oil had not significant inhibitory effects on tested isolates Geotrichum candidum. However, these oils affected the growth of colonies throughout the cultivation. Fennel EO had 100 % inhibition only on the growth of one isolates of Geotrichum candidum to the 3rd day of cultivation. Basil EO had the stimultating effect on the growth of two isolates of G. candidum (KMi 322 and KMi 189). Our results showed that clove and thyme essential oil could be used as a natural preservative useful in the food industry.
Keywords: essential oils, inhibitory effect, Geotrichum candidum
INTRODUCTION phase of five essential oils on the growth of different strains of Geotrichum candidum isolated from moldy milk products. The genus Geotrichum is classified as microscopic filamentous fungi (Pitt and Hocking, 2009, Samson et al., 2010), but also as yeast (Boutrou and Guéguen, Scientific hypothesis 2004) and some characterize it as a filamentous fungus that resembles yeast (Pottier et al., 2008). G. candidum is generally associated with milk and milk The aim of the present research was to determine the inhibitory effect of five products. Its occurrence in food has no clear meaning. Its presence in certain dairy essential oils on the growth of different Geotrichum candidum isolates. products such as butter, a cream, fresh cheese is considered undesirable (Laurenčík et al., 2008).Moulds are common contaminants of milk and milk MATERIAL AND METHODS products and contribute to their spoilage. Today, it is very important to find out the protection of products of natural origin as an alternative to synthetic fungicides. Fungal isolation and identification The promising alternative is the use of the essential oils. Essential oils from plants have great potential as a new source of fungicide to control the pathogenic fungi Five strains (Table 1) from different moldy milk products of domestic origin were (Císarová et al., 2016a). Essential oils have deserved much attention in the past used. These strains belong to the Collection of Fungi of Department of decades for their antimicrobial activity, since many of them have demonstrated Microbiology; Faculty of Biotechnology and Food Sciences SUA in Nitra, efficacy against food-borne pathogenic and spoilage microorganisms (Bassanetti Slovakia. Isolates of the genus Geotrichum were identified to the species level et al., 2017). Essential oils have been identified as natural food additives which according to morphological characteristics based on microscopy. To determine can find useful application in food preservation (Davidson et al., 2013). particular species, diagnostic literature was used as follows: Pitt and Hocking Nowadays, natural biological preservatives are mainly extracted from edible (2009), Samson et al., (2010). Strains were inoculated in three points on the spices, and the essential oils from these spices have been found to be effective identification media MEA (malt extract agar). Inoculated media were incubated at preservatives (Burt, 2004). Natural flavours, this is primarily the essential oils, as 25 ± 1 °C, 7 days in the dark. well as individual aromatic components that are derived from essential oils by physical methods. Gas chromatogra-phy coupled with mass spectrometry (GC– Table 1 Origin of the strains Geotrichum candidum MS) and flame ionizationdetector (GC-FID) are used for chemical composition Strains Origin analysis ofisolated oils (Matos, 2012; Smelcerovic et al., 2013; Frolova et al., G. candidum KMi 329 sour cream 2019). Both the essential oil and some of its components, e.g. carvacrol, thymol, G. candidum KMi 120 raw cow's milk gamma-terpinene, alpha-terpinene, or para-cymene, have well-documented G. candidum KMi 189 base for production of the brand biological activity. Carvacrol exerts antimicrobial (Kotan et al., 2012), antifungal, G. candidum KMi 322 unsalted cheese antioxidant, analgesic, and antiinflammatory effects (Moghadam, 2015). Another G. candidum KMi 172 cheese made from cows' milk phenolic compound present in the essential oil, i.e. thymol, has antimicrobial, expectorant and disinfectant effect (Moghtader, 2012). Limited information is available on the antifungal activity of plant extracts against Geotrichum candidum. The aim of the present research was to determine the inhibitory effect of vapor
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Essential oils Statistical analysis
The essential oils used in this study were obtained from clove (Syzygnium All analyses were performed in triplicate and the results were expressed as the aromaticum L.), basil (Oscimum basilicum L.), rosemary (Rosmarinus officinalis mean of the data obtained in each replicate. Statistical analyses were performed L.), fennel (Foeniculum vulgare L.), thyme (Thymus vulgaris L.). The used oils with descriptive statistics. were supplied by Calendula company a.s. (Nová Ľubovňa, Slovakia) and Hanus (Nitra, Slovakia). All essential oils were extracted by hydrodistillation and stored RESULTS AND DISCUSSION in air-tight sealed glass bottles at 4°C. In our study were isolated the genus Geotrichum from different moldy milk Antifungal activity of essential oils products of domestic origin. Based on phylogenetic and morphologilcal studies were identified to the species of Geotrichum candidum. The colonies of all The antifungal activity of selected essential oils was investigated by isolates had relatively rapidly growth on MEA with diameters between 50–65 microatmosphere method. The test was performed in sterile Petri dishes (Ø 90 mm) mm, softer, yeast-like texture (Fig. 1A). Conidiophores were undifferentiated containing 15 ml of CYA. Evaluation by filter paper was made by the method hyphae, fragmented almost to form arthroconidia, which were hyaline and adapted from Guynot et al., (2003). Essential oils were tested in concentration cylindrical (Fig. 1B). 0.625 μL.cm-3 of air. A round sterile filter paper (Ø 9 cm) was placed in the lid of Petri dish and 50 μL of essential oil was pipetted by micropipette to the paper. Dishes were kept in inverted position. Filter paper discs impregnated with sterilized distilled water were used as a control to confirm no solvent effect of bioactivity. Each isolate was inoculated in the center of Petri dishes with needle. Dishes were tightly sealed with parafilm and incubated for seven days at 25 ±1 °C (three replicates were used for each treatment). Diameters (Ø mm) of the growing colonies were measured at the 3rd, 7th, 11th and 14th day with a ruler. The antifungal activity was expressed in terms of percentage of mycelial growth inhibition and calculated according to the following formula:
Mycelial growth Inhibition: MGI % = [(dc-dt)/dc] *100 Where dc=average (mm) increase in mycelial growth in control, dt=average (mm) increase in mycelial growth in treatment (Marandi et al., 2011).
GC-MS analysis of essential oils
Essential oils constituent were identified and the relatively composition of the oil Figure 1 Geotrichum candidum - 7 days of cultivation at 25 ± 1 °C in the dark, was determined by gas chromatography followed by mass spectrometry (GC-MS). colonies on MEA (A), conidia (B) Prior to the analysis, essential oils were diluted in hexane to a concentration of l μL/mL. Analyses were carried out using an Agilent 7890A GC coupled to an Currently, there is a growing interest in using new methods in food industry. Agilent MSD5975C MS detector (Agilent Technologies, Palo Alto, CA, USA) These methods include the use of essential oils as preservatives to prolong food with a HP-5MS column (30 m × 0.25 mm, 0.25 mm film thickness). One microliter durability (Adelakun et al., 2016). Many species effectively inhibit the growth of the sample was injected in split mode 1:12, at an injector temperature of 250°C of some pathogens such as e.g. Escherichia, Shigella, Enterococcus, Bacillus, and at electron ionization energy of 70 eV. Analysis were measured in SCAN Pseudomonas, Streptococcus, because of their excellent antimicrobial properties mode, mass range was 40-400m/z. Starting at 60°C, the oven temperature was (Burt, 2004). However, essential oils are also characterized by their antifungal increased at a rate of 3°C/min to a maximum of 231°C, where it was kept constant activity. This activity can be maintained even in the gas phase (Reyes-Jurado for 10 min. The identification of constituents was based on a comparison of their et al., 2019). The aim of our paper was to determine the activity of volatile mass spectra and relative retention indices (RI) against the National Institute of components of selected essential oils - clove (Syzygnium aromaticum L.), basil Standards and Technology Library (NIST, USA), as well as authentic analytical (Oscimum basilicum L.), rosemary (Rosmarinus officinalis L.), fennel standards and data from the literature. Relative proportions were calculated by (Foeniculum vulgare L.) and thyme (Thymus vulgaris L.) on the growth of five dividing individual peak area by total area of all peaks. The response factor was Geotrichum candidum isolates. Geotrichum candidum isolates reacted not taken into account. Only compounds over 1% were included. Peaks under 1% differently to the presence of the EOs. Among the essential oils, the ones that were not counted. The following standards were used: α-pinene, Camphene, β- totally inhibited growth of all tested isolates were clove and thyme regardless of Pinene, p-Cymene, (R)-(+)-Limonene, 1,8-cineole, terpinolene, linalool, geraniol, time of the days of cultivation. Basil, rosemary and fennel were also able to (−)-Bornyl acetate, Trans-anetole, Eugenol, β-caryophyllene, α-caryophyllene and inhibit the growth of mould, but depending of the isolates and time of incubation. Caryophyllene oxide. The results are presented in Table 2.
Table 2 Mycelial growth inhibition (%) of Geotrichum candidum by tested essential oils after 14th days of cultivation Tested isolates Essential oils Mycelial growth inhibition (%)
KMi 329 KMi 120 KMi 189 KMi 322 KMi 172 Clove 100 100 100 100 100 Basil 0 1.51 -7.59 -2.62 7.81 Thyme 100 100 100 100 100 Rosemary 2.81 -9.56 19.62 12.67 6.94 Fennel 8.43 -1.09 10.97 13.68 28.19
The rosemary and fennel essential oil showed only partial inhibitory effects on the when the thyme and clove essential oils showed 100% antifungal activity growth of G. candidum KMi 329 (Fig. 2). Basil EO had no effect on the growth of regardless of the culture day. Kalemba and Kunicka (2003) report that the this isolate after 14 days of cultivation. The most resistant isolate was G. candidum antibacterial activity of thyme, cinnamon and rosemary has been demonstrated KMi 120. Its growth was stimulating by two EOs - rosemary and fennel after 14 against undesirable pathogens of Agrobacterium tumefaciens and Erwinia days of cultivation. In the presence of basil EO its growth was slightly inhibited carotovora. Božik et al., (2016) investigated the antifungal action of thyme (but (1.51%). Also the stimulating effect was detected in the presence of basil essential also other essential oils) on the growth of three species of the genus Aspergillus oil on the last day of cultivation (14th) for both, G. candidum Kmi 189 (-7.59%) (A. parasiticus, A. flavus and A. clavatus). This study confirmed the 90% and KMi 322 (-2.62%). The isolates G. candidum KMi 322 and KMi 189 have also antimicrobial action of thyme essential oil at a concentration of 500 µl/l air for the similar responses to the presence of fennel essential oil. The most sensitive isolate growth of Aspergillus microscopic filamentous fungi. Very strong inhibitory effect was the G. candidum KMi 172. The fennel essential oil showed the most potency of thyme is results based oil (Císarová et al., 2016a) was confirmed against A. after thyme and clove EOs (28.19%) Basil and rosemary had only a partial niger and A. tubingensis. This essential oil was able to completely inhibit the inhibitory effect on the growth of the strain throughout the cultivation period. growth of all tested isolates using a minimum inhibitory dose of 125 µl.1-l. The Essential oils that have the strongest antifungal activity include clove, oregano, 100 % inhibition effect of thyme, red thyme, mint, and savory essential oils (625 cinnamon and thyme oils (Taylor, 2015). This allegation has also been confirmed μL.L-1 of air) against Rhizopus spp. has shown Tančinová et al. (2018). Based on
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our results, basil essential oil showed partial inhibitory effects on the growth of all tested strains. In the case of the two tested isolates (KMi 189 and KMi 322), there were also stimulatory effects. However, Kocić-Tnackov et al., (2012) report that basil essential oil is effective against the Penicillium species, in particular P. aurantiogriseum, P. glabrum, P. chrysogenum and P. brevicompactum. Based on the growth of tested isolates during the cultivation days under treatments with EOs, we constructed growth curves for each isolate. The results showed that EOs inhibit the tested isolates of G. candidum differently in depend of the isolates and time of cultivation. From day 7 of cultivation, the diameter of the colonies increased gradually. When using rosemary essential oil, the growth of colonies has increased since the beginning of the cultivation, and on the last day of the cultivation we also noticed the stimulating effects on mycelium growth.
Figure 4 Antifungal activity of tested EOs (0.625 μL.cm-3) to G. candidum KMi 189
Fennel had a potent inhibitory effect by day 3 of cultivation on the growth of isolate KMi 322. The basil EO had the lowest antimicrobial effect on the growth of this isolate during the cultivation period, with stimulatory effect on the last day of cultivation (Fig. 5). The fennel EO showed 100 % inhibition effect on 3rd day of cultivation on the growth of isolate KMi 172, but the inhibitory effect gradually decreased over the next days of cultivation (Fig. 6).
Figure 2 Antifungal activity of tested EOs (0.625 μL.cm-3) to G. candidum KMi 329
Fig. 3 shows the growth curves of G. candidum KMi 120. In the presence of fennel essential oil on day 3 of cultivation, the colony size was low. From day 7 of cultivation, the diameter of the colonies increased gradually. When using rosemary essential oil, the growth of colonies has increased since the beginning of the cultivation, and on the last day of the cultivation we also noticed the stimulating effects on mycelium growth.
Figure 5 Antifungal activity of tested EOs (0.625 μL.cm-3) to G. candidum KMi 322
Figure 3 Antifungal activity of tested EOs (0.625 μL.cm-3) to G. candidum KMi 120
Fig. 4 shows the average efficiency of essential oils for the growth of G. candidum KMi 189. The stimulating effect was detected in the presence of basil essential oil on the last day of cultivation, with the lowest growth inhibitory effect of this isolate. Císarová et al., (2016b) observed the ability of basil to inactivate the growth of representatives of the genus Aspergillus. In this study, the antifungal Figure 6 Antifungal activity of tested EOs (0.625 μL.cm-3) to G. candidum KMi effect of this oil showed up only for a limited period of time, because from the 7th 172 day the growth of the isolates did not suppress. The antifungal activity of essential oils depends largely on their chemical composition (Coban et al., 2018). Therefore, in our study, we subjected tested essential oils to qualitative and quantitative analysis by gas chromatography. Qualitative and quantitative analysis of the tested EOs is listed in Table 3.
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Table 3 Qualitative and quantitative analysis (%) of the used essential oils by GC-MS RIb Component Clove Basil Thyme Rosemary Fennel 938 α-pinenea 2.5 10.3 2.1 953 Camphenea 4.8 981 β-Pinenea 7.2 1029 p-Cymenea 39.1 3.0 1033 (R)-(+)-Limonenea 2.6 5.1 1034 1,8-cineolea 3.5 42.9 1090 terpinolenea 4.5 1101 linalola 1.2 5.1 1148 (−)-Isopulegol 13.1 1192 α-terpineol 1.1 1200 Estragol 88.6 4.4 1257 geraniola 1.1 1287 (−)-Bornyl acetatea 1.3 1289 Trans-anetolea 79.9 1298 (+)-Menthofuran 43.1 1363 Eugenola 82.3 1419 β-caryophyllenea 6.0 3.6 1454 α-caryophyllenea 1.6 1531 acetyleugenol 7.0 1574 Caryophyllene oxidea 1.3 total 98.90 97.80 97.70 98.00 99.90 Legend: a – Identification confirmed by co-injection of authentic standard; b – RI: identification based on Kovatś retention indices (HP-5MS capillary column) and mass spectra
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INHIBITORY EFFECT OF ESSENTIAL OILS FROM SOME LAURACEAE SPECIES ON THE GROWTH OF PENICILIUM COMMUNE
Denisa Foltinová1, Dana Tančinová*1, Miroslava Císarová2
Address(es): prof. Ing. Dana Tančinová, PhD., 1Slovak University of Agriculture, Faculty of Biotechnology and Food Sciences, Department of Microbiology, Tr. A. Hlinku 2, 949 76 Nitra, Slovak republic. 2University of SS. Cyril and Methodius, Department of Biology, Faculty of Natural Sciences, Nám. J. Herdu 2, SK-91701 Trnava, Slovak republic.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2019.9.special.385-389
ARTICLE INFO ABSTRACT
Received 5. 9. 2019 The aim of this study was to determine the inhibitory effect of four essential oils (EOs) on the growth of seven strains of Penicillium Revised 16. 10. 2019 commune isolated from moldy milk products by contact vapour. Next objective was to determine the minimum inhibitory doses (in vitro -1 Accepted 22. 10. 2019 and probit analyses) of EOs, which at concentration 625 μl.l of air completely inhibited the growth of all strains. The antifungal activity Published 8. 11. 2019 was evaluated by the micro-atmosphere method. Cinnamon, cinnamon bark and litsea cubeba EOs completely inhibited the growth of strains during cultivation at 25 °C and 5 °C. Laurel EO had no effects on the growth of P. commune strains. EOs that completely inhibit the growth of all strains were used to determine their minimum inhibitory doses (MIDs). The best results were obtained for cinnamon EO Regular article (MIDs 15.625 µl.L-1 of air) after all days at 25 °C of incubation. Litsea cubeba EO had the highest MIDs value (from 250 to 62,5 μl.l-1 of air). It was found that EOs have different effects on individual strains of P. commune. The tested strains (KMi 370 and 402) showed differences among themselves even though they came from the same products (sour cream). According to probit analyses, the most effective tested EOs were cinnamon EO and the least effective was litsea cubeba EO. The antitoxinogenic effect of tested EOS was evaluated by TLC method. Also in this study, cinnamon EO was the most effective against CPA production by P. commune. This oil affected the production of CPA by strain KMi177 at 31.25 μl.l–1 of air completely, and by strain KMi 403, respectively.
Keywords: P. commune, essential oils, antifungal activity, vapor phase, cyclopiazonic acid
INTRODUCTION and proteins. So their antimicrobial effect can be reduced, and this is the reason for use height concentration of EOs in food application. But EOs have intense aroma Mould growth on cheese represents both a quality and a food safety problem and and their utilization at high concentrations in order to compensate their interaction several moulds may destroy cheese. The genus Penicillium is the major with food components could result in sensory defects (Hyldgaard et al., 2012). In contaminant followed by the genus Aspergillus (Kure and Skaar, 2019). order to rectify this shortcoming, various approaches have been proposed. For Penicillium spp. is the predominant contaminant of dairy products (Hansen et al., these reasons, the aim of the presented study is to assess the antimycotic activity 2003). It is because it’s well-adapted to grow in the cheese matrix. The growth of of some EOs against P. commune strains spoiling selected dairy products from molds in cheese is associated with undesirable taints and odors, liquefaction of the Slovakia and evaluating their activity at lowest concentration by using Minimum curd and in some cases, production of mycotoxins. P. commune (42%) is one of inhibitory doses. the most widespread microscopic filamentous fungus from genus Penicillium, which is able to involved the degradation of cheese (Kure et al., 2001). Moreover, MATERIAL AND METHODS P. commune is able to produce cyklopiazonic acid. Some of the mycotoxins have been shown to be stable under normal processing conditions of cheese (Martín Eseential oils analysis and Liras, 2017; Casquete et al., 2019). Natural preservatives have proven popularity such that interest continues in substituting chemical additives with The essential oils (EOs) used in this study were obtained from cinnamon natural. Therefore, production of safe food without or with low amounts of (Cinnamomum zeylanicum L.), cinnamon bark (Cinnamomum zeylanicum L.), synthetic preservatives is one of the most important challenges in the food industry laurel (Laurus nobilis L.) and litsea cubeba (Litsea deccanensis L.) by (Khorshidian et al., 2018). Essential oils (EOs) are mixtures of aromatic oily hydrodistillation. The essential oils were supplied by Calendula company a.s. liquids extracted from different parts of plants, usually by steam distillation. (Nová Ľubovňa, Slovakia) and Hanus (Nitra, Slovakia). All EOs were stored in Several of them are characterized by antibacterial and antifungal activities. This air-tight sealed glass bottles at 4°C. Essential oils constituent were identified and activity is depending on their constituents, mainly due to their content in terpenes the relatively composition of the oil was determined by gas chromatography and phenylpropanoids (Seow et al., 2014; Nikkhah et al., 2017). Several essential followed by mass spectrometry (GC-MS) s described by Božik et al. (2017). The oils can be applied as natural antimicrobial agents in order to inhibit microbial identification of constituents was based on a comparison of their mass spectra and deterioration of cheeses and extending the shelf-life. Major compounds including relative retention indices (RI) against the National Institute of Standards and thymol, carvacrol, eugenol, carvone and cinnamaldehyde are mainly responsible Technology Library (NIST, USA), as well as authentic analytical standards and for exerting antimicrobial activity through various mechanisms such as increasing data from the literature. Relative proportions were calculated by dividing the cell permeability, change of membrane fatty acids and effect on membrane individual peak area by total area of all peaks. The response factor was not taken proteins. Few data are available about the application of EOs for their antifungal into account. Only compounds over 1% were included. Peaks under 1% were not activity in food such as cheese. Despite of the suitable efficacy of essential oils in counted. The used standards are listed in Table 2. restriction of growth and survival of microorganisms in cheese, some limitations have been recognized in their application (Khorshidian et al., 2018). Essential oils constituents could be interacting with food components such as fat, carbohydrate
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Fungal isolation and identification Agar). MEA (Malt extract agar), YES (Yeast extract agar) and CREA (Creatine sucrose agar). Inoculated media were incubated at 25 ± 1 °C, 7-17 days in the dark. Seven isolates (Table 1) from different moldy milk products were used. Isolates of After identification the strains belong to Penicillium commune were saved to the the genus Penicillium were identified to the species level according to Collection of Fungi of Department of Microbiology; Faculty of Biotechnology and morphological characteristics based on microscopy. To determine particular Food Sciences SUA in Nitra, Slovakia. For further analysis the 5 days old culture species, diagnostic literature was used as follows: Blakeslee (1915), Frisvad and of Penicillium commune strains were used. Thrane (1995), Pitt and Hocking (2009), Samson et al., (2010). Isolates were inoculated in three points on the identification media CYA (Czapek Yeast Extract
Table 1 Origin of the strains Penicillium commune and identification media and temperature used Strains Origin Cultivation temperature and identification media used P. commune KMi 177 cheese flavored with pepper P. commune KMi 270 smoked cheese (block) CYA25: incubation period 7 – 14 days in the dark at 25 ± 1°C P. commune KMi 276 smoked cheese (slices) MEA: incubation period 7 – 14 days in the dark at 25 ± 1°C P. commune KMi 277 smoked cheese (slices) CREA: incubation period 7 – 14 days in the dark at 25 ± 1°C P. commune KMi 370 sour cream YES: incubation period 7 – 14 days in the dark at 25 ± 1°C P. commune KMi 402 sour cream P. commune KMi 403 parenica (pasta filata)
Antifungal activity of essential oils and minimum inhibitory doses (MIDs) We performed all analyses in three replicates by TLC method followed the methodology described in Císarová et al. (2015). The antifungal activity of tested EOs was evaluated by using the vapour phase of oils. The test was performed in sterile Petri dishes (PD) (Ø 90 mm) for the first Statistical analysis analysis and three sectors Petri dishes for the MIDs analysis. The PDs contained 15 mL of CYA. The used method was modified from Guynot et al. (2003) and is The antifungal effect of EOs at concentration 625 µl.l-1 of air was performed in described in Císarová et al. (2016). Firstly EOs were tested in higher triplicate and the MIDs were performed in six repetitions. The results were concentration (625 µl/L-1 of air). After preparation the PDs were tightly saled with expressed as the mean of the data obtained in each replicate. Statistical analyses parafilm and incubated for 14 days at 25 ± 1°C and 35 days at 5 ± 1°C. The were performed with descriptive statistics. diameters (in mm) of the growing colonies were measured at the 3rd, 7th, 11th, 14th, The ability of strains to grow in the presence of EO was coded to binomial scale (1 21th, 28th and 35th day and strains incubated at 25 ± 1°C were measured at 3rd, 7th, - growth observed, 0 - without growth). All data were processed by probit analysis 11th, 14th day with a digital caliper. After incubation, the minimum inhibitory doses in Statgraphics Centurion XVI (Statgraphics) software. Doses that inhibit the (MIDs) of EOs with the most significant activity were recorded. EOs dissolved in growth in 50 % respectively 90 % of cases (MID50 and MID90) were reversely DMSO were prepared at different concentrations (500, 250, 125, 62.5, 31.25 and predicted from regression equation. 15.625). The used method and MIDs evaluation are described in Tančinová et al. (2019).For each fungal strain the inoculum was prepared. The spore suspension RESULTS AND DISCUSSION contained 106 spore/.ml-1. 5 µl of this suspension was inoculated on PDs with CYA medium. Cultivation was carried out at the 25 ± 1°C and measured after 7th and Chemical analysis of essential oils 14th day. In this study, the antifungal properties of four essential oils from family Lauraceae Anti-toxicogenic effect of essential oils were evaluated. Essential oils are a mixture of many complex compounds, which may vary depending on individual chemical compounds or their concentrations. The inhibitory of essential oils on the production of mycotoxins of tested strains Their antimicrobial activity also depends on their chemical composition (Omonijo was studied after 7 days of cultivation at 25 °C on individual species treated by the et al., 2018). The most effective components of essential oils are predominantly gas diffusion method with EOs. The concentrations of EOs used depended on the terpenes, terpenoids and phenolic substances (Wiese et al., 2018; Olmedo et al., activity of the concrete EO at the results from minimum inhibitory doses (MIDs). 2018; Pichersky a Raguso, 2018). Based on the above we evaluated the chemical The range of 625 to 15.625 μl.l-1 of air was used. We used only those samples of composition of tested EOs by the GC-MS analysis. The identified compounds of treated strains by EOs whose visible colony growth was not completely inhibited. EOs are listed in Table 2.
Table 2 Essential oils - chemical composition (in %) determined by gas chromatography coupled with mass spectrometry Component Cinnamon Cinnamon bark Laurel Litsea cubeba 1 α-pinenea 5.4 1.5 2 β-phellandrene 9.8 1.6 3 β-Pinenea 4.0 1.1 4 p-Cymenea 2.2 5 (R)-(+)-Limonenea 14.6 6 1,8-cineolea 52.5 1.6 7 linalola 1.9 1.1 8 mentola 2.6 9 α-terpineol 1.5 10 asarone 1.0 11 Cinnamaldehydea 76.0 63.33 12 β-citrall 31.4 13 α-citralla 38.0 14 α-terpineole acetate 12.2 15 Eugenola 20.0 16 β-caryophyllenea 4.2 2.6 1 17 cumarine 2.4 18 Citronelyl propionatea 4.2 1.6 19 o-methoxycinnamaldehyde 11.6 2.2 total 94.8 94.9 96.9 96.0 Legend: a – Identification confirmed by co-injection of authentic standard
The major components according to the concrete essential oil were: cinnamon - Antifungal activity of essential oils Cinnamaldehyde (76.00 %); cinnamon bark - Cinnamaldehyde (63.3 %) and Eugenol (20.00 %); laurel - Eucalyptol (52.5 %) and litsea cubeba - α-citrall (38.00 The antifungal activity of four EOs against seven strains of P. commune was %) and limonene (14.6 %). Our results are similar with other authors (Lins et al., determined, using micro-atmosphere method, firstly at the concentration of 625 2019; Wang et al., 2018; Fidan et al., 2019, Huang et al., 2019). μl.l-1 of air. Three essential oils: cinnamon, cinnamon bark and litsea cubeba completely inhibited the growth of all strains during cultivation at 25 °C and 5 °C. Laurel essential oil had different effects on the growth of P. commune strains.
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Inhibitory effect of this EO on the growth of the all strains of P. commune during the cultivation period at 25 ± 1°C is showed in Figure 1A and during the cultivation period at 5 ± 1°C is showed in Figure 1B.
Figure 1 Growth of tested strains P. commune during the cultivation period at 25 ± 1°C (A) and at 5 ± 1°C (B) under treatment by laurel EO
The strain KMi 403 was the most resistant to antifungal effect of laurel EO at both, It could be means that laurel can be able to inhibit growth of fungi but only in a 25 °C after 14 days and at 5 °C after 35 days of cultivation. In addition its growth contact with mycelium of tested strain. was after 14 days of cultivation at 25 °C stimulating in compared with control set. The most sensitive strain was KMi 177 regardless of time of cultivation Evaluation of minimum inhibitory doses (MIDs) temperature. The laurel EO was able to inhibit its growth significantly. But is interesting, that this strain is obtained from a cheese flavored by pepper, due this Three essential oils, concretely: cinnamon, cinnamom bark and litsea cubeba fact it can be weakened. The similar result obtained Massa et al. (2018). They inhibited growth of tested strains completely at all temperatures (25 °C and 5 °C) studied antifungal activity of some essential oils (included laurel EO) on growth of during all days of cultivation. Therefore, lower concentrations (500-15.625 µl.L-1 C. glabrata. The results showed that laurel EO had no inhibitory effect on the of air) of these essential oils were used to determine the minimum inhibitory doses growth of this yeast. But authors Sevindik et al. (2019) tested essential oil from (MIDs) on the tested fungi. MIDs determined by the micro-atmosphere method are Laurel nobilis flowers and Laurel nobilis leaves by microdilution method against summarized in Table 3. some strain of bacteria and yeast. Their results showed that those essential oils acted better against yeasts than against bacteria at relatively low concentrations (range between 0.097 - 3.125 µl/ml-1).
Table 3 Minimum inhibitory doses (µl.L-1 of air) of essential oils in vapor phase effective against the tested P. commune strains on CYA at 25 °C after 7 and 14 days, respectively. MIDs (µl.L-1 of air) after 7 days of cultivation Essential oils Tested strain number 177 270 276 277 370 402 403 Cinnamon 62.5 - 31.25 62.5 62.5 - - Cinnamon bark 62.5 31.25 31.25 62.5 62.5 31.25 31.5 Litsea cubeba 125 125 62.5 125 125 250 250 MIDs (µl.L-1 of air) after 14 days of cultivation Tested strain number 177 270 276 277 370 402 403 Cinnamon 125 125 62.5 62.5 62.5 - - Cinnamon bark 125 125 125 62.5 62.5 31.25 31.5 Litsea cubeba 125 125 125 250 125 125 250
The lowest MIDs against the tested Penicillium strains was found in cinnamon statistically significant (p ˂ 0.005), except MIDs values for litsea cubeba EO (MIDs 15.625 µl.L-1 of air) for three tested strains KMi 270, KMi 402 and KMi against strain KMi 370 (p 0.348) after 7 and 14 days of cultivation. 403 after 7 days of cultivation and for strains KMi 402 and 403 after 14 days of cultivation at 25 °C. Also Císarová et al. (2016) reported a relatively good effect Inhibitory effect of EOs on CPA production of cinnamon essential oil. They found that cinnamon EO inhibited the growth of tested Aspergillus strains at MIDs in the range of 31.5 µl.L-1 of air to 125 µl.L-1 of All strains of P. commune were tested in vitro for the production of cyclopiazonic air. Gómez et al. (2018) tested Origanum vulgare and Cinnamomum zeylanicum acid (CPA) by TLC method. Production of CPA was confirmed for four strains essential oils and their major active constituents, carvacrol and cinnamaldehyde, (KMi 177, 277, 370 and 403). Anti-toxinogenic effect was determined in 4 EOs, respectively, for inhibiting species of the genus Aspergillus and aflatoxin which did not inhibit the growth of the tested isolates completely at the different production in maize extract medium under different environmental conditions. concentration. The used concentration was selected according the results from They reported that the effectiveness of EOs and their main constituents to inhibit MIDs evaluation. The results are summarized in Table 5. fungal growth and aflatoxin production in contact assays was lower than in vapour The anti-toxinogenic potential of essential oils has been already demonstrated in phase assays. previous studies (Císarová et al., 2015; Císarová et al., 2016; Foltinova et al., In our study, the lowest effective essential oil was litsea cubeba with the higher 2017).Treatments with cinnamon EO showed some potential of fungal toxic MIDs for four tested strains - for 2 strains (KMi 402 and 403) after 7 days of inhibition. This oil affected the production of CPA by strain KMi177 at 31.25 μl.l– cultivation and for 2 strains (KMi 277 and 403) after 14 days of cultivation with 1 of air completely, and by strain KMi 403, respectively. Production of CPA by MIDs value 250 µl.L-1 of air. But many authors described Litsea cubeba EOs like strains KMi 370 was completely inhibited by this oil at all tested concertation the very potent antifungal and antibacterial agents (Li et al., 2016; Hu et al., 2019) (31.25 – 15.625 μl.l–1). Cinnamon bark EO was able to inhibited production of –1 Using probit analysis, predicted MIDs90 and MIDs50 were calculated. The MID CPA completely only in strain KMi 403 at the concentration 31.25 μl.l of air. values were calculated only for those essential oils that did not inhibit growth of Litsea cubeba had very low effect on the production of CPA by tested strains. the tested strains of P. commune completely at reduced concentrations (500-15.625 Laurel was the least effective EO because its inhibitory effect on the growth of µl.L-1 of air). The results are shown in Table 4. tested strains was not detected even at the highest concentration (625 μl.l–1 of air), The most effective tested essential oils were cinnamon and cinnamon bark EOs, as so as antitoxinogenic activity. -1 less effective litsea cubeba EO. The highest MID90 (162.29 µl.L of air) was The strains used in our study were isolated directly from the moldy dairy products determinate for litsea cubeba against strains KMi 277 after 14 days of cultivation. from Slovakia. For the use of EOs in food matrix, it is necessary to use very low -1 Also the highest MID50 (150.53 µl.L of air) was determinate for litsea cubeba, but concentrations, in order to not negatively affect the organoleptic properties of food, against strains KMi 403 after 7 days of cultivation. All results obtained were especially dairy products. Therefore, it is necessary to test essential oils directly on
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food. Although essential oils are not always able to inhibit fungal growth at low concentrations completely, they could be used in foods to inhibit their toxic metabolites.
-1 Table 4 The minimum inhibitory doses (MID50 and MID90) of tested essential oils expressed as µl.L of air of air estimated by probit analyses for tested strains of P. commune
Cinnamon Cinnamon bark Litsea cubeba Strains MIDs 7 days 14 days 7 days 14 days 7 days 14 days
MID 27.53 21.88 27.53 21.88 66.50 66.50 KMi177 50 MID90 32.73 52.56 32.73 52.56 78.94 78.94 p Value 0.000 0.000 0.000 MID nd nd 13.3 13.3 56.12 66.50 KMi 270 50 MID90 nd nd 20.98 20.98 64.97 78.94 p Value nd 0.005 0.000 MID 13.4 18.21 13.4 140.63 nd 66.50 KMi 276 50 MID90 20.98 45.68 20.98 162.29 nd 78.94 p Value 0.000 0.000 0.000 MID 15.87 18.21 15.87 18.21 nd 140.63 KMi 277 50 MID90 36.49 45.68 36.49 45.68 nd 162.29 p Value 0.000 0.000 0.000 MID nd nd nd nd 58.03 58.03 KMi 370 50 MID90 34.25 34.25 34.25 34.25 64.23 64.23 p Value 0.041 0.041 0.348 MID nd nd 13.4 18.21 119.80 119.80 KMi 402 50 MID90 nd nd 20.98 45.68 135.99 135.99 p Value nd 0.000 0.000 MID nd nd 13.4 18.21 131.21 94.47 KMi 403 50 MID90 nd nd 20.98 45.68 150.53 104.73 p Value nd 0.000 0.000 Legend: nd-MID not determined
Table 5 Inhibitory effect of tested EOs at different concentrations on CPA production by tested strains of P. commune Tested strain number Essential oils Tested concentration (μl.l–1 of air) 177 277 370 403 CPA 15.625 3*/1n 3/3 3/0 - Cinnamon 31.25 3/0 3/3 3/0 3/0 15.625 3/3 3/3 3/2 3/1 Cinnamon bark 31.25 3/3 3/3 3/1 3/0 15.625 3/3 3/3 3/3 3/3 31.25 3/3 3/3 3/3 3/3 Litsea cubeba 62.5 3/1 3/3 3/2 3/2 125 - - - 3/1 Laurel 625 3/3 3/3 3/3 3/3 Legend: * - repetition tested, n - number of positive production
CONCLUSION REFERENCES
In this study, the inhibitory effect of selected essential oils cinnamon BLAKESLEE, A. F. 1915. Linder᾿s roll tube method of separating cultures. (Cinnamomum zeylanicum L.), cinnamon bark (Cinnamomum zeylanicum L.), Phythopathology, 5, 68-69. https://doi.org/10.5962/bhl.title.109120 laurel (Laurus nobilis L.) and litsea cubeba (Litsea deccanensis L.) on the growth BOŽIK, M., CÍSAROVÁ, M., TANČINOVÁ, D., KOUŘIMSKÁ, L., HLEBA, L., of P. commune strains (7 strains) was evaluated. Our results showed that cinnamon, & KLOUČEK, P. 2017. Selected essential oil vapours inhibit growth of cinnamon bark and litsea cubeba EO had a 100% inhibition effect on the growth Aspergillus spp. in oats with improved consumer acceptability. Industrial crops of all tested strains at the higher concentration (625 µl.L-1 of air) throughout the and products, 98, 146-152. https://doi.org/10.1016/j.indcrop.2016.11.044 cultivation days. Laurel EO had no effect on the growth of tested strains. The MIDs CASQUETE, R., BENITO, M. J., ARANDA, E., MARTÍN, A., HERNÁNDEZ, assay confirmed the most significant efficacy of the two oils, namely cinnamon A., DE GUÍA CÓRDOBA, M. 2019. Cyclopiazonic acid gene expression as and cinnamon bark. Litsea cubeba had no significant effect. Is interestingly, the strategy to minimizing mycotoxin contamination in cheese. Fungal Biology. tested strains (KMi 370 and 402) showed differences among themselves even https://doi.org/10.1016/j.funbio.2019.06.011 though they came from the same products (sour cream). Strain KMi 402 was much CÍSAROVÁ, M., TANČINOVÁ, D., BRODOVÁ, M. 2015. The inhibitory effect more sensitive to essential oils than strain KMi 370. We also achieved the same of essential oils on the growth of genus Penicillium isolated from peanuts by results in testing the potential ability of essential oils to inhibit mycotoxin contact vapor. Journal of Microbiology, Biotechnology & Food Sciences, 4(1), 6- production, where cinnamon was the most effective and laurel had no effect on 11. https://doi.org/10.15414/jmbfs.2014.4.special1.6-11 CPA production. Our findings confirm that although essential oils are not able to CÍSAROVÁ, M., TANČINOVÁ, D., MEDO, J., KAČÁNIOVÁ, M. 2016. The in inhibit fungal growth at the lowest concentrations, they are capable of inhibiting vitro effect of selected essential oils on the growth and mycotoxin production of mycotoxins production. Essential oils can be used in agro industries instead of Aspergillus species. Journal of Environmental Science and Health, Part B, 51(10), synthetic pesticides or preservatives, to control plant diseases causing severe 668-674. https://doi.org/10.1080/03601234.2016.1191887 destruction to crops and they can be proposed as potential antimicrobial agents for FIDAN, H., STEFANOVA, G., KOSTOVA, I., STANKOV, S., DAMYANOVA, food commodity preservation in the near future. S., STOYANOVA, A., ZHELJAZKOV, V. D. 2019. Chemical Composition and Antimicrobial Activityof Laurus nobilis L. Essential Oils from Bulgaria. Acknowledgments: This work was supported by APVV-15-0543, KEGA Molecules, 24(4), 804. https://doi.org/10.3390/molecules24040804 015SPU-4/2018 and Research Center AgroBioTech.The authors thank Eva FOLTINOVÁ, D., TANČINOVÁ, D., CÍSAROVÁ, M. 2017. Influence of Sádovská for her responsible approach in the preparation and realization of the essential oils on the growth of Aspergillus flavus. Potravinárstvo: Slovak Journal experiment. of Food Sciences, 11(1), 322-331. https://doi.org/10.5219/725
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FRISVAD, J. C., THRANE, U. 1995. Filamentous fungi in foods and feeds: SAMSON, R. A., HOUBRAKEN, U., THRANE, U., FRISVAD, J. C., ecology, spoilage and mycotoxins production. Handbookof applied mycology, 3, ANDERSEN, B. 2010. Food and Indoor Fungi. Utrecht, Netherlands : CBS- 31-68. KNAW Fungal Biodiversity Centre, 390 p. ISBN 978-90-70351-82-3. GÓMEZ, J. V., TARAZONA, A., MATEO-CASTRO, R., GIMENO- SEOW, Y.X., YEO, C., CHUNG, H. L., YUK, H. G. 2014. Plant Essential Oils ADELANTADO, J. V., JIMÉNEZ, M., MATEO, E. M. 2018. Selected plant as Active Antimicrobial Agents. Critical Reviews in Food Science and Nutrition, essential oils and their main active components, a promising approach to inhibit 54, 625-644. https://doi.org/10.1080/10408398.2011.599504 aflatoxigenic fungi and aflatoxin production in food. Food Additives & SEVINDIK, E., AYDIN, S., OKAN, K., EFE, F. 2018. Antibacterial Activity of Contaminants, 35(8), 1581-1595. Essential Oils from Laurus nobilis L. Flowers and Leaves Grown in the West https://doi.org/10.1080/19440049.2017.1419287 Anatolian Area. Fresenius Environmental Bulletin, 28(9), 6555-6559. GUYNOT, M. E., RAMOS, A. J., SETO, L., PURROY, P., SANCHIS, V., https://doi.org/10.1080/0972060x.2018.1505556 MARIN, S. 2003. Antifungal activity of volatile compounds generated by essential TANČINOVÁ, D., MEDO, J., MAŠKOVÁ, Z., FOLTINOVÁ, D., & ÁRVAY, J. oils against fungi commonly causing deterioration of bakery products. Journal of 2019. Effect of essential oils of Lamiaceae plants on the Penicillium commune. The Applied Microbiology, 94(5), 893-899. https://doi.org/10.1046/j.1365- Journal of Microbiology, Biotechnology and Food Sciences, 8(4), 1111. 2672.2003.01927.x PMid:12694455 https://doi.org/10.15414/jmbfs.2019.8.4.1111-1117 HANSEN, M. E., LUND, F., CARSTENSEN, J. M. 2003. Visual clone WANG, Y., ZHANG, Y., SHI, Y. Q., PAN, X. H., LU, Y. H., CAO, P. 2018. identification of Penicillium commune isolates. Journal of Microbiological Antibacterial effects of cinnamon (Cinnamomum zeylanicum) bark essential oil on Methods, 52(2), 221-229. https://doi.org/10.1016/s0167-7012(02)00160-4 Porphyromonas gingivalis. Microbial pathogenesis, 116, 26-32. HU, W., LI, C., DAI, J., CUI, H., & LIN, L. 2019. Antibacterial activity and https://doi.org/10.1016/j.micpath.2018.01.009 mechanism of Litsea cubeba essential oil against methicillin-resistant WIESE, N., FISCHER, J., HEIDLER, J., LEWKOWSKI, O., DEGENHARDT, J., Staphylococcus aureus (MRSA). Industrial Crops and Products, 130, 34-41. ERLER, S. 2018. The terpenes of leaves, pollen, and nectar of thyme (Thymus https://doi.org/10.1016/j.indcrop.2018.12.078 vulgaris) inhibit growth of bee disease-associated microbes. Scientific reports, HUANG, H., CHEN, R., MA, H., YUAN, Z. 2019. Quality attributes and chemical 8(1), 14634. https://doi.org/10.1038/s41598-018-32849-6 composition of commercial cinnamon oils. Quality Assurance and Safety of Crops & Foods, 11(1), 89-94. https://doi.org/10.3920/qas2018.1348 HYLDGAARD, M., MYGIND, T., MEYER, R. L. 2012. Essential oils in food preservation: mode of action, synergies, and interactions with food matrix components. Frontiers in microbiology, 3, 12. https://doi.org/10.3389/fmicb.2012.00012 KHAN, A., AHMAD, A., AKHTAR, F., YOUSOUF, S., XESS, I., KHAN, L. A., MANZOOR, N. 2010. Ocimum sanctum essential oil and its active principles exert their antifungal activity by disrupting ergosterol biosynthesis and membrane integrity. Research in Microbiology. 161, 816-823. https://doi.org/10.1016/j.resmic.2010.09.008 KHORSHIDIAN, N., YOUSEFI, M., KHANNIRI, E., MORTAZAVIAN, A. M. 2018. Potential application of essential oils as antimicrobial preservatives in cheese. Innovative Food Science & Emerging Technologies, 45, 62-72. https://doi.org/10.1016/j.ifset.2017.09.020 KURE, C. F., SKAAR, I. 2019. The fungal problem in cheese industry. Current Opinion in Food Science. 29, 14. https://doi.org/10.1016/j.cofs.2019.07.003 KURE, C. F., WASTESON, Y., BRENDEHAUG, J., & SKAAR, I. 2001. Mould contaminants on Jarlsberg and Norvegia cheese blocks from four factories. International Journal of Food Microbiology, 70(1-2), 21-27. https://doi.org/10.1016/s0168-1605(01)00520-7 LI, Y., KONG, W., LI, M., LIU, H., ZHAO, X., YANG, S., YANG, M. 2016. Litsea cubeba essential oil as the potential natural fumigant: inhibition of Aspergillus flavus and AFB1 production in licorice. Industrial Crops and Products, 80, 186-193. https://doi.org/10.1016/j.indcrop.2015.11.008 LINS, L., DAL MASO, S., FONCOUX, B., KAMILI, A., LAURIN, Y., GENVA, M., DELEU, M. 2019. Insights into the Relationships Between Herbicide Activities, Molecular Structure and Membrane Interaction of Cinnamon and Citronella Essential Oils Components. International journal of molecular sciences, 20(16), 4007. https://doi.org/10.3390/ijms20164007 MARTÍN, J. F., LIRAS, P. 2016. Secondary metabolites in cheese fungi. Fungal Metabolites, 1-23. https://doi.org/10.1007/978-3-319-19456-1_37-1 MASSA, N., CANTAMESSA, S., NOVELLO, G., RANZATO, E., MARTINOTTI, S., PAVAN, M., BONA, E. 2018. Antifungal activity of essential oils against azole-resistant and azole-susceptible vaginal Candida glabrata strains. Canadian journal of microbiology, 64(10), 647-663. https://doi.org/10.1139/cjm- 2018-0082 NIKKHAH, M., HASHEMI, M., HABIBI NAJAFI, M.,B., FARHOOSH, R. 2017. Synergistic effects of some essential oils against fungal spoilage on pear fruit. International Journal of Food Microbiology. 18, 285-294. https://doi.org/10.1016/j.ijfoodmicro.2017.06.021 OLMEDO, R., RIBOTTA, P., GROSSO, N. R. 2018. Antioxidant Activity of Essential Oils Extracted from Aloysia triphylla and Minthostachys mollis that Improve the Oxidative Stability of Sunflower Oil under Accelerated Storage Conditions. European journal of lipid science and technology, 120(8), 1700374. https://doi.org/10.1002/ejlt.201700374 OMONIJO, F. A., NI, L., GONG, J., WANG, Q., LAHAYE, L., & YANG, C. 2018. Essential oils as alternatives to antibiotics in swine production. Animal Nutrition, 4(2), 126-136. https://doi.org/10.1016/j.aninu.2017.09.001 PICHERSKY, E., RAGUSO, R. A. 2018. Why do plants produce so many terpenoid compounds. New Phytologist, 220(3), 692-702. https://doi.org/10.1111/nph.14178 PITT, J. I., HOCKING, A. D. 2009. Fungi and food spoilage. 3rd ed. London, New York : Springer Science & Business Media, LLC, 519. ISBN 978-0-387-92206-5. https://doi.org/10.1007/978-0-387-92207-2
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BIOLOGICAL ACTIVITY OF ESSENTIAL OILS AGAINST STAPHYLOCOCCUS SPP. ISOLATED FROM HUMAN SEMEN
Miroslava Kačániová1,2, Eva Ivanišová3, Miroslav Ondrejovič4, Daniela Chmelová4, Margarita Terentjeva5, Simona Kunová6, Eva Tvrdá7
Address(es): 1Slovak University of Agriculture in Nitra, Faculty of Horticulture and Landscape Engineering, Department of Fruit Sciences, Viticulture and Enology, Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia, phone number: +421 37 641 4985 2University of Rzeszow, Faculty of Biology and Agriculture, Department of Bioenergy and Food Technology, Zelwerowicza St. 4, PL-35601 Rzeszow, Poland, 3Slovak University of Agriculture, Faculty of Biotechnology and Food Sciences, Department of Technology and Quality of Plant Products, Tr. A. Hlinku 2, SK-94976 Nitra, Slovakia, 4University of Ss. Cyril and Methodius Faculty of Natural Sciences, Department of Biotechnology,, Nám. J. Herdu 2, Trnava, 917 01, Slovakia 5Latvia University of Life Sciences and Technologies, Institute of Food and Environmental Hygiene, Faculty of Veterinary Medicine, Latvia K. Helmaņa iela 8, LV- 3004, Jelgava, Latvia, 6Slovak University of Agriculture, Faculty of Biotechnology and Food Sciences, Department of Food Safety and Hygiene, Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia, phone number: +421 37 641 5807 7Slovak University of Agriculture, Faculty of Biotechnology and Food Science, Department of Animal Physiology, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2019.9.special.404-408
ARTICLE INFO ABSTRACT
Received 30. 6. 2019 The antimicrobial and antioxidant activities of 5 commercial plant essential oils (Citrus paradisi peel oil, Citrus reticulata peel oil, Revised 23. 9. 2019 Juniperus communis fruit oil, Eucalyptus globulus leaf oil, and Cananga odorata flower) were determined. Essential oil samples were Accepted 7. 10. 2019 analysed by GC-FID chromatography (Agilent 6890N). Furthermore, the plant essential oils were tested using antioxidant activity and Published 8. 11. 2019 antimicrobial activity test against 19 species of Staphylococcus spp. isolated from human semen and identified with MALDI-TOF MS Biotyper. Antioxidant activities test using DPPH method revealed that Cannaga odorata had the higher antioxidant activity and the essential oil showed a positive effect. This activity is probably due the high content of benzyl benzoate and caryophyllene, which are the Regular article dominant compounds in this kind of oil. The lowest antioxidant activity was found for Juniperus communis essential oil. Antimicrobial activities showed that all tested essential oils inhibited the growth of 19 staphylococci studied. However, C. paradise, C. reticulata, J. communis and C. odorata had the best antimicrobial effect against S. hominis, whereas E. globulus essential oil has the best antimicrobial activity against S. aureus and S. capiti.
Keywords: antioxidant activity, antimicrobial activity, bacteria, spermatozoa, GC-FID chromatography
INTRODUCTION with the internal parts divided into segments. The number of natural species is unclear, as many of the named species are hybrids. Grapefruit (Citrus paradisi. Some bacteria are deleterious to the spermatozoa in a concentration-dependent L) belongs to the Citrus genus, a taxa of flowering plants in the family Rutaceae. manner. The most extensively studied is the effect of Escherichia coli on human The grapefruit is believed to have arisen from the pomelo or shaddock (Citrus spermatozoa survival. This Gram-negative bacterium reduces sperm motility grandis) or as a hybrid between pomelo and sweet orange. Mandarin fruits through sperm adhesion and agglutination (Monga and Roberts, 1994). (Citrus reticulata, family Rutaceae) are one of the most abundant edible citruses Staphylococcus aureus (S. aureus) is one of the most common pathogens causing in the fresh fruit markets. Mandarin peel oil is usually obtained through the cold- both human and animal infections. Transmission of S. aureus to humans via pressing process. There are three different types of cold-pressed mandarin oil contaminated food continues to be a health public concern (Qiuchun et al., distinguished by the colour and maturity of the fruits: the so-called “green oil” is 2019). Staphylococcus aureus (S. aureus) is an important zoonotic pathogen, obtained from immature fruits and mainly used as fragrance, the “yellow oil” is which can infect both humans and animals. It is widely distributed in the nature obtained from mature fruits, being used as flavour and fragrance, while and is present in air, water and feed; it also exists on the surface of the human completely matured ones are used to produce the “red oil”, mainly used as body, in the nasal cavity, on animal fur, and in the digestive tract among other flavour (Reeve and Arthur, 2002). Main components of mandarin peel oil are sites. S. aureus has been responsible for several infectious diseases including limonene (up to 95 wt.%) and other terpenes, like γ-terpinene. Oxygenated tissue and skin infections, pneumonia, sepsis, mastitis, arthritis, and soft tissue components (from 0.2 to 1.5 wt.%) like linalool, decanal and citral have the infections (David and Daum, 2010; Tong et al., 2015). Livestock products can highest contribution to the aroma fraction (Lota et al., 2001). Essential oils are act as a source of S. aureus zoonotic infections, and handling or consuming of the odorous, volatile products of the secondary metabolism of an aromatic plant, contaminated food could potentially result in transmission to humans (Feingold which are often concentrated in a particular organ of the plant such as leaves, et al., 2012; Papadopoulos et al., 2018). stems, bark or fruit and are stored in secretory cells, cavities, canals, epidermic Citrus essential oils (O) have been applied in many products, such as foods, cells or glandulartrichomes (Gilles et al., 2010). Juniperus (Cupressaceae) is a beverages, cosmetics and medicines, as flavouring agents as well as for plant widely cultivated in the northern hemisphere. This species grows as trees or aromatherapy. They are also used for their germicidal, antioxidant and shrubs. Traditionally, Juniperus has shown several applications, mainly related to anticarcinogenic properties (Uysal et al., 2011). The active constituents of citrus its medicinal properties and a highly specific flavour which are associated to its Essential oils (Eos), such as limonene, α‐pinene, β‐pinene and α‐terpinolene volatile oil components (Carpenter et al., 2014). Most of the organs of this plant exhibit a wide spectrum of antimicrobial activity, as convinced by many studies contain essential oils, but it is mainly extracted from the berries, needles, in other plants (Cristóbal-Luna, et al., 2018Citrus fruits are a distinctive berries branches and roots (Foudil-Cherif and Yassaa, 2012). The eucalyptus oils can
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be found in the leaves of more than 300 species of this genus and less than 20 of were obtained from “Oxoid”. The results were interpreted according to EUCAST these have ever been exploited commercially for the production of essential oils (2019). rich in 1,8-cineole by pharmaceutical and cosmetic industries (Elaissi et al., 2011 Ylang-ylang (Cananga odorata) is one of the plants that are exploited at a large Disc diffusion method scale for its essential oil which is an important raw material for the fragrance industry. The essential oils extracted via steam distillation from the plant have We used the agar disc diffusion method for the determination of antimicrobial been used mainly in cosmetic industry but also in food industry. Traditionally, C. activities of the essential oil. Briefly, a suspension of the tested microorganism odorata is used to treat malaria, stomach ailments, asthma, gout, and rheumatism. (0.1 mL of 105 cells per ml) was spread on the solid media plates. Filter paper The essential oils or ylang-ylang oil is used in aromatherapy and is believed to be discs (6 mm in diameter) were impregnated with 15 µl of the oil and placed on effective in treating depression, high blood pressure, and anxiety (Tan et al., the inoculated plates. They were inoculated onto the surface of Mueller Hinton 2015). Agar (MHA, Oxoid, Basingstoke, United Kingdom). These plates, after The aim of this study was to determine the chemical and biological properties of remaining at 4 °C for 2 hours, were incubated anaerobically at 37 °C for 24 h. selected essential oils. From a biological point of view, we studied the The diameters of the inhibition zones were measured in millimeters. All the tests antimicrobial and antioxidant activity of essential oils against staphylococci from were performed in duplicate. sperm identified with MALDI-TOF MS Biotyper. Statistical analyses MATERIAL AND METHODS All measurements and analyses were carried out in triplicate. Experimental data Essential oils were evaluated by basic statistical variability indicators using the Microsoft™ Excel® program. Dependency rate between the tested traits was expressed using Samples of commercial pure essential oils: Citrus paradisi peel oil, Citrus the linear correlation analysis. reticulata peel oil, Juniperus communis fruit oil, Eucalyptus globulus leaf oil, and Cananga odorata flower oil, obtained via steam distillation were purchased from RESULTS AND DISCUSSION Aromatika (Russia). The essential oil samples were stored in glass vials with teflon-sealed caps at laboratory temperature in the absence of light. Different kinds of essential oils from various plant material (Citrus paradisi, Citrus reticulata, Juniperus communis, Eucalyptus globulus, and Cananga Gas chromatography odorata) were tested. The most intensive peak in C. paradisi essential oil was registered at 15.83 min with area percentage of 87 % (Fig. 1). Okunowo et al. Essential oil samples were analysed by Agilent 6890N (Agilent Technologies, (2013) identified 19 compounds in C. paradise essential oil and the most Santa Clara, CA, USA) with FID detector. System control and data analysis were abundant compound in sample was D-limonene (75.1 %), following β-myrcene processed using the Agilent ChemStation software Rev. B.04.03-SP1 (Agilent (7.3 %), α-pinene (2.1 %), caryophyllene (1.9 %), octanal (1.7 %), β- Technologies, Santa Clara, CA, USA). The chromatographic separation was phellandrene (1.2 %) and decanal (1.1 %). performed in the DB-23 column (0.25 mm i.d., 30 m long, 0.25 µm film FID1 A, (ACID VIOLET\09092016000069.D) thickness) (Agilent Technologies), and 5 µl of the sample was injected. The pA injector temperature was 250 °C, and the FID temperature is set at 250 °C. The 8000 carrier gas (nitrogen) flow was 1.1 ml/min (constant flow) with a split ratio 1:100 7000 and a temperature program from 40 °C to 80 °C at 3 °C/min, from 80 °C to 180 °C at 5 °C/min, and from 180 °C to 220 °C at 8 °C/min and finally held at 220°C 6000 for 15 min. 5000
Radical scavenging activity – DPPH method 4000
3000 Radical scavenging activity of essential oils was measured using 2,2-diphenyl-1- picrylhydrazyl (DPPH) according to methodic by Sanchéz-Moreno et al., (1998) 2000 with slight modification. The sample (0.1 ml) was mixed with 3.9 ml of DPPH 1000 solution (0.025 g DPPH in 100 ml methanol). Absorbance of the reaction mixture was determined using the spectrophotometer Jenway (6405 UV/Vis, England) at 0 515 nm. The scavenging activity percentage (AA%) was determined according 0 10 20 30 40 50 min formula: Figure 1 Gas chromatograph trace for Citrus paradisi essential oil sample.
AA% = [(A0 - AAT)/A0 × 100] The analysis of essential oil from C. reticulata showed the dominant peak at 15.89 min. (85 % of area percentage) (Fig. 2). Similar peak was observed at GC where A0 is absorbance of control reaction (DPPH radical); A1 is the absorbance in presence of sample of C. paradisi essential oil sample (Fig. 1). Limonene (80.3 %), γ-terpinene (4.7 %), myrcene (2.1 %), α-pinene (1.2 %) and octanal (1.0 %) were identified in C. Antimicrobial activity reticulata Blanco (Ponkan) peel oil (Sawamura et al., 2004). FID1 A, (ACID VIOLET\09092016000070.D) pA Microorganisms 8000 Nineteen different kinds of staphylococci were isolated from human semen. The 7000 semen samples were obtained from 30 healthy donors by masturbation into sterile container. Only ejaculates showing normal semen parameters were used. The 6000 samples underwent liquefaction at 37 °C for 30 min. Experiments were 5000 performed within 1 h from sample collection. The samples were inoculated on Tryptone Soya agar (TSA) and Levine agar (LA). After incubation the bacteria 4000 were selected for further confirmation with MALDI-TOF MS Biotyper. Isolated 3000 staphylococci were tested for antibiotics resistance against tobramycin 10 msg. Following six human sperm isolates were tested: Staphylococcus aureus (4 2000 isolates), S. capiti (3 isolates), S. epidermidis (4 isolates), S. haemoliticus (4 1000 isolates), S. hominis (4 isolates). The bacteria species were maintained in Mueller 0 Hinton Agar (Merck, Germany). The mother cultures of each staphylococci were 0 10 20 30 40 50 min set up 24 h before the assays in order to reach the stationary phase of growth. The Figure 2 Gas chromatograph trace for Citrus reticulata essential oil sample. tests were assessed by inoculating Petri dishes from the mother cultures with proper sterile media. The main aim was to obtain the microorganism 5 -1 The major peaks determined for J. communis essential oil sample were identified concentration of 10 colony forming units cfu.mL . at 10.75 min (66.6 %), 13.24 min (16.7 %), and 15.3 min (2.9 %) (Fig. 3). Falasca et al. (2016) found that the most abundant monoterpene identified in J. Antibiotic susceptibility testing communis fresh berries was α-pinene (13.43-32.34 %), following sesquiterpenes, namely Germacrene D (12.29-20.65%), β-Caryophyllene (7.72-11.77%), γ- he antibiotic susceptibility test was performed by using Disc Diffusion Method. Cadinene (3.86-5.47 %), α-humulene (5.21-7.87 %), germacrene B (4.84-9.33 Four different forms of sensitivity discs with 10 mcg concentrations were used %), and bicyclogermacrene (2.50-4.18 %). Orav et al. (2010) found that the for studying the in vitro sensitivity of isolates: tobramycin.(TOB). These discs
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main compounds in essential oil from J. communis were α-pinene, germacrene D, Misharina and Samusenko (2008) found that essential oils exhibit antioxidant (E)-β-caryophyllene, and β-myrcene. activity that largely depends on their composition in their study on the
FID1 A, (ACID VIOLET\09092016000071.D) antioxidant properties of essential oils from lemon, grapefruit, coriander, clove, pA and their mixtures. Furthermore, Misharina et al. (2011) found that the 10000 antioxidant activity of lemon peel essential oils depends on the system composition and concentrations of essential oils. Individual citral and limonene
8000 displayed the lowest antioxidant activity, whereas the activity of their mixture was higher, which explains the synergetic effects in the antioxidant activity of the components. The antioxidant activity of essential oils is revealed in Table 1. In 6000 our study the best antioxidant activity varied from 0.997% of Juniperus communis to 73.137% of Cananga odorata. The essential oil of Citrus paradise 4000 showed 58.96% of antioxidant acitivty. C. grandis oils and cold-pressed C. paradisi oil displayed weak DPPH radicals scavenging capability. DPPH scavenging capacity of cold-pressed C. paradisi oil was less than 20% (Ou et al. 2000 2015). Distilled C. paradisi oil exhibited the potent DPPH scavenging capacity among 4 citrus oils; the EC50 value was more than 40 mg/mL. This is consistent 0 with previous studies where 34 kinds of 10 mg/mL citrus oils obtained from 0 10 20 30 40 50 min Japan and Korea and Italy exhibited weak DPPH radical scavenging effect Figure 3 Gas chromatograph trace for Juniperus communis essential oil sample. ranging from 12% to 17.7%. There are studies that reported antioxidant activity of galbuli EO from J. communis (Höferl et al., 2014) and J. excelsa (Emami et Essential oil isolated from E. globulus leafs showed the dominant peak at 17.95 al., 2007). Our results showed very faint activity of J. communis. The DPPH min (81.7 %) and some minor peaks at 15.5 min (7.3 %) and 18.23 min (6.4 %) scavenging activity was highest in E. citriodora (82.1%), followed by E. (Fig. 4). Sacchetti et al. (2005) observed that the major compounds in essential camaldulensis (81.9%) and E. microtheca (81.8%) as compared to positive oil from E. globulus were 1,8-cineole (52.6 %), α-pinene (20.0 %), and α- control BHT (Ghaffar et al., 2015). We found in our study E. globulus 31.63 % Phellandrene (6.2 %). of antioxidant activity. DPPH assay was used to evaluate the antioxidant property FID1 A, (ACID VIOLET\09092016000072.D) of C. odorata extracts to determine the free radical scavenging properties of the pA extracts. The ethyl acetate extract of the stem bark of C. odorata revelled to exhibit the maximum % of DPPH inhibition (79 %) when compared to other investigations (Kusuma et al., 2014). Our results showed similar values of the 8000 antioxidant activity.
6000 Table 1 Antioxidant activity of essential oils Essential oil Antioxidant activity %
4000 Citrus reticulata 3.40 Citrus paradisi 58.96
2000 Juniperus communis 1.00 Eucalyptus globulus 31.63 Cananga odorata 73.14 0
0 10 20 30 40 50 min Figure 4 Gas chromatograph trace for Eucalyptus globulus essential oil sample. Table 2 summarizes the antimicrobial activity of the five EOs studied against 5 isolated Staphyloccus species isolated from human semen with MALDI TOF MS The last sample of essential oils was isolated from C. odorata flower. The gas Biotyper. Citrus EOs are complex mixtures of different compounds and their chromatograph showed the dominant peaks at 24.38 min (22.98 %), 43.92 min antimicrobial activity depends on their chemical composition. Methicillin- (18.28 %), and 30.42 min (7.7 %). Minor peaks were registered at 27.49 min (4.6 resistant S. aureus was found to be the most sensitive to the three Eos of Citrus %), 29.1 min (4.9 %) and 32.79 min (3.4 %) (Fig. 5). Sacchetti et al. (2005) sp. because its growth was affected (Boudries et al., 2017). Martinez et al. analyzed the chemical composition of essential oil from C. odorata and found (2003) found that mandarin oil (Citrus reticulata Blanco) variety Dancy showed that the most abundant compounds were benzyl benzoate (33.61 %), linalool an antibacterial activity against B. subtilis, S. aureus and L. monocytogenes. (24.5 %), benzyl salicylate (12.89 %), benzyl acetate (9.77 %), and methyl Whereas Espina et al. (2011) found that the inhibition zones of mandarin EO salicylate (2.79 %). The main components of cananga oil were trans- against S. aureus, E. coli O157:H7 and P. aeruginosa were 18.8, 20.0 (mm) and carryophyllene (39.03 %), α-humulene (11.59 %), α- bergamotene (11.29 %) and no inhibition, respectively, which is almost in accordance with our results. In our germacrene (10.94 %) (Mahfud et al., 2017). study the most sensitive Staphylococcus was S. homins MK-39, resistant to FID1 A, (ACID VIOLET\09092016000073.D) tobramycin against Citrus reticulata. Twenty milligram per milliliter cold- pA pressed C. paradisi oil and distilled C. grandis oil exhibited very strong
5000 inhibitory effects against S. aureus (Ou et al., 2015). The most sensitive staphylococci to C. paradisi were S. hominis MK-38 and MK-39 resistant to tobramycin. Zheliazkov et al. (2017) found a strong antimicrobial activity of J. 4000 communis EO against Staphylococcus aureus subsp. aureus. The strongest antimicrobial activity of J. communis essential oil was found against S. hominis 3000 MK-38. Salari et al. (2006) used Eucalyptus globulus leaf extract to evaluate their activity on 56 isolates of Staphylococcus aureus. The EOs extracted from all
2000 seven Eucalyptus spp. showed antibacterial activity against S. aureus. The best antimicrobial activity of E. globulus in our study were found against S. aureus MK-24 and S. capiti MK-26 and MK-27. Recent work from Indonesia showed 1000 that the stem bark extracts of C. odorata exhibited a great antimicrobial property using the agar well disc diffusion assay (Kusuma et al., 2014). C. odorata 0 showed the best antimicrobial activity against S. hominis MK-38 and MK-39. 0 10 20 30 40 50 min Figure 5 Gas chromatograph trace for Cananga odorata essential oil sample.
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Table 2 Antimicrobial activity of essential oils in mm Citrus Juniperus Eucalyptus Cananga TOB Citrus paradisi reticulata communis globulus odorata S. aureus MK-21 20 6.33±1.15 4.67±1.15 3.33±1.53 4.67±0.58 11.33±0.58 S. aureus MK-22 15 5.67±0.58 4.33±0.58 3.67±1.53 4.67±1.15 9.67±0.58 S. aureus MK-23 25 4.67±0.58 4.67±0.58 3.33±0.58 5.33±0.58 10.67±0.58 S. aureus MK-24 15 4.33±0.58 4.67±0.58 4.33±0.58 5.67±0.58 11.33±0.58 S. capiti MK-25 30 4.33±0.58 3.67±0.58 3.33±0.58 4.33±0.58 8.33±0.58 S. capiti MK-26 20 4.67±0.58 5.33±1.15 5.67±0.58 5.67±1.15 8.67±1.15 S. capiti MK-27 20 5.67±0.58 4.67±0.58 5.67±1.15 5.67±0.58 9.67±1.53 S. epidermidis MK-28 21 5.33±0.58 2.67±0.58 3.33±1.53 4.33±0.58 9.33±1.15 S. epidermidis MK-29 25 4.67±0.58 2.33±0.58 4.33±0.58 4.67±0.58 9.67±0.58 S. epidermidis MK-30 28 4.67±0.58 3.67±1.53 5.33±0.58 4.67±0.58 8.67±2.08 S. epidermidis MK-31 20 4.67±0.58 2.67±0.58 4.67±0.58 4.67±0.58 7.33±1.15 S. haemoliticus MK-32 20 3.33±0.58 4.67±0.58 3.33±0.58 2.33±0.58 7.67±0.58 S. haemoliticus MK-33 20 3.33±1.53 5.33±0.58 3.67±0.58 2.67±0.58 7.33±0.58 S. haemoliticus MK-34 20 2.67±1.15 4.67±0.58 3.33±0.58 2.67±1.15 7.67±0.58 S. haemoliticus MK-35 25 4.67±1.53 3.67±1.15 3.67±1.15 4.67±1.15 8.67±0.58 S. hominis MK-36 15 7.67±0.58 11.33±1.15 8.33±0.58 4.33±1.15 18.67±1.15 S. hominis MK-37 20 7.33±0.58 11.33±0.58 8.33±0.58 4.67±0.58 18.67±2.08 S. hominis MK-38 20 7.67±0.58 12.67±0.58 9.33±1.15 5.33±0.58 20.33±0.58 S. hominis MK-39 15 8.67±0.58 12.67±2.08 7.67±0.58 4.67±0.58 20.33±0.58
CONCLUSION hemisphaerica. and Juniperus oblonga., Pharmaceutical Biology, 45(10), 769- 776, https://doi.org/10.1080/13880200701585931 In view of their potential as inhibitors of pathogenic microbial growth as well as ESPINA, L., SOMOLINOS, M., LORÁN, S., CONCHELLO, P., GARCÍA, D., their antioxidant activity, the Citrus paradisi peel oil, Citrus reticulata peel oil, PAGÁN R. 2011. Chemical composition of commercial citrus fruit essential oils Juniperus communis fruit oil, Eucalyptus globulus leaf oil, and Cananga odorata and evaluation of their antimicrobial activity acting alone or in combined flower essential oils may be recommended for formulation of plant based processes. Food Control, 22(6), 896–902. preservatives for enhancement of shelf life of food items by controlling their https://doi.org/10.1016/j.foodcont.2010.11.021 losses from bacterial contamination and lipid peroxidation during storage. EUCAST, 2019. Clinical breakpoints and dosing of antibiotics. Additionally, the abundance of Citrus paradisi peel and Citrus reticulata peel as http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/ by- products from food industry and Juniperus communis fruit, Eucalyptus Breakpoint_tables/v_9.0_Breakpoint_Tables.pdf globulus leaf, and Cananga odorata flower as renewable raw materials makes the FALASCA, A., CAPRARI, C., DE FELICE, V., FORTINI, P., SAVIANO, G., use of essential oils obtained from these plant sources economically ZOLLO, F., IORIZZO, M., 2016. GC-MS analysis of the essential oils of adventogenous for practical application, thus they can be considered as potential Juniperus communis L. berries growing wild in the Molise region: Seasonal possible alternatives to synthetic preservatives. variability and in vitro antifungal activity. Biochemical Systematics and Ecology, 69, 166-175. https://doi.org/10.1016/j.bse.2016.07.026 Acknowledgments: This study was supported by the KEGA grant no. 009spu- FEINGOLD, B.J., SILBERGELD, E.K., CURRIERO, F.C., VAN CLEEF, B.A., 4/2018 and by the APVV grant no. APVV-15-0544 HECK, M.E., KLUYTMANS, J.A. 2012. Livestock density as risk factor for livestock-associated methicillin-resistant Staphylococcus aureus, the REFERENCES Netherlands. Emerging Infectious Diseases, 18(11), 1841–1849. https://doi.org/10.3201/eid1811.111850 BOUDRIES, H., LOUPASSAKI, S., LADJAL ETTOUMI, Y., SOUAGUI, S., FOUDIL-CHERIF, Y., YASSAA, N. 2012. Enantiomeric and non-enantiomeric BACHIR BEY, M., NABET, N., CHIKHOUNE, A., MADANI, K., CHIBANE, monoterpenes of Juniperus communis L. and Juniperrus oxycedrus needles and M. 2017. Chemical profile, antimicrobial and antioxidant activities of Citrus berries determined by HS-SPME and enantioselective GC/MS. 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ANTIMICROBIAL AND ANTIOXIDANT PROPERTIES OF ESSENTIAL OILS AGAINST BACTERIAL STRAINS ISOLATED FROM HUMAN SEMEN
Simona Kunová1, Eva Tvrdá2, Eva Ivanišová3, Miroslav Ondrejovič4, Daniela Chmelová4, Margarita Terentjeva5, Miroslava Kačániová6,7
Address(es): 1Slovak University of Agriculture, Faculty of Biotechnology and Food Sciences, Department of Food Safety and Hygiene, Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia, phone number: +421 37 641 5807. 2Slovak University of Agriculture, Faculty of Biotechnology and Food Science, Department of Animal Physiology, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia. 3Slovak University of Agriculture, Faculty of Biotechnology and Food Sciences, Department of Technology and Quality of Plant Products, Tr. A. Hlinku 2, SK-94976 Nitra, Slovakia. 4University of Ss. Cyril and Methodius Faculty of Natural Sciences, Department of Biotechnology,, Nám. J. Herdu 2, Trnava, 917 01, Slovakia. 5Latvia University of Life Sciences and Technologies, Institute of Food and Environmental Hygiene, Faculty of Veterinary Medicine, Latvia K. Helmaņa iela 8, LV- 3004, Jelgava, Latvia. 6Slovak University of Agriculture in Nitra, Faculty of Horticulture and Landscape Engineering, Department of Fruit Sciences, Viticulture and Enology, , Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia, phone number: +421 37 641 4985. 7University of Rzeszow, Faculty of Biology and Agriculture, Department of Bioenergy and Food Technology, Zelwerowicza St. 4, PL-35601 Rzeszow, Poland.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2019.9.special.409-413
ARTICLE INFO ABSTRACT
Received 30. 6. 2019 The aim of our study was to work the antimicrobial and antioxidant activities of Citrus paradisi peel, Citrus reticulata peel, Juniperus Revised 23. 9. 2019 communis fruit, Eucalyptus globulus leaf, and Cananga odorata flower essential oils. Essential oil samples were analysed by GC Accepted 7. 10. 2019 chromatography coupled with FID detector (Agilent 6890N). The plant essential oils were tested regarding their antioxidant activity and + - Published 8. 11. 2019 antimicrobial activity against 13 species of Gram positive (G ) and Gram negative (G ) bacteria isolated from human semen and identified by MALDI-TOF MS Biotyper. Isolated G+ and G- were tested for antibiotics resistance Among tested bacteria, the lowest antibiotic susceptibility for Corynebacterium species were observed for ciprofloxacin in three cases, for gentamycin in three cases, for Regular article vancomycin in two cases and for tetracycline in five cases. Antioxidant activities tested using DPPH method reveals that Cannaga odorata had the higher antioxidant activity and the lowest was found at J. communis essential oil. Antimicrobial activities showed that all the essential oils inhibited on the entire 13 species of Gram positive (G+) and Gram negative (G-) bacteria being used. However, C. odorata have the best antimicrobial effect against Corynebacteria. Good antimicrobial activity of C. paradise was found against Corynebacteria also. Strong antibacterial activity of C. odorata against E. coli, E. faecium, P. aglomerans, P. fulva and S. agalactiae were found.
Keywords: biological activity, Gram positive bacteria, Gram negative bacteria, plants, semen samples
INTRODUCTION of the pathogenic role of these species in human infections and of the mechanisms of pathogenesis continues to be elucidated (Long et al., 2018). Recent studies have shown that the simple presence of bacteria in semen samples Escherichia coli are Gram-negative bacteria that are found in food and the may compromise the sperm quality. The bacteria responsible for semen environment. Most E. coli are harmless, but some cause diseases such as contaminations generally originate from the urinary tract of patients or can be diarrhoea and respiratory pneumonia (Kalita et al., 2014). Most people infected transmitted by the partner via sexual intercourse (Purvis and Christiansen, with the Shiga toxin-producing E. coli have stomach cramps, diarrhoea (usually 1993). The most frequently isolated microorganism in male patients with genital bloody), vomiting and high fever, and more serious haemolytic uremic syndrome tract infections or semen contamination is Escherichia coli. The negative (HUS). Escherichia coli is one of the world's best-characterized organisms, influence of this species on sperm quality is partially due to its effect on motility because it has been extensively studied for over a century. Typically, a (Diemer et al., 2003). The influence of gram-positive uropathogenic bacteria on commensal bacterium, E. coli resides in the lower intestines of a slew of animals. sperm morphology and function has been poorly investigated until now. Aerobic Outside of the lower intestine, E. coli can adapt and survive in a very different set cocci are present in about 50% of semen samples of male partners in infertile of environmental conditions. Biofilm formation allows E. coli to survive, and couples. Enterococcus faecalis was isolated from 53% of patients, micrococci even thrive, in environments that do not support the growth of planktonic from 20% and alpha-haemolytic streptococci from 16% of the infected samples. populations. E. coli can form biofilms virtually everywhere: in the bladder during Increased prevalence of genital tract infections caused by E. faecalis is associated a urinary tract infection, on in-dwelling medical devices, and outside of the host with compromised semen quality in terms of sperm concentration and on plants and in the soil. Streptococcus is one of the major pathogens causing morphology. The presence of micrococci and alpha-haemolytic streptococci does mastitis in dairy cows, mainly S. agalactiae, S. dysgalactiae, and S. uberis. The not appear to exert any detrimental effect on sperm quality (Qiang et al., 2007). proportion of cow mastitis caused by Streptococcus is as high as 30%. Notably, Corynebacterium species, which are recognized as members of the normal Streptococcus is prevalent in many countries (Coffey et al., 2006). In adults, S. human flora, were isolated from the skin, mucous membranes, and agalactiae may cause meningitis or septicaemia as well as localized infections gastrointestinal tract. Corynebacterium species have occurred predominantly such as subcutaneous abscesses, urinary tract infection or arthritis. The drivers among patients with prosthetic and other medical devices. Currently, 88 valid behind emergence of S. agalactiae disease in adults are poorly understood studies have been published on Corynebacterium species, and further definition (Delannoy et al., 2013).
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Essential oils and plant products have had a tremendous application in food, cosmetic and folk medicine, and are continuously investigated for their Antibiotic susceptibility testing antimicrobial activities. Essential oils (EOs) obtained from plants are a complex mixture of some compounds such as hydro-carbons, alcohols, esters, aldehydes The antibiotic susceptibility test was performed by using Disc Diffusion Method. and have been reported to exhibit inhibitory activities against a wide spectrum Four different forms of sensitivity discs with 10 mcg concentrations were used food spoilage microorganism (Uysal et at., 2011). Citrus is one of the most for studying the in vitro sensitivity of isolates: ciprofloxacin, gentamycin, consumed fruits in the world and contain a high amount of useful by-products vancomycin, tetracycline, tigecycline and imipenem, ampicillin and which include essential oil. Grapefruit (Citrus paradisi) belongs to the Citrus chloramphenicol. These discs were obtained from “Oxoid”. The results were genus, a taxa of flowering plants in the family Rutaceae. The peel of Citrus fruits interpreted according to EUCAST (2019). is a rich source of flavanones and many polymethoxylated flavones, which are very rare in other plants. These compounds, not only play an important Antimicrobial activity physiological and ecological role, but are also of commercial interest because of their multitude of applications in the food and pharmaceutical industries (Ahmad Disc diffusion method et al., 2006). The berries of Juniperus communis contain 0.2–3.42% of volatile oil, depending on specific parameters of cultivation such as the climate, the soil, We used the agar disc diffusion method for the determination of antimicrobial the altitude, and the degree of ripeness. Traditionally the oil is collected by activities of the essential oil. Briefly, a suspension of the tested microorganism extraction using organic solvent: methanol, n-hexane, but the main used process (0.1 mL of 105 cells per ml) was spread on the solid media plates. Filter paper is steam distillation of the crushed, dried, partially dried, or fermented berries discs (6 mm in diameter) were impregnated with 15 µl of the oil and placed on (Carpenter et al., 2012). the inoculated plates. They were inoculated onto the surface of Mueller Hinton Essential oils are the odorous, volatile products of the secondary metabolism of Agar (MHA, Oxoid, Basingstoke, United Kingdom). These plates, after an aromatic plant, which are often concentrated in a particular organ of the plant remaining at 4 °C for 2 hours, were incubated anaerobically at 37 °C for 24 h. such as leaves, stems, bark or fruit and are stored in secretory cells, cavities, The diameters of the inhibition zones were measured in millimeters. All the tests canals, epidermic cells or glandular trichomes. Approximately 3000 essential oils were performed in duplicate. are known, 300 of which are commercially important especially for the pharmaceutical, agronomic, food, sanitary, cosmetic and perfume industries. A Radical scavenging activity – DPPH method number of studies have demonstrated the antimicrobial properties of Eucalyptus essential oils against a wide range of microorganisms. These studies, however, Radical scavenging activity of essential oils was measured using 2,2-diphenyl-1- are focused on a few Eucalyptus species, especially Eucalyptus globulus oil, picrylhydrazyl (DPPH) according to Sanchéz-Moreno et al., (1998) with slight which has been shown to have a wide spectrum of antimicrobial activity (Tyagi modification. The sample (0.1 ml) was mixed with 3.9 ml of DPPH solution and Malik, 2011). (0.025 g DPPH in 100 ml methanol). Absorbance of the reaction mixture was Cananga (Cananga odorata) is known for its fragrant flowers . In addition, determined using the spectrophotometer Jenway (6405 UV/Vis, England) at 515 cananga is also known as a medicinal plant, the leaves as a remedy for itch, dried nm. The scavenging activity percentage (AA%) was determined according flowers as malaria drugs, fresh flowers for aroma therapy, as well as the bark as a formula: remedy for ulceration. Essential oil contained in cananga flowers is used as a AA% = [(A0 - AAT)/A0 × 100] fragrance ingredient. Cananga oil has quite a high economic value in the world market. In addition to economic value, cananga trees also have ecological value, where A0 is absorbance of control reaction (DPPH radical); A1 is the absorbance where this trees can be used for slope stability due to the type of roots are strong in presence of sample (Mahfud et al., 2017). The main aim of our study were evaluate the chemical composition, antimicrobial Gas chromatography and antioxidant activity of five essential oils against Gram positive and Gram negative bacteria isolated from human semen. In our study we also identified Essential oil samples were analysed by Agilent 6890N chromatograph (Agilent bacterial strain with mass spectrometry and tested these strains for antibiotic Technologies, Santa Clara, California, USA) with FID detector. System control resistance. and data analysis were processed using the Agilent ChemStation software Rev. B.04.03-SP1 (Agilent Technologies). The chromatographic separation was MATERIAL AND METHODS performed in the DB-23 column (0.25 mm i.d., 30 m long, 0.25 µm film thickness) (Agilent Technologies), and 5 µl of the sample was injected. The Essential oils injector temperature was 250 °C, and the FID temperature was set at 250 °C. The carrier gas (nitrogen) flow was 1.1 ml/min (constant flow) with a split ratio 1:100 Citrus paradisi peel oil, Citrus reticulata peel oil, Juniperus communis fruit oil, and a temperature program from 40 °C to 80 °C at 3 °C.min-1, from 80 °C to 180 Eucalyptus globulus leaf oil, and Cananga odorata flower oil, were purchased °C at 5 °C.min-1, and from 180 °C to 220 °C at 8 °C.min-1 and finally held at from Aromatika (Russia). Samples were obtained via steam distillation as pure 220°C for 15 min. essential oils. The essential oil samples were stored in glass vials with teflon- sealed caps at laboratory temperature in darkness. Statistical analyses
Microorganisms All measurements and analyses were carried out in triplicate. Experimental data were evaluated by basic statistical variability indicators using the Microsoft™ Thirteen different kinds of Gram positive (G+) and Gram negative (G-) bacteria Excel® program. Dependency rate between the tested traits was expressed using were isolated from human ejaculates. The semen samples were obtained from 30 the linear correlation analysis. healthy donors by masturbation into sterile container. Only ejaculates showing normal semen parameters were used. The samples underwent liquefaction at 37 RESULTS AND DISCUSSION °C for 30 min. Experiments were performed within 1 h from sample collection. Semen samples were inoculated on Tryptone Soya agar (TSA) and Levine agar Some previous studies have identified bacteria in semen as being a potential (LA). After incubation , bacteria were selected for further confirmation with factor in male infertility (Anitha et al., 2006; Weng et al., 2014). Similar results MALDI-TOF MS Biotyper. Isolated G+ and G- species were tested for antibiotics were obtained in our study (Table 1). Kiessling et al. (2008) performed PCR resistance against ciprofloxacin, gentamycin, vancomycin, tetracycline, amplification of bacterial rDNA on 34 semen samples, and identified gram- tigecycline and imipenem, ampicillin and chloramphenicol 10 msg. Three G- positive anaerobic cocci, Corynebacterium spp., Staphylococcus, Lactobacillus, bacteria (Escherichia coli, Pantotea aglomerans, P. fulva) and ten G+ bacteria Streptococcus spp., Pseudomonas spp., Haemophilus and Acinetobacter spp. as (Corynebacterium autimocus, four strains of C. glucuromoliticum, C. singulaere the largest groups in different specimens. Corynebacterium is an emerging and two strains of Streptococcus agalactiae) were tested. The bacteria species multidrug-resistant bacteria (Hahn et al., 2016). In our study Corynebacterium were maintained in Mueller Hinton Agar (Merck, Germany). The mother cultures species were resistant for ciprofloxacin on three case, for gentamycin on three of each bacteria tested were set up 24 h before the assays in order to reach the case, for vancomycin on two case and for tetracycline on five case. stationary phase of growth. The tests were assessed by inoculating Petri dishes from the mother cultures with proper sterile media. The main aim was to obtain the microorganism concentration of 105 colony forming units (cfu).mL-1.
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Table 1 Antibiotic resistance of bacterial strains isolated from human semen Microorganisms CIP GEN VAN TET TGC IMP AMP CHL Corynebacterium autimocus S S S R - - - - Corynebacterium glucuromoliticum S S R R - - - - Corynebacterium glucuromoliticum S R R R - - - - Corynebacterium glucuromoliticum S R S S - - - - Corynebacterium glucuromoliticum S R S R - - - - Corynebacterium singulare R S S R - - - - Enterococcus faecalis R - - - S R S - Enterococcus faecalis S - - - S S S - Escherichia coli R S - S - R - - Pantotea aglomerans S S - S - S - - Pantotea fulva S S - S - S - - Streptococcus agalactiae - - S R S - - R Streptococcus agalactiae - - S R S - - S Legend: CIP-ciprofloxacin; GEN-gentamycin; VAN-vancomycin; TET-tetracycline; TGC-tigecycline; IMP- imipenem; AMP-ampicillin; CHL-chloramphenicol
In table 2 we can observe the antimicrobial activity of five essential oils against The pinene-type of essential oil showed moderate antimicrobial activity against bacteria isolated from human sperm. Our results show that the best antibacterial Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, activity against Corynebacterium species for Cananga odorata essential oil were Streptococcus pyogenes, Corynebacterium spp. and Campylobacter jejuni with found on C. autimocus, C. glucoromoliticu and C. singulare. The study of MIC >50% (Karapandzova et al., 2015). However, a slight antagonism effect antibacterial properties proved the sensitivity of all wild strains tested to EO: E. was then observed when ylang-ylang oil was used together with thyme oil against coli; Bacillus sp.; Pseudomonas sp.; K. oxytoca; Corynebacterium sp.; Nocardia Escherichia coli ATCC 25922, the inhibition zone was reduced by 48.9% when sp.; S. aureus; Enterobacter sp.; E. agglomerans and Streptococcus group D. It compared to thyme oil alone (Kon and Rai, 2012). Similarly, another study showed, however, some differences in sensitivity profile, and Gram-positive revealed that blended essential oil preparation which is comprised of lavender, species are more sensitive to the EO, which is very likely to be explained by the clary sage, and ylang-ylang oils in the ratio 3:4:3 displayed a strong antibacterial lower structural complexity of their cell walls. Traditional antibiotics act on a and antifungal activities against Staphylococcus aureus ATCC 6538, single cell site, and thus can develop bacterial resistance, there is then the EO as Staphylococcus epidermidis, Escherichia coli ATCC 25923, Pseudomonas an alternative to the use of conventional antibiotics. The results open perspectives aeruginosa ATCC 9027, and Candida albicans ATCC 10231 (Tadtong et al., for future use in hospital settings (Cole et al., 2014). 2012).
Table 2 Antimicrobial activity of five essential oils against bacteria isolated from human sperm Citrus Juniperus Eucalyptus Cananga Citrus paradisi reticulata communis globulus odorata Corynebacterium autimocus 8.33±0.58 7.33±0.58 6.67±1.15 7.67±0.58 11.33±1.15 Corynebacterium glucuromoliticum 6.67±1.53 5.33±0.58 4.33±0.58 5.67±0.58 10.33±0.58 Corynebacterium glucuromoliticum 8.67±1.15 3.33±0.58 3.67±0.58 2.67±0.58 8.33±0.58 Corynebacterium glucuromoliticum 8.33±0.58 5.33±0.58 2.33±0.58 3.33±0.58 6.67±1.53 Corynebacterium glucuromoliticum 7.33±0.58 5.33±0.58 3.67±0.58 4.33±0.58 8.33±0.58 Corynebacterium singulare 5.33±0.58 5.67±0.58 7.33±0.58 7.67±0.58 9.67±1.53 Enterococcus faecalis 5.33±0.58 7.33±0.58 4.67±0.58 7.67±0.58 8.67±0.58 Enterococcus faecalis 5.67±0.58 3.67±0.58 5.67±0.58 3.67±0.58 5.67±0.58 Escherichia coli 5.33±0.58 6.67±0.58 8.00±1.00 9.33±1.53 11.00±1.73 Pantotea aglomerans 11.00±1.00 8.33±0.58 6.67±0.58 5.67±0.58 14.33±1.15 Pantotea fulva 5.67±0.58 2.67±0.58 3.67±0.58 11.33±1.15 10.67±0.58 Streptococcus agalactiae 5.33±0.58 4.33±1.53 6.33±1.15 9.33±1.53 11.33±1.15 Streptococcus agalactiae 4.33±0.58 5.33±0.58 4.67±0.58 8.33±0.58 14.33±0.58
The antioxidant activity of all five essential oils is shown table 3. The best Table 3 Antioxidant activity of essential oils antioxidant activity of essential oil was found on Canangaodorata. The Essential oil Antioxidant activity % antioxidant activity of C. odorata extracts was evaluated using DPPH assay to Citrus reticulata 3.40 determine the free radical scavenging abilities of the extracts. The result of the study revealed that the ethyl acetate extract of the stem bark of C. odorata Citrus paradisi 58.96 exhibited the highest percentage of DPPH inhibition (79%) as compared to other Juniperus communis 0.100 tested plant extracts (Tan et al., 2015). The most important applications of citrus Eucalyptus globulus 31.63 peel essential oils is the presence of some bioactive compounds in them which Cananga odorata 73.14 serve as alternatives to the synthetic antioxidants (Tepe et al., 2006; Viuda- Martos et al., 2008; Choi et al., 2000). The antioxidant activity of the oil attributable to electron transfer made juniper berry essential oil a strong Different kinds of essential oils from various plant material (Citrus paradisi, antioxidant, whereas the antioxidant activity attributable to hydrogen atom Citrus reticulata, Juniperus communis, Eucalyptus globulus, and Cananga transfer was lower (Höferl et al., 2014). The Eucalyptus oil extracted from the odorata) were compared by GC-FID chromatography. The most intensive peak leaves of Eucalyptus globulus family Myrtaceae was screened for the presence of in C. paradisi essential oil was registered at 15.83 min with area percentage of 87 phytochemicals and their effect on 2, 2-diphenyl-1-picryl-hydrazyl radical % (Fig. 1, red). The analysis of essential oil from C. reticulata showed the (DPPH) and Nitric oxide free radical. Phytochemical screening of the plants dominant peak at 15.89 min. (85 % of area percentage) (Fig. 1, green). The major showed the presence of flavonoids, terpenoids, saponins and reducing sugars. peaks determined for J. communis essential oil sample were identified at 10.75 Eucalyptus globulus is not having any cardiac glycosides and anthraquinones. min (66.6 %), 13.24 min (16.7 %), and 15.3 min (2.9 %) (Fig. 1, blue). Essential The free radical scavenging activity of the different concentrations of the leaf oil oil isolated from E. globulus leafs showed the dominant peak at 17.95 min (81.7 (10, 20, 40, 60 and 80% (v/v) in DMSO) of E. globulus increased in a %) and some minor peaks at 15.5 min (7.3 %) and 18.23 min (6.4 %) (Fig. 1, concentration-dependent manner. In DPPH method, the oil in 80% (v/v) pink). The last sample of essential oils was isolated from C. odorata flower. The concentration exhibited 79.55 ± 0.82%. In nitric oxide radical scavenging assay gas chromatograph showed the dominant peaks at 24.38 min (22.98 %), 43.92 method, it was found that 80% (v/v) concentration exhibited 81.54 ± 0.94% min (18.28 %), and 30.42 min (7.7 %). Minor peaks were registered at 27.49 min inhibition (Mishra et al., 2010). (4.6 %), 29.1 min (4.9 %) and 32.79 min (3.4 %) (Fig. 1, khaki). The study revealed that the main components identified from the oxygenated fraction of ylang-ylang essential oil were p-methylanisole, methyl benzoate and benzyl benzoate, benzyl acetate, geranyl acetate, cinnamyl acetate and (E,E)-
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farnesyl acetate, linalool, geraniol, and benzyl salicylate (Tan et al., 2015). The against wild strains of hospital origin. Brazilian Journal of Microbiology, 45(3), chemical composition of the essential oils obtained from the citrus peels, by 821-828. https://dx.doi.org/10.1590/S1517-83822014000300009 hydrodistillation, was analyzed by Gas chromatography–mass spectrometry (GC- DELANNOY, C. M., CRUMLISH, M., FONTAINE, M. C., POLLOCK, J., MS). 12 compounds were identified and limonene was the common major FOSTER, G., DAGLEISH, M. P., TUMBULLI, J.F., ZADOKS, R. N. 2013. component for the three essential oils (77- 97%) (Boudries et al., 2017). The 80 Human Streptococcus agalactiae strains in aquatic mammals and fish. BMC components identified in the Citrus peel essential oil were all terpenes and related microbiology, 13(41), https://doi.org/10.1186/1471-2180-13-41 compounds, which comprised of 11 monoterpenes (tota1 60 μg.g-1 of oil), 8 DIEMER, T., HUWE, P., LUDWIG, M., SCHROEDER-PRINTZEN, I., oxygenated monoterpenes (277 μg.g-1), 25 sesquiterpenes (1.940 μg.g-1), 29 MICHELMANN, H.W., SCHIEFER, H.G., WEIDNER, W. 2003. Influence of oxygenated sesquiternpenes (9.420 μg.g-1), and 7 esters (175 μ.g-1). The major autogenous leucocytes and Escherichia coli on sperm motility parameters in chemical compounds in the Eucalyptus camaldulensis leaves oil were spathulenol vitro. Andrologia, 35(2), 100–105. https://doi.org/10.1046/j.1439- (1.900 μg.g-1), α-14-oxymuurolene (1,830 μg.g-1), β-bisabolene (1.190 μg.g-1), 0272.2003.00523.x caryophyllene oxide (1.010 μg.g-1), γ-patchoulene (848 μg.g-1), aristolone (797 EL-GHORAB, A., EL-MASSRY , K.F., ANJUM , F.M., SHAHWARB, M.K., μg.g-1), α-atlantone (561 μg.g-1), β-piotol (332 μg.g-1), (E)-caryophyllene (328 SHIBAMOTO, T. 2009. The Chemical Composition and Antioxidant Activity of μg.g-1), and β-copaen-4α-ol (313 μg.g-1) (El-Ghorab et al., 2009). Using GC/FID Essential Oil of Pakistani Eucalyptus camaldulensis Leaves. Journal of Essential and GC/MS, 70 compounds were identified in the essential Juniperus communis Oil Bearing Plants, 12(3), 262-272. L. oil (altogether, about 96% of the volatiles). As main components, the https://doi.org/10.1080/0972060X.2009.10643719 monoterpene hydrocarbons α-pinene (51.4%), myrcene (8.3%), sabinene (5.8%), EUCAST, 2019. Clinical breakpoints and dosing of antibiotics. limonene (5.1%) and β-pinene (5.0%) were found. Concluding, the essential oil http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/ mainly comprises mono- and sesquiterpene hydrocarbons (80.4% and 9.6%, Breakpoint_tables/v_9.0_Breakpoint_Tables.pdf respectively); oxygenated derivatives are only minor constituents of this essential HAHN, W.O., WERTH, B.J., BUTLER-WU, S.M., RAKITA, R.M. 2016. oil (Höferl et al., 2014). Multidrug-Resistant Corynebacterium striatum Associated with Increased Use of *FID1 A, (ACID VIOLET\09092016000071.D) *FID1 A, (ACID VIOLET\09092016000069.D) Parenteral Antimicrobial Drugs. Emerging infectious diseases, 22(11), 1908– *FID1 A, (ACID VIOLET\09092016000070.D) *FID1 A, (ACID VIOLET\09092016000072.D) *FID1 A, (ACID VIOLET\09092016000073.D) 1914. https://doi.org/10.3201/eid2211.160141 pA HÖFERL, M., STOILOVA, I., SCHMIDT, E., WANNER, J., JIROVETZ, L., TRIFONOVA, D., KRASTEV, L., KRASTANOV, A. 2014. Chemical 10000 composition and antioxidant properties of juniper berry (Juniperus communis L.) essential oil. Action of the essential oil on the antioxidant protection of 8000 Saccharomyces cerevisiae model organism. Antioxidants, 3, 81-98. https://doi.org/10.3390/antiox3010081 6000 CHOI, H.S., H.S. SONG, H. UKEDA AND M. SAWAMURA. 2000. Radicalscavenging activities of citrus essential oils and their components:
4000 Detection Using l,l-Diphenyl-2-picrylhydrazyl. Journal of Agriculture and Food Chemistry, 48(9), 4156-4161. https://doi.org/10.1021/jf000227d KALITA, A., HU, J., TORRES, A.G. 2014. Recent advances in adherence and 2000 invasion of pathogenic Escherichia coli. Current Opinion in Infectious Diseases, 27(5), 459-464. https://doi.org/10.1097/qco.0000000000000092 0 KARAPANDZOVA, M., STEFKOV, G., CVETKOVIKJ, I., KULEVANOVA, 0 10 20 30 40 50 min Figure 1 Gas chromatograph for Juniperus communis (blue), Citrus paradisi S., SELA, F. 2015. Chemical composition and antimicrobial activity of essential (red), Citrus reticulata (green), Eucalyptus globulus (pink), and Cananga oils of Juniperus excelsa Bieb. (Cupressaceae) grown in R. Macedonia. odorata (khaki) essential oil samples Pharmacognosy Research, 7(1), 74. https://doi.org/10.4103/0974-8490.147212 KIESSLING, A.A., DESMARAIS, B.M., YIN, H.Z., LOVERDE, J., EYRE, CONCLUSION R.C. 2008. Detection and identification of bacterial DNA in semen. Fertility and Sterility, 90, 1744-1756. https://doi.org/10.1016/j.fertnstert.2007.08.083 C. odorata showed the best antioxidant activity when compared with other KON, K., RAI, M. 2012. Antibacterial activity of Thymus vulgaris essential oil essential oils. The antimicrobial activity of C. odorata has a very good effect alone and in combination with other essential oils. Nusantara Bioscience. 4(2), against main bacterial species isolated from human semen. The best antimicrobial 50-56. https://doi.org/10.13057/nusbiosci/n040202 activity of Cananga odorata essential oil in our study were found against LONG, S.S., CHARLES, G., PROBER, M.D., FISCHER, M. 2018. Principles Pantotea aglomerans and Streptococcus agalactiae. Overpopulation is known to and Practice of Pediatric Infectious Diseases (Fifth Edition), 1688 p. ISBN: be a global issue and public health concern. This activity is probably due the 978-0-323-40181-4 higher level of benzyl benzoate and carryophyllene which were detected in this MAHFUD, M., PUTRI, D.K.Y., DEWI, I.E.P., KUSUMA, H.S. 2017. Extraction kind of oils by many authors. Currently, medicinal plants have also received huge of essential oil from cananga (Cananga odorata) using solvent-free microwave attention for its use as contraceptives due to their little side effects. C. odorata extraction: a preliminary study. Rasayan Journal of Chemistry, 10(1), 86-91, 86- was also found to possess spermicidal activity in both in vitro and in vivo studies. 91 ISSN 0974-1496. https://doi.org/10.7324/rjc.2017.1011562 MISHRA, A.K., SAHU, N., MISHRA, N., GHOSH, A.K., JHA, S., Acknowledgments: This study was supported by the Slovak Research and CHATTOPADHYAY, P. 2010. Phytochemical Screening and Antioxidant Development Agency under the grant APVV-15-0544. Activity of essential oil of Eucalyptus leaf. Pharmacognosy Journal, 2(16), 25- 28. https://doi.org/10.1016/S0975-3575(10)80045-8 REFERENCES PURVIS, K., CHRISTIANSEN, E. 1993. Infection in the male reproductive tract. Impact, diagnosis and treatment in relation to male infertility. International ANITHA, P., INDIRA, M. 2006. Impact of feeding ethanolic extract of root bark Journal of Andrology , 16(1), 1-13. https://doi.org/10.1111/j.1365- of Cananga odorata (Lam) on reproductive functions in male rats. Indian 2605.1993.tb01146.x Journal of Experimental Biology. 44(12), 976-980. QIANG, H., JIANG, M.S., LIN, J.Y., HE, W.M. 2007. Influence of enterococci BOUDRIES, H., LOUPASSAKI, S., LADJAL ETTOUMI, Y., SOUAGUI, S., on human sperm membrane in vitro. Asian Journal of Andrology, 9(1), 77–81. BACHIR BEY, M., NABET, N., CHIKHOUNE, A., MADANI, K., CHIBANE, https://doi.org/10.1111/j.1745-7262.2007.00219.x M. 2017. Chemical profile, antimicrobial and antioxidant activities of Citrus SÁNCHÉS-MORENO, C., LARRAURI, A., SAURA-CALIXTO, F. 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ANTAGONISTIC EFFECT OF GUT MICROBIOTA OF HONEYBEE (APIS MELLIFERA) AGAINST CAUSATIVE AGENT OF AMERICAN FOULBROOD PAENIBACILLUS LARVAE
Miroslava Kačániová1,2, Jaroslav Gasper3, Margarita Terentjeva4
Address(es): 1Slovak University of Agriculture in Nitra, Faculty of Horticulture and Landscape Engineering, Department of Fruit Sciences, Viticulture and Enology, Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia, phone number: +421 37 641 4715. 2University of Rzeszow, Faculty of Biology and Agriculture, Department of Bioenergy and Food Technology, Zelwerowicza St. 4, PL-35601 Rzeszow, Poland. 3Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Microbiology, Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia. 4Latvia University of Life Sciences and Technologies, Institute of Food and Environmental Hygiene, Faculty of Veterinary Medicine, Latvia K. Helmaņa iela 8, LV- 3004, Jelgava, Latvia.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2019.9.special.478-481
ARTICLE INFO ABSTRACT
Received 30. 6. 2019 The aim of our study was to isolate and identify the microorganisms of gut microbiota of honeybees with MALDI-TOF MS Biotyper Revised 23. 9. 2019 and to identify their anti-Paenibacillus larvae affect. In our study, 200 samples of bee’s gut originated from honeybee of Slovak origin Accepted 10. 10. 2019 were tested. A total of 23 species of 17 bacterial genera (10 Gram-negative and 6 Gram-positive, one yeast) were identified in gut of Published 8. 11. 2019 bees with MALDI-TOF Mass Spectrometry. The bacterial strains Bacillus cereus, B. megatherium, B. oleronius, B. thuringiensis, Delftia acidovorans, Escherichia coli, Enterococcus cloacae, Issatchenkia orientalis, Klebsiella oxytoca, Lactobacillus acidophilus, L. crispatus, L. kunei, Morganella morgani, Pantotea aglomerans, P. ananatis, Pseudomonas oryzihabitans, Rahnella aquatilis, R. Regular article terrigena, Raoutella ornithinolytica, Serratia liquefaciens, S. marcescens, Sphingomonas parapaucimobilis, Staphylococcus epidermidis were representative part of gut microflora of bees. Bees isolates of Paenibacillus larvae and Paenibacillus larvae CCM 4438 were tested for antimicrobial activity. The best antimicrobial activity against P. larvae isolated from bee’s gut showed L. kunkei, L. crispatus and L. acidophilus. The lower antibacterial activity against P. larvae was found in Raoutella ornithinolytica, Pantotea ananatis and Serratia liquefaciens isolated from bees’ gut. The strongest antimicrobial activity of L. kunkei, L. acidophilus and L. crispatus and the lowest antimicrobial activity of Pantotea ananatis and Rahnella aquatilis were found against P. larvae CCM 4438.
Keywords: GIT microbiology, mass spectrometry, Apis mellifera, antimicrobial activity, Paenibacillus larvae
INTRODUCTION incurable, and the significant economic loss in apiculture are attributed due to high mortality rates (Sabate et al., 2009). The Western honeybee (Apis mellifera) is the most important insect pollinator The aim of this study was to study microbiota isolated from gut of local honeybee around the globe. Human food consumption relies on insect pollination and (Apis mellifera) of East Slovakia with MALDI-TOF MS Biotyper and to evaluate almost 90% of crops depends on honeybee pollination services (Klein et al., the effect of isolated gut bacteria from to the P. larvae. 2007). Honeybee is important not only for its services and products consumed by humans, because the honeybee plays unique role in sustaining of natural plant MATERIAL AND METHODS biodiversity in ecosystem (Potts et al., 2010). Honeybees are social insects living in colonies consisted of different generations, taking cooperative brood care and a Samples reproductive division of labor. The colony consists of female worker bees which number could be between 15000-50000 in different seasons with a peak in the Altogether, 100 samples of the Slovak worker honeybees examined in this study. summer, male drones usually present in the spring and one reproducing female Additionally, a total of 100 gastrointestinal samples from the Slovak beekeeper queen bee (Vojvodic et al., 2013). Honeybees share a diverse microbiome with located in the east, middle and west part of Slovakia were collected (Košice, different bacterial taxa, ranging from Gram-positive bacteria to alpha-, beta-, and Poprad, Liptovský Hrádok, Nitra, Brezno). The specimens were obtained from gamma-proteobacteria (Gilliam, 1997; Jeyaprakash et al., 2003). Only some of the hive. All samples were placed in sterile sample containers and transported to those bacterial species are pathogenic to bees, most of them have never been the laboratory on ice for microbiological investigations. The workers were reported in honeybee disease and their impacts on honeybee are still unknown. decapitated, and the midgut and rectum were removed. The content of gut was Gut bacteria is associated with stimulation of the immunity of honeybee larvae, weighed to obtain a 0.1 g of sample material. because in this stage the organism is especially vulnerable to infection, and anti- pathogen immune responses. Feeding larvae with non-pathogenic gut bacteria Microbiological analyses containing diet stimulated the expression of genes involved in immune response (Evans and Lopez, 2004). Supplement of probiotic bacteria into diet of the Gram-positive, Gram-negative bacteria and yeasts in bee gut were detected. A honeybee larval food decreased the number of infections with Paenibacillus content of gut was streaked onto MacConkey agar (MCA, Merck, Germany) larvae (Forsgren et al., 2010). It has been concluded that the addition of which was incubated for 24-48 h at 37°C, aerobically. For cultivation of Gram- probiotic bacteria will improve the honeybee immune response and protection positive and Gram-negative microorganisms, the inoculated Tryptone soya agar against pathogens. Paenibacillus larvae, a Gram-positive sporulated bacterium, and Enterococcus selective agar (TSA, ESA, Oxoid, UK) was incubated for 48- causes the American foulbrood disease is of broad distribution, extremely 72 h at 30°C. Also blood agar (BA, Oxoid, UK) and de Man, Rogosa and Sharpe contagious diseases and could kill the honeybee colonies (Williams, 2000). agar (MRS, Oxoid, UK) were incubated for 48-72 h at 37°C anaerobically. For Infectious dose of a 24-h-old larva ill dose is 10 spores. Since disease is mostly
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detection of yeasts, sample was plated onto Malt extract agar (MEA, Merck) and Table 1 Isolated species of microorganisms from gut of honeybees inoculated agar was incubated for 5 days at 25°C aerobically. Family Genera Species
Bacillaceae Bacillus Bacillus cereus Sample preparation and MALDI-TOF MS measurement Bacillaceae Bacillus Bacillus megatherium Prior to identification, the bacterial colonies were sub-cultured on TSA agar Bacillaceae Bacillus Bacillus oleronius (Tryptone Soya Agar, Oxoid, UK) at 37°C for 18-24 h. One colony of eight Bacillaceae Bacillus Bacillus thuringiensis bacterial cultures was selected. The identification was performed with Maldi Comamonadaceae Delftia Delftia acidovorans TOF MS Biotyper as described by Kačániová et al. (2017). Totally, a number of 534 isolates were identified with score higher than 2. Enterobacteriaceae Escherichia Escherichia coli Enterococaceae Enterococcus Enterococcus cloacae Detection of antibacterial activity of different isolates of bacteria against Saccharomycetaceae Issatchenkia Issatchenkia orientalis Paenibacillus larvae Enterobacteriaceae Klebsiella Klebsiella oxytoca
Lactobacillaceae Lactobacillus Lactobacillus acidophilus The bacterial strains of Bacillus cereus, B. megatherium, B. oleronius, B. thuringiensis, Delftia acidovorans, Escherichia coli, Enterococcus cloacae, Lactobacillaceae Lactobacillus Lactobacillus crispatus Issatchenkia orientalis, Klebsiella oxytoca, Lactobacillus acidophilus, L. Lactobacillaceae Lactobacillus Lactobacillus kunkei crispatus, L. kunkei, Morganella morgani, Pantotea aglomerans, P. ananatis, Enterobacteriaceae Morganella Morganella morgani Pseudomonas oryzihabitans, Rahnella aquatilis, R. terrigena, Raoutella Enterobacteriaceae Pantotea Pantotea aglomerans ornithinolytica, Serratia liquefaciens, S. marcescens, Sphingomonas parapaucimobilis, Staphylococcus epidermidis, Paenibacillus larvae were Enterobacteriaceae Pantotea Pantotea ananatis isolated from gut microflora of bees. Paenibacillus larvae CCM 4438 was Pseudomonadaceae Pseudomonas Pseudomonas oryzihabitans originated from the Czech Collection of microorganisms (Brno, Czech Republic). Enterobacteriaceae Rahnella Rahnella aquatilis Bacterial strains after 24 h of incubation in MRS broth and TSA broth were Enterobacteriaceae Rahnella Rahnella terrigena centrifuged at 5500 g for 10 min at 4°C and 0.1 mL of the supernatant was used for detection of antibacterial activity. The suspension of tested bacteria isolated Enterobacteriaceae Raoutella Raoutella ornithinolytica from gut of bees in saline (0.1 mL of 105 cfu.mL-1) were spread on Mueller Enterobacteriaceae Serratia Serratia liquefaciens Hinton Agar (MHA, Oxoid). Filter paper discs (6 mm in diameter) were Enterobacteriaceae Serratia Serratia marcescens impregnated with 15 µL of supernatant and placed on the inoculated agars. Agars Sphingomonadaceae Sphingomonas Sphingomonas parapaucimobilis were incubated at 4°C for 2 h and at 37°C for 24 h. All the tests were performed in triplicate. Filter discs impregnated with a 10 μL of distilled water were used as Staphylococcaceae Staphylococcus Staphylococcus epidermidis the negative, but antibiotic (amikacin 10 µg and gentamicin 10 µg) as the positive control (Kačániová et al., 2018). Table 2 Number of isolates identified with MALDI-TOF MS Biotyper in bees’ gut No. of RESULTS AND DISCUSSION Microorganisms No. of isolates in % isolates The composition of the gut microbiota of social insects has been shaped changed Delftia acidovorans 42 18.1 by coevolution. The social behaviour of honeybees creates favorable conditions Escherichia coli 21 9.1 for exchange with microorganisms, which are going to be transmitted either Klebsiella oxytoca 18 7.73 between colonies members or different generations (Engel and Moran, 2013a). The gut microbial composition of honeybees is unique with microorganisms Morganella morgani 15 6.44 present to be niche-adapted bacterial species with a high degree of genetic Pantotea aglomerans 10 4.29 diversity (Engel and Moran, 2013b). Pantotea ananatis 12 5.15 Pseudomonas oryzihabitans 10 4.29 Isolated bacterial group Rahnella aquatilis 15 6.44 Total count of microorganisms bacteria in bees gut ranged from 5.25 to 6.58 log Rahnella terrigena 14 6.01 cfu.g-1. Enterococci count ranged from 4.25 to 4.87 Raoutella ornithinolytica 18 7.73 -1 -1 log cfu.g . Coliforms bacteria counts were from 4.55 to 5.80 log cfu.g , lactic Serratia liquefaciens 20 8.58 acid bacteria counts - from 2.22 to 2.51 log cfu.g-1 and yeast from 1.18 to 1.23 log cfu.g-1. Kačániová et al. (2004) found in the midgut and rectum of the honeybee Serratia marcescens 22 9.44 that counts of aerobic microorganisms were significantly lower than counts of Sphingomonas parapaucimobilis 16 6.87 anaerobes (105-106 vs. 108-109 viable cells per g of intestinal content). Total Gram-negative bacteria 233 numbers of anaerobic microorganisms were almost identical with the count of Microorganisms Total Percentage of bacterial species anaerobic Gram-positive acid resistant rods. A higher number of coliform bacteria and Bacillus spp. was detected in the rectum (105 per g). Anaerobic and Bacillus cereus 22 7.94 aerobic microorganisms, coliforms, enterococci, Bacillus spp., Pseudomonas spp. Bacillus megatherium 5 1.81 and yeasts were found in all bees; lactobacilli, staphylococci and moulds were Bacillus oleronius 8 2.89 not found. Bacillus thuringiensis 12 4.33
Microorganisms identified with MALDI-TOF MS Biotyper Enterococcus cloacae 27 9.75 Lactobacillus acidophilus 46 16.61 A total of 23 species of 17 bacterial genera (10 Gram-negative and 6 Gram Lactobacillus crispatus 52 18.77 positive, 1 yeast) were identified in gut of bees with MALDI-TOF Mass Lactobacillus kunkei 69 24.91 Spectrometry. Gram-negative, Gram-positive and yeast isolates comprised Staphylococcus epidermidis 36 12.99 43.63% (233 isolates), 51.87% (277 isolates) and 4.49% (24 isolates), Gram-positive bacteria 277 respectively. Isolated species of microorganisms from bees’ gut are shown in Table 1. Percentage of isolated species is at Figure 1. Issatchenkia orientalis 24 100 Number of isolates of each species for Gram-positive, Gram-negative bacteria Yeasts 24 and yeasts are shown in Table 2. The most abundant Gram-negative bacteria were Total 534 Delfia acidovorans, Serratia marcescens, Escherichia coli and Serratia liquefaciens. Lactobacillus spp. was the most abundant within 3 different species of Gram-positive bacteria with Lactobacillus acidophilus, L. crispatus and L. The microbiota associated with honeybee is complex, and previously mainly kunkei were the most distributed. yeasts, Gram-positive bacteria (Lactobacillus rigidus apis, S. constellatus, Bacillus spp., Streptococcus, and Clostridium spp.), and Gram-negative or Gram- variable bacteria (Achromobacter, Citrobacter, Enterobacter, Erwinia, Escherichia coli, Flavobacterium, Klebsiella, Proteus, and Pseudomonas) were identified (Mohr and Tebbe, 2006). The presence of Enterobacteriaceae, represented by Pantoea spp. and Enterobacter gergoviae were identified (Rada et al., 1997, Ebrahimi and Lotfalian, 2005; Chahbar and Mahamed, 2014). Distribution of the same
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genera of Enterobacteriaceae was explained with trophallaxie is made between Enterococcus faecalis with Melissococcus plutonius was reported as developing the worker bees (Hansen et al., 2004). Enterococcus group is not abundant in (Evans and Schwarz, 2011). honeybee intestinal samples in comparison to Lactobacillus spp. Coinfection of
Figure 1 Composition of honeybees gut microbiota
The anti-Paenibacillus larvae activity Endogenous administration of LAB inhibited Melissococcus plutonius, which causes European Foulbrood (Forsgren et al., 2009, Vasquez et al., 2012). All tested microorganisms exhibited the antimicrobial activity against According to their works, the effectiveness of endogenous LAB administration Paenibacillus larvae. The strongest antimicrobial activity was shown by against the bacterial pathogens increased when a mixture of LAB was used, but Lactobacillus spp., while the weakest antimicrobial activity was exhibited by this antimicrobial action has never been attributed to bacteriocin action. Lactic Enterobacteriaceae (Table 3). The best antimicrobial activity against P. larvae acid bacteria strains of the bee gut, namely Lactobacillus AJ5, IG9, A15 and were expressed by L. kunkei, L. crispatus and L. acidophilus. Raoutella CRL1647 were able to inhibit S. aureus ATCC29213, B. cereus C1, E. coli ornithinolytica, Pantotea ananatis and Serratia liquefaciens were less active O157:H7, L. monocytogenes and P. larvae; but strain A15 failed to inhibit two of against P. larvae isolated from bees’ gut. The strongest antimicrobial activity of eight P. larvae strains. E. faecium SM21 exhibited activity only against L. L. kunkei, L. acidophilus and L. crispatus and the weakest antimicrobial activity monocytogenes (Audisio et al., 2011). of Pantotea ananatis and Rahnella aquatilis were found against P. larvae CCM 4438. In vitro inhibition of P. larvae has been previously reported (Evans and Armstrong, 2005; Alippi and Reynaldi, 2006; Sabaté et al., 2009). Endogenous strains of lactic acid bacteria of bees did not inhibit honeybee pathogenic P. larvae or L. sakei subsp. sakei JCM 1157 used as the indicator bacteria. The bacteriocin-producing exogenous bacterial strains - E. durans A5- 11, E. faecalis KT2W2G and L. lactis subsp. lactis KT2W2L - inhibited P. larvae strains (Janashia et al., 2016). The bacteriocin-producing strains were absolutely inactive against Lactobacillus spp. group with different patterns were observed for other bacterial groups tested (Sabate et al., 2009).
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Table 3 Antimicrobial activity of microorganisms isolated from gut of honeybees EVANS, J.D., ARMSTRONG, T.M. 2005. Inhibition of the American foulbrood Paenibacillus larvae bacterium, Paenibacillus larvae larvae, by bacteria isolated from honeybees. Species Paenibacillus larvae CCM 4438 Journal of Apicultural Research, 44(4), 168-171. mean±SD mean±SD https://doi.org/10.1080/00218839.2005.11101173 EVANS, J.D., SCHWARZ, R.S. 2011. Bees brought to their knees: microbes Bacillus cereus 14.67±0.58 15.33±0.58 affecting honeybee health, Trends in Microbiology, 19(12), 614-620, Bacillus megatherium 13.67±1.53 12.67±1.15 http://dx.doi.org/10.1016/j.tim.2011.09.003 Bacillus oleronius 13.67±1.53 13.33±0.58 FORSGREN, E., OLOFSSON, T.C., V́ ASQUEZ, A., FRIES, I. 2010. Novel Bacillus thuringiensis 13.67±1.53 13.67±0.58 lactic acid bacteria inhibiting Paenibacillus larvae in honeybee larvae. Apidologie, 41(1), 99-108. https://doi.org/10.1051/apido/2009065 Delftia acidovorans 16.67±1.53 15.33±0.58 GILLIAM, M. 1997. Identification and roles of non-pathogenic microflora Escherichia coli 16.33±1.53 14.67±2.52 associated with honeybees. FEMS Microbiology Letters, 155(1), 1-10. Enterococcus cloacae 15.33±2.52 14.33±1.15 https://doi.org/10.1016/s0378-1097(97)00337-6 Issatchenkia orientalis 15.67±2.08 13.67±1.53 HANSEN, D.S., AUCKEN, H.M., ABIOLA, T., PODSCHUM, R., 2004. Klebsiella oxytoca 13.67±1.53 12.67±1.15 Recommended test panel for differentiation of Klebseila species on the basis of a trilateral interlaboratory evaluation of 18 biochemical tests. Journal of Clinical Lactobacillus acidophilus 20.33±0.58 19.67±1.53 Microbiology, 42(8),3665-3669. https://doi.org/10.1128/jcm.42.8.3665- Lactobacillus crispatus 20.67±1.15 19.33±1.15 3669.2004 Lactobacillus kunkei 24.33±0.58 23.67±1.53 JANASHIA, I., CHOISET, Y., RABESONA, H., HWANHLEM, N., Morganella morgani 11.33±0.58 11.67±0.58 BAKURADZE, N., CHANISHVILI, N., HAERTLÉ, T. 2016. Protection of honeybee Apis mellifera by its endogenous and exogenous lactic flora against Pantotea aglomerans 9.67±0.58 9.33±1.53 bacterial infections, Annals of Agrarian Science, 14(3), 177-181, Pantotea ananatis 7.67±0.58 6.67±0.58 http://dx.doi.org/10.1016/j.aasci.2016.07.002 Pseudomonas oryzihabitans 11.33±1.15 10.67±0.58 JEYAPRAKASH, A., HOY, M.A., ALLSOPP, M.H. 2003. Bacterial diversity in Rahnella aquatilis 8.67±0.58 6.67±1.53 worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences. Journal of Invertebrate Rahnella terrigena 8.33±1.53 7.33±1.15 Pathology, 84(2), 96-103. https://doi.org/10.1016/j.jip.2003.08.007 Raoutella ornithinolytica 6.33±1.53 7.67±0.58 KAČÁNIOVÁ, M., CHLEBO, R., KOPERNICKÝ, M., TRAKOVICKÁ, A. Serratia liquefaciens 7.67±0.58 8.33±0.58 2004. Microflora of the Honeybee Gastrointestinal Tract. Folia Microbiologica, Serratia marcescens 8.33±0.58 7.33±0.58 49(2), 169-171. https://doi.org/10.1007/bf02931394 Sphingomonas KAČÁNIOVÁ, M., GASPER, J., TERENTJEVA, M., KUNOVÁ, S., KLUZ, M., 11.33±0.58 10.67±0.58 parapaucimobilis PUCHALSKI, C. 2018. Antibacterial Activity of Bees Gut Lactobacilli against Staphylococcus epidermidis 12.67±1.15 13.67±1.53 Paenibacillus Larvae In Vitro. Advanced research in life sciences, 2(1), 2018, 7- 10. https://doi.org/10.1515/arls-2018-0020 KAČÁNIOVÁ, M., KLŪGA, A., KÁNTOR, A., MEDO, J., ŽIAROVSKÁ, J., CONCLUSION PUCHALSKI, C., TERENTJEVA, M. 2019. Comparison of MALDI-TOF MS Biotyper and 16S rDNA sequencing for identification of Pseudomonas species Bacillus cereus, B. megatherium, B. oleronius, B. thuringiensis, Delftia isolated from fish. Microbial Pathogenesis, 132, 313-318. acidovorans, Escherichia coli, Enterococcus cloacae, Issatchenkia orientalis, https://doi.org/10.1016/j.micpath.2019.04.024 Klebsiella oxytoca, Lactobacillus acidophilus, L. crispatus, L. kunkei, KLEIN, A.M., VAISSIERE, B.E., CANE, J.H., STEFFAN-DEWENTER, I., Morganella morgani, Pantotea aglomerans, P. ananatis, Pseudomonas CUNNINGHAM, S.A., KREMEN, C., TSCHARNTKE, T. 2007. Importance of oryzihabitans, Rahnella aquatilis, R. terrigena, Raoutella ornithinolytica, pollinators in changing landscapes for world crops. Proceeding of Royal Society, Serratia liquefaciens, S. marcescens, Sphingomonas parapaucimobilis, 274, 303-313. Staphylococcus epidermidis strains associated with adult worker bees were MOHR, K.I., TEBBE, C.C. 2006. Diversity and phylotype consistency of isolated and characterized to extend the knowledge on microorganisms inhibiting bacteria in the guts of three bee species (Apoidea) at an oilseed rape field. the bees gut. The best antimicrobial activity against both P. larvae isolated from Environmental Microbiology, 8(2), 258-272. https://doi.org/10.1111/j.1462- bees’ gut were shown by L. kunkei, L. crispatus and L. acidophilus. 2920.2005.00893.x POTTS, S.G., BIESMEIJER, J.C., KREMEN, C., NEUMANN, P., Acknowledgement: This study was written during realization of the project SCHWEIGER, O., KUNIN, W.E., 2010. Global pollinator declines: Trends, REVITAL No.26210120038 supported by the Operational Programme Research impacts and drivers. Trends in Ecology and Evolution, 25(6), 345-353. and Development funded from the European Regional Development Fund and https://doi.org/10.1016/j.tree.2010.01.007 European Community project no. 26220220180: Building the Research Centre RADA, V., MÁCHOVÁ, M., HUK, J., MAROUNEK, M., DUKOVÁ, D. 1997. „AgroBioTech“. Microflora in the honeybee digestive tract counts, characteristics. Apidologie. 28(6), 357-365. https://doi.org/10.1051/apido:19970603 REFERENCES SABATÉ, D.C., CARRILLO, L. AUDISIO, M.C. 2009. Inhibition of Paenibacillus larvae and Ascosphaera apis by Bacillus subtilis isolated from ALIPPI, A.M., REYNALDI, F.J 2006. Inhibition of the growth of Paenibacillus honeybee gut and honey samples. Research in Microbiology, 160(3), 193-199. larvae the causal agent of American Foulbrood of honeybees by selected strains https://doi.org/10.1016/j.resmic.2009.03.002 of aerobic spore forming bacteria isolated from apiarian sources. Journal of VÁSQUEZ, A., FORSGREN, E., FRIES, I., PAXTON, R.J., FLABERG, E., Invertebrate Pathology, 91(3), 141-146. https://doi.org/10.1016/j.jip.2005.12.002 SZEKELY, L., OLOFSSON, T.C. 2012. Symbionts as major modulators of AUDISIO, M.C., TORRES, M.J., SABATÉ, D.C., IBARGUREN, C. APELLA, insect health: lactic acid bacteria and honeybees, PLoS One, 7(3), e33188, M.C. 2011. Properties of different lactic acid bacteria isolated from Apis http://dx.doi.org/10.1371/journal.pone.0033188 mellifera L. bee-gut. Microbiological Research, 166(1), 1-13, VOJVODIC, S., REHAN, S.M., ANDERSON, K.E. 2013. Microbial Gut https://doi.org/10.1016/j.micres.2010.01.003 Diversity of Africanized and European Honeybee Larval Instars. PLoS One, 8(8): CHAHBAR, N., MAHAMED, A.L. 2014. Contribution to identification of the e72106. https://doi.org/10.1371/journal.pone.0072106 microfloraof the digestive tractand pollenof Algerian honeybees: Apis mellifera WILLIAMS, D.L. 2000. A veterinary approach to the European honeybee (Apis intermissa and Apis mellifera sahariensis. International Journal of Current mellifera). The Veterinary Journal, 160(1), 61-73. Microbiology and Applied Science, 3(6) 601-607. https://doi.org/10.1053/tvjl.2000.0474 EBRAHIMI, A., LOTFALIAN, S., 2005. Isolation and antibiotic resistance patterns of Escherichia coli and coagulase positive Staphylococcus aureus from bee’s digestive tract. Iranian Journal of Veterinary Research, 6(2), 1-3. ENGEL, P., MORAN, N.A. 2013a. The gut microbiota of insects – diversity in structure and function. FEMS Microbiology Reviews, 37(5), 699-735. https://doi.org/10.1111/1574-6976.12025 ENGEL, P., MORAN, N.A. 2013b. Functional and evolutionary insights into the simple yet specific gut microbiota of the honeybee from metagenomic analysis. Gut Microbes, 4(1), 60-65. https://doi.org/10.4161/gmic.22517 EVANS, J.D., LOPEZ, D.L. 2004. 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MICROBIOTA OF THE TRADITIONAL SLOVAK SHEEP CHEESE “BRYNDZA”
Miroslava Kačániová1,2*, Simona Kunová3, Jana Štefániková4, Soňa Felšöciová5, Lucia Godočíková5, Elena Horská6, Ľudmila Nagyová6, Peter Haščík7, Margarita Terentjeva8
Address(es): 1Slovak University of Agriculture in Nitra, Faculty of Horticulture and Landscape Engineering, Department of Fruit Sciences, Viticulture and Enology, Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia, phone number: +421 37 641 4715. 2University of Rzeszow, Faculty of Biology and Agriculture, Department of Bioenergy and Food Technology, Zelwerowicza St. 4, PL-35601 Rzeszow, Poland. 3Slovak University of Agriculture, Faculty of Biotechnology and Food Sciences, Department of Food Safety and Hygiene, Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia, phone number: +421 37 641 5807. 4Slovak University of Agriculture in Nitra, AgroBioTech - Research Center, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia. 5Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Microbiology, Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia, phone number: +421 37 641 5812. 6Slovak University of Agriculture, Faculty of Economics and Management, Department of Marketing and Trade, Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia, phone number: +421 37 641 4102. 7Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Technology and Quality of Animal Products, Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia, phone number: +421 37 641 4708. 8Latvia University of Life Sciences and Technologies, Institute of Food and Environmental Hygiene, Faculty of Veterinary Medicine, Latvia K. Helmaņa iela 8, LV- 3004, Jelgava, Latvia.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2019.9.special.482-486
ARTICLE INFO ABSTRACT
Received 30. 6. 2019 The aim of the present study was to describe microbiota of the traditional Slovak cheese „Bryndza“. A total of 30 cheese samples were Revised 17. 10. 2019 collected from seven different farms during in 2019. The microbiota studies included the total bacterial count, coliforms, enterococci, Accepted 22. 10. 2019 lactic acid bacteria, and yeasts. The total bacterial counts were cultivated on plate count agar at 30°C in aerobic conditions, lactic acid Published 8. 11. 2019 bacteria on MRS, APT and MSE at 37°C in anaerobic conditions, coliform on VRBL at 37 °C in aerobic condition and yeasts on MEA at 25°C in aerobic condition. Gram-positive, Gram-negative and yeasts isolates were identified by MALDI-TOF MS profiling. Totally, a number of 870 isolates were identified with score higher than 2. Hafnia alvei and Klebsiella oxytoca were the most frequently identified Regular article species of Gram-negative and Lactococcus lactis and Lactobacillus paracasei from Gram-positive bacteria. Dipodascus candidum and Yarrowia lipolitica were the most distributed yeasts. Lactic acid bacteria group was represented by Lactobacillus, Lactococcus and Pediococcus. The most abundant genera of lactic acid bacteria were Lactobacillus with 7 species. This study describs the indigenous microbiota of the traditional raw milk cheeses from Slovakia. Our results provide useful information on occurrence of valuable microbial strains for the industrialization of producing of the traditional cheese products.
Keywords: Gram-positive and Gram-negative bacteria, yeasts, „Bryndza“, MALDI-TOF MS Biotyper
INTRODUCTION The risks and benefits of traditional cheeses, which are frequently produced from raw milk, could be detected objectively by studying the microbiota of cheese Raw milk was found to be contaminated with non-pathogenic and pathogenic inhabiting the product (Bhowmik and Marth, 1990). The microbial diversity microorganisms. Pathogens possibly present in raw milk may be originated from and the benefits related to consumption of raw milk cheese depends on both the sick or apparently health animals or as a contamination from the environment or milk microbiota and traditional manufacturing practices, including a quality of personnel during the collection or storage of milk. Contamination from animals inoculation practices. Traditional processing from farming to cheese making can appears directly, eg. an endogenous infection then the milk is contaminated helps to maintain the diversity of microbiota of individual cheeses and the directly from the blood stream (systemic infection) or from udder in case of between lots of cheeses throughout processing (Litopoulou-Tzanetaki et al., mastitis. Milk cross-contamination could be a result from contamination of 1989). More than 400 lactic acid bacteria species, Gram and catalase-positive faeces, the skin or the environment (Claeys et al., 2013). D'Amico and Donnelly bacteria, Gram-negative bacteria, yeasts and moulds have been detected in raw (2010) did not find significant difference between the total microbial counts in milk. The cheese surface is inhabited by numerous species of bacteria, yeasts and raw milk from goats, sheep and cows. The total microbial counts goat and sheep moulds, but the cheese cores reveals the smaller degree of biodiversity with a milk were variable depending on milking, the number of milking sessions making number of lactic acid bacteria species are numerically dominant (Montel et al., up the milk mix, the type of milking system and herd size (Alexopoulos et al., 2014). Raw milk can contain pathogenic bacteria that have been raising public 2011). health concern and many of raw milk related outbreaks were describe since the Consumer health has become a priority concern for food production. Sheep milk beginning of dairy industry. The most common pathogenic bacteria found in raw is expected to be an excellent source of nutrients (Balthazar et al., 2017). Sheep milk and milk products were Salmonella, Listeria monocytogenes, milk is rarely consumed as itself: mostly it is used for production of cheese and Staphylococcus aureus and Escherichia coli (Markov et al., 2011). yogurt (Haenlein and Wendorff, 2006). Consequently, the milk quality has Matrix-assisted laser desorption/ionization time of flight mass spectrometry direct impact to the production of high‐quality products and high cheese yield per (MALDI-TOF MS) is a tool in microbiological diagnostics which allow the liter of milk used in the cheese manufacture. Functional volume of milk used in identification of identification of microorganisms in various matrixes. the traditional manufacturing process depends on the type of cheese (Santillo Conventional identification methods rely on biochemical reactions and require and Albenzio, 2015). additional pre-testing and incubation. In comparison, MALDI-TOF MS can
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directly identify bacteria and yeast from their colonies in very short period of Isolation of coliform bacteria time. This new and methodically simple approach reduces the cost of materials and duration of diagnostics (Wieser et al., 2012). Identification of Violet red bile lactose agar (VRBGA, Sigma-Aldrich®, St. Louis, USA) for microorganisms by MALDI-TOF MS is done by comparing the peptide mass enumeration of coliforms bacteria was used. Inoculated plates were incubated at fingerprint (PMF) of tested organism with previously described PMFs from the 37°C for 24-48 h and then examined for the characteristics of typical colonies. database, or by matching the masses of biomarkers of tested microorganisms with the proteome database. In PMF matching, the MS spectrum of unknown Isolation of enterococci microbial isolates is compared with the MS spectra of known microbial isolates in the database (Murray, 2012). Enterococcus selective agar (ESA, Sigma-Aldrich®, St. Louis, USA) for The aim of our study was to determine microbiota of traditional sheep cheese enumeration of enterococci was used. Inoculated plates were incubated at 37°C with mass spectrometry MALDI-TOF MS Biotyper. for 24-48 h and then examined for the characteristics of typical colonies.
MATERIAL AND METHODS Isolation of Lactic Acid Bacteria (LAB)
Samples MRS (Main Rogose agar, Oxoid, UK), MSE (Mayeux, Sandine and Elliker in 1962, Oxoid, UK), and APT (All Purpose TWEEN® agar, Oxoid, UK) agars were There were 30 samples of the Slovak national cheese „Bryndza“ examined in our used for enumeration of LAB including lactobacilli, leuconostocs and lactic acid study. Additionally, a total of 30 sheep milk cheese samples from the Slovak streptococci as well as other microorganisms with high requirements for thiamine producers located in Slovakia were collected (Bukovina, Turčianské Teplice, (Sigma-Aldrich®, St. Louis, USA). Inoculated agars were incubated at 30°C for Važec, Zvolenská Slatina). All samples were placed in sterile sample containers 72 h anaerobically and then the bacterial growth was evaluated. and transported to laboratory on ice for microbiological investigations. Samples were kept in a refrigerator (4±1°C) until the testing began. The primary dilution Isolation of yeasts of the milk products was made for preparing the samples for testing: a 5 ml of sample material was added to 45 ml of 0.87 % sterile saline. Then the serial Malt extract agar (Sigma-Aldrich®, St. Louis, USA) and acid base indicator dilutions (10−2 to 10−4) were done and a 100 µl of each dilution was plated out. bromocresol green (Sigma-Aldrich®, St. Louis, USA) (0.020 g.L-1) were used for yeasts identification. Inoculated plates were incubated at 25°C for 5 days aerobically and then the growth was evaluated. Sample preparation and MALDI-TOF MS measurement
Prior to identification, the bacterial colonies were subcultured on TSA agar (Tryptone Soya Agar, Oxoid, UK) at 37°C for 18-24 h. One colony of eight bacterial isolate was selected. Subsequently, the identification was performed using the Maldi TOF MS Biotyper as was described by Kačániová et al. (2019). Totally, a number of 870 isolates were identified with a score higher than 2.
RESULTS AND DISCUSSION
Traditional bryndza is sharp, salty, greyish, grated and pin-rolled, crumbly, semi- spreadable 100% sheep cheese. There is no close equivalent in taste and texture among sheep, cow or goat cheeses. Unique food and drinks make up a significant part of Slovak culture, as the country produces several products which cannot be found or replicated in any other part of the world; bryndza cheese is one of those products (EC, 2008). The numbers of microorganisms in sheep cheese in our study is shown in table 1. Total count of bacteria in bryndza ranged from 3.83 to -1 -1 3.78 log cfu.g . Enterococci were from 2.97 to 3.24 log cfu.g in the studied Figure 1 Map of Slovakia (www.google.com) samples. Coliform bacteria counts ranged from 3.07 to 3.85 log cfu.g-1, lactic acid bacteria counts ranged from 3.05 to 3.13 log cfu.g-1. The counts of yeasts ranged Determination of total count of microorganisms from 2.19 to 2.54 log cfu.g-1.
Plate count agar (PCA, Sigma-Aldrich®, St. Louis, USA) for total microbial count enumeration was used. Inoculated plates were incubated at 30°C for 24-48 h and then examined for the characteristics of bacterial colonies.
Table 1 The number of isolated group of microorganisms from sheep cheese „Bryndza“ in cfu.g-1 Sample Coliforms Enterococcus Total Bacterial Lactic Acid Yeasts Counts Bacteria 1. 3.63±0.05 3.05±0.03 3.68±0.18 3.05±0.04 2.37±0.13 2. 3.42±0.03 2.97±0.02 3.78±0.12 3.07±0.07 2.37±0.14 3. 3.85±0.06 3.01±0.02 3.83±0.09 3.09±0.07 2.30±0.20 4. 3.07±0.02 3.08±0.06 3.73±0.13 3.13±0.02 2.54±0.62 5. 3.72±0.02 3.04±0.07 3.70±0.09 3.11±0.05 2.19±0.07 6. 3.54±0.02 3.07±0.04 3.72±0.12 3.10±0.04 2.63±0.06 7. 3.81±0.03 3.13±0.02 3.78±0.05 3.11±0.02 2.19±0.07 8. 3.71±0.11 3.24±0.22 3.67±0.13 3.09±0.03 2.19±0.10 9. 3.60±0.04 3.13±0.02 3.62±0.11 3.11±0.01 2.20±0.09 10. 3.52±0.12 3.11±0.05 3.72±0.12 3.13±0.02 2.30±0.09
A total of 40 species of 10 microbial families and 20 genera (14 Gram-negative (G-), 17 Gram-positive (G+) and 9 yeasts species) were identified in sheep cheese by MALDI-TOF Mass Spectrometry. The G-, G + and yeasts comprised 25.86% (225 isolates), 49.43% (430 isolates) and 24.71% (215 isolates), respectively. Isolated species of bacteria from cheese „Bryndza” are shown in table 2.
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Table 2 Isolated species of microorganisms from sheep cheese „Bryndza” Altogether, nine yeast species were isolated from sheep cheese. Candida spp. Family Genera Species were the most abundant yeast genus, while Dipodascus candidum was the most frequently isolated yeast species (table 5). Moraxellaceae Acinetobacter Acinetobacter baumannii
Moraxellaceae Acinetobacter Acinetobacter tandoii Table 3 Isolated species of Gram-negative bacteria from sheep cheese „Bryndza” Bacillaceae Bacillus Bacillus pumilus No. of isolates Microorganism No. of isolates Saccharomycetaceae Candida Candida catenulata in % Saccharomycetaceae Candida Candida krusei Acinetobacter baumannii 15 6.7 Saccharomycetaceae Candida Candida lusitaniae Acinetobacter tandoii 6 2.7 Saccharomycetaceae Candida Candida rugosa Citrobacter braakii 8 3.6 Saccharomycetaceae Candida Candida utilis Citrobacter koseri 7 3.1 Enterobacteriaceae Citrobacter Citrobacter braakii Enterobacter cloacae 21 9.3 Enterobacteriaceae Citrobacter Citrobacter koseri Enterobacter ludwigii 15 6.7 Dipodascaceae Dipodascus Dipodascus candidum Escherichia coli 15 6.7 Dipodascaceae Dipodascus Dipodascus silvicola Hafnia alvei 45 20.0 Enterobacteriaceae Enterobacter Enterobacter cloacae Klebsiella oxytoca 25 11.1 Enterobacteriaceae Enterobacter Enterobacter ludwigii Klebsiella pneumoniae ssp. ozaenae 10 4.4 Klebsiella pneumoniae ssp. Enterococcaceae Enterococcus Enterococcus faecalis 15 6.7 pneumoniae Enterococcaceae Enterococcus Enterococcus faecium Raoultella ornithinolytica 20 8.9 Enterococcaceae Enterococcus Enterococcus hirae Serratia liquefaciens 15 6.7 Enterobacteriaceae Escherichia Escherichia coli Stenotrophomonas maltophilia 8 3.6 Enterobacteriaceae Hafnia Hafnia alvei
Enterobacteriaceae Klebsiella Klebsiella oxytoca Table 4 Isolated species of Gram positive microorganisms from sheep cheese Klebsiella pneumoniae ssp. Enterobacteriaceae Klebsiella „Bryndza” ozaenae No. of isolates Klebsiella pneumoniae ssp. Microorganism No. of isolates Enterobacteriaceae Klebsiella in % pneumoniae Bacillus pumilus 22 5.1 Lactobacillaceae Lactobacillus Lactobacillus brevis Enterococcus faecalis 25 5.8 Lactobacillaceae Lactobacillus Lactobacillus harbinensis Enterococcus faecium 20 4.7 Lactobacillaceae Lactobacillus Lactobacillus johnsonii Enterococcus hirae 15 3.5 Lactobacillaceae Lactobacillus Lactobacillus plantarum Lactobacillus paracasei ssp. Lactobacillus brevis 34 7.9 Lactobacillaceae Lactobacillus paracasei Lactobacillus harbinensis 35 8.1 Lactobacillaceae Lactobacillus Lactobacillus paraplantarum Lactobacillus johnsonii 30 7.0 Lactobacillaceae Lactobacillus Lactobacillus suebicus Lactobacillus plantarum 25 5.8 Lactobacillus paracasei ssp. Streptococcaceae Lactococcus Lactococcus lactis ssp lactis 15 3.5 Streptococcaceae Lactococcus Lactococcus lactis paracasei Lactobacillus paraplantarum 35 8.1 Microbacteriaceae Microbacterium Microbacterium liquefaciens Lactobacillus suebicus 15 3.5 Lactobacillaceae Pediococcus Pediococcus acidilactici Lactococcus lactis ssp. lactis 50 11.6 Saccharomycetaceae Pichia Pichia cactophila Lactococcus lactis 55 12.8 Enterobacteriaceae Raoultella Raoultella ornithinolytica Microbacterium liquefaciens 9 2.1 Enterobacteriaceae Serratia Serratia liquefaciens Staphylococcus aureus ssp. Pediococcus acidilactici 20 4.7 Staphylococcaceae Staphylococcus aureus Staphylococcus aureus ssp. aureus 10 2.3 Staphylococcaceae Staphylococcus Staphylococcus pasteuri Staphylococcus pasteuri 15 3.5 Stenotrophomonas Xanthomonadaceae Stenotrophomonas maltophilia Table 5 Isolated yeasts from sheep cheese „Bryndza” Dipodascaceae Yarrowia Yarrowia lipolytica Yeast species No. of isolates No. of isolates in % Altogether, 14 species of Gram-negative bacteria were isolated. Klebsiella spp. Candida catenulata 15 6.98 were represented by three species and were the most widespread bacterial genus. Candida krusei 25 11.63 The most distributed bacterial species were Hafnia alvei, Klebsiella oxytoca and Candida lusitaniae 25 11.63 Enterobacter cloacae (table 3). Candida rugosa 15 6.98 Altogether, 17 bacterial species of Gram-positive bacteria were isolated from Candida utilis 10 4.65 sheep cheese. Lactobacillus spp. were the most widespread genus represented Dipodascus candidum 50 23.26 with 7 species. Lactococcus lactis was the most frequently isolated bacterial Dipodascus silvicola 25 11.63 species (table 4). Pichia cactophila 5 2.32 Previous culture-independent studies showed the diversity of bacteria and fungi Yarrowia lipolytica 45 20.93 and the changes is their population during the production of bryndza (Chebeňová-Turcovská et al., 2011; Pangallo et al., 2014). Interactions Galactomyces/Geotrichum, and yeasts Yarrowia lipolytica, Kluyveromyces lactis between the lactic acid bacteria and Galactomyces/Geotrichum group and and Debaryomyces hansenii were the main representative of eukaryotic coagulase-positive staphylococci were studied as well (Hudecová et al., 2011, microbiota in study of Šaková et al., 2015. Yarrowia lipolytica was identified as Medveďová and Valík, 2012). The culture-depended methods showed that the one of the most abundant species in the present study. bryndza samples contained lactococci, lactobacilli and Galactomyces/Geotrichum Composition and activity of microflora is believed to have a great impact on the in high numbers. Majority of lactobacilli were identified as Lactobacillus flavour of bryndza cheese. The compounds contained in ewes’ milk and from the paracasei and Lb. plantarum and lactococci as Lactococcus lactis with PCR- products of fermentation of the substrate by microflora were responsible for based identification methods. Culture-independent analysis revealed that typical sensory characteristics (Sádecká et al., 2014). In previous studies, Lactococcus spp. followed by Streptococcus spp. and Leuconostoc spp were the Lactobacillus spp. (Berta et al., 1990), Lactococcus spp., Streptococcus spp., most abundant bacterial genera (Šaková et al., 2015). Enterococcus spp., Kluyveromyces marxianus and Galactomyces geotrichum were identified as the main microorganism of bryndza cheese (Görner, 1980;
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Palo and Kaláb, 1984; Görner and Valík, 2004). Enterococci (Jurkovič et al., probiotic cultures or producers of bacteriocins, and pathogenic coliforms 2006b), staphylococci (Mikulášová et al., 2014) and fungal species (Laurenčík responsible for safety problems (Lauková and Czikková, 2001; Lauková et al., et al., 2008) were identified in bryndza cheese as well. 2003; Jurkovič et al., 2006a; Jurkovič et al., 2006b). Lactobacillus spp., Lactococcus spp., Streptococcus spp., Enterococcus spp., Lactobacillus species identified in bryndza cheese in this study, i. e. Lb. brevis, Kluyveromyces marxianus and Geotrichum candidum are believed to influence are well known microorganism in cheeses and may contribute to flavour the characteristic organoleptic properties of the Slovakian bryndza cheese (Valík, development by peptidolytic activities and aminoacid catabolism (Olson, 1990; 2004; EC, 2007). There are only limited studies on microbiota of Slovakian Poveda et al., 2002; Wouters et al., 2002; Kieronczyk et al., 2003). bryndza cheese with mostly classical microbiological methods were applied. Modern methods have been used for characterization of enterococci, potential
Figure 2 Microbiota of sheep cheese “Bryndza” of Slovak origin
CONCLUSION Acknowledgments: Work was supported by the grants APVV-16-0244 "Qualitative factors affecting the production and consumption of milk and Microbiologically, the 40 species of 20 bacterial genera of three main groups of cheese". microorganisms were identified with MALDI-TOF Mass Spectrometry. The Gram-negative, Gram-positive bacteria and yeasts comprised 25.86% (225 REFERENCES isolates) 49.43% (430 isolates) and 24.71% (215 isolates), accordingly. Fast microbial identification is in high demand in industry for improving of HACCP- Alexopoulos, A., Tzatzimakis, G., Bezirtzoglou, E., Plessas, S., Stavropoulou, E., based procedures, reduce biocide consumption and to avoid the distribution of Sinapsis, E. & Abas, Z. (2011). Microbiological quality and related factors of contaminated products. The speed and precision of microbial identification with sheepmilk produced in farms of NE Greece. Anaerobe, 17(6), 276-279. MALDI-TOF-MS were well described for clinical isolates, but the present study https://doi.org/10.1016/j.anaerobe.2011.03.011 show that the methods could be applicable for dairy producing and industrial Balthazar, C.F., Pimentel, T.C., Ferrão, L.L., Almada, C.N., Santillo, A., applications. Albenzio, M., Mollakhalili, N., Mortazavian, A.M., Nascimento, J.S., Silva,
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DIVERSITY OF BACTERIA IN SLOVAK AND FOREIGN HONEY, WITH ASSESSMENT OF ITS PHYSICO- CHEMICAL QUALITY AND COUNTS OF CULTIVABLE MICROORGANISMS
Vladimíra Kňazovická1, Michal Gábor2, Martina Miluchová2, Marek Bobko3, Juraj Medo*4
Address(es): Ing. Juraj Medo, PhD. 1National Agricultural and Food Centre, Research Institute for Animal Production Nitra, Institute of Apiculture Liptovsky Hradok, Gasperikova 599, 033 80 Liptovsky Hradok, Slovakia. 2Slovak University of Agriculture in Nitra, Faculty of Agrobiology and Food Resources, Department of Genetics and Animal Breeding Biology, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia. 3Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Technology and Quality of Animal Products, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia. 4Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Microbiology, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia, phone number: +421 37 641 5810.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2019.9.special.414-421
ARTICLE INFO ABSTRACT
Received 23. 8. 2019 The aim of the study was to assess microbial microbiome of 30 honey samples and compare potential differences between the samples Revised 7. 10. 2019 from apiaries and commercial trade in Slovakia and foreign countries (Latvia, Switzerland, India, Japan and Tanzania) as well as to Accepted 10. 10. 2019 indicate their physico-chemical and basic microbiological quality. Study of each sample consisted of physico-chemical analysis (water Published 8. 11. 2019 content, pH, free acidity and electrical conductivity), basic microbiological analysis performed by dilution plating method (total plate count, sporulating aerobic microorganisms, bacteria from Enterobacteriaceae family, preliminary lactic acid bacteria and microscopic fungi) and metagenomic analysis for bacterial diversity evaluation. Seven samples did not meet with legislative limits of physico-chemical Regular article parameters. Average values of cultivable microbial groups ranged at level 1-2 log CFU.g-1, while bacteria from Enterobacteriaceae family were not detected in any samples. Sporulating microorganisms occurred most often, in 77% of samples and yeasts were proven in 60% of samples. Bacterial diversity, determined by metagenomic analysis, was varied. We distinguished 2 groups – group A and group B. Group A contained mainly fresh (produced in 2018) Slovak and Swiss honey and we found mainly genus Lactobacillus followed by genus Bombella in them. Group B contained mainly older Slovak honey (produced in 2017) and commercial foreign samples, in which production year is difficult to know. Group B samples were interesting because of human bacteria presence with genus Prevotella dominance. Redundancy analysis showed significant connection of the electric conductivity and microbial assemblage, that indicates important influence of botanical origin to microbial representation in honey.
Keywords: Bee product, Electrical conductivity, Lactic acid bacteria, Metagenomics, Prevotella sp.
INTRODUCTION markets in Portugal; they were centred on Bacillaceae spores and fungi; spores of Clostridium perfringens were not detected in any sample, Bacillus cereus were Honey is a natural product of bees traditionally used as food product and medicine identified in 13.7%; yeasts and microscopic filamentous fungi (MFF) were all over the world including Slovakia, Romania and Russia (Guziy et al., 2017; detected in 88.8% samples with identification of 3 MFF genera: Aspergillus, Šedík et al., 2019). Consumption of honey almost doubled in last decade in Penicillium and Mucor and 2 genera of yeasts: Saccharomyces and Candida. Slovakia. Comparing with other food, ripe high quality honey is considered to be Tolba et al. (2007) identified bacilli in 7 honey samples mainly from Northern a food with a minimal level of microbial contamination with many beneficial Ireland. They found Bacillus pumilus, B. licheniformis, B. subtilis, B. fusiformis effects in human nutrition and with no or limited risks for human health. Most of and Paenibacillus motobuensis. Amir et al. (2010) analysed occurrence of MFF in the potential microorganisms relevant for food safety are expected to be in inactive 19 honeys of blossom, blended and honeydew origin from Algeria. Microscopic forms as they cannot survive in honey because of its properties including filamentous fungi were found mainly in samples with low water content (16.2 and hyperosmolarity (Bovo et al., 2018). Essentially, honey is a supersaturated solution 17%) and included it that this fact was influenced by xerophilic properties of honey comprising approximately 80% sugars by weight, predominantly fructose and and MFF. Honey, especially in the fresh state, is interesting by presence of lactic glucose, with sucrose, maltose, and many other sugars at much lower acid bacteria (LAB) presence. For the first time Olofsson et Vásquez (2008) found concentrations (Cooper, 2014). Israili (2014) concluded antimicrobial activity of LAB in the honey stomach (crop) and in the fresh honey as well, and they honey as follows: A large number of in vitro and limited clinical studies have suggested that honey can be considered a fermented food product because of the confirmed the broad-spectrum antimicrobial (antibacterial, antifungal, antiviral LAB involved in honey production. In the last time, numerous studies about and antimycobacterial) properties of honey, which may be attributed to the acidity microorganisms in bees were published with main focus to bee intestinal (presence of acids, low pH), osmotic effect, high sugar concentration, presence of microbiome. Maes et al. (2016) performed bee-cage experiments with different bacteriostatic and bactericidal factors (hydrogen peroxide, antioxidants, lysozyme, diet and demonstrated that typically occurring alterations in diet quality play a polyphenols, phenolic acids, flavonoids, methylglyoxal and bee peptides) and significant role in colony health and establishment of a dysbiotic gut microbiome. increase in cytokine release and to immune modulating and anti-inflammatory According to Bonilla-Rosso et Engel (2018), simple sugars such as glucose and properties of honey. fructose present in nectar and pollen, and complex polysaccharides such as pectin In general, sporulating bacteria and yeast belong to common inhabitants of honey. from the pollen wall are apparent substrates for bacterial fermentation. Martins et al. (2003) tested 80 multifloral honey samples from retail public
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Properties as well as quantity and diversity of microorganisms of honey are The aim of the study was to analyse the honey samples in term of their basic influenced by many factors. Some of them can be partially or fully managed during physico-chemical properties, counts of cultivable microorganisms and the production process while others not. Snowdon et Cliver (1996) described metagenomic analysis to detect diversity of bacteria. primary sources of microorganisms in honey, including pollen, the digestive tract of honey bees, dust, air, dirt and flowers as well as secondary contamination, which MATERIAL AND METHODS can be taken place during and after honey extraction, including humans, equipment, containers, wind, dust, insects, animals or water. Blossom honey comes Material from nectar of plants and honeydew honey is originated from honeydew produced mainly by aphids. Gilliam et al. (1983) found that nectar is not a major source of Totally, we tested 30 honey samples: 24 were available in Slovakia (18 with Slovak microorganisms for honey bees, but nectars of some flowers, they tested, contained origin and 1 with origin in EU and non-EU countries) and 6 were obtained from Staphylococcus sp. as well as gram-positive and gram-negative rod-shaped foreign countries: from Latvia (mix EU and non-EU countries honey), Switzerland bacteria, actinomycetes and fungi. Bacteria are probably added during the process India, Japan and Tanzania. From botanical point of view, 24 samples were blossom by which nectar becomes honey and the nectar sugars probably act as inducers for (4 false acacia, 1 rape, 1 sunflower, 1 buckwheat and 17 multifloral), 5 samples the resident microbiota in honey stomach (crop), with enhancing their numbers were honeydew and 1 sample was blended. According to way of obtaining, 24 (Olofsson et Vásquez, 2008). Honeybees collect honeydew, which is product from samples were from apiaries (22 directly from beekeepers and 2 from farmers’ aphids inhabiting green parts of plants and at the same time, with honeydew, they market), and 6 samples were from commercial trade. Detailed characterization is may collect other attached structures, such as the hyphae or fungal spores of plant in the table 1. pathogens and microalgae (Escuredo et al., 2012). According to Snowdon et Cliver (1996), bacteria or yeasts are principally found Physico-chemical analysis in comb honey and sometimes also filamentous fungi are present, while information about presence and persistence of viruses and parasites are not The physico-chemical analysis consisted of water content, pH, free acidity and available. However some human enteric viruses, such as hepatitis A, sustain dry electrical conductivity. These parameters were measured according IHC (2009): conditions and could be expected to persist in honey. water content by refractometric method, potentiometric pH measurement by pH- Traditionally number of microorganisms particularly bacteria and microscopic meter, free acidity by titration to pH 8.3 and electrical conductivity (EC) by fungi were assessed by cultivation of them on artificially prepared media in conductometric method. Individual measurements were performed at least 2 times laboratory. However such conditions are not responded to requirement of many with average expression. microbial species. Microbiology has experienced a transformation during the last As the electrical conductivity is one of main descriptive criteria to differentiate decade that has altered microbiologists’ view of microorganisms and how to study blossom and honeydew honey where honeydew (and nectar only of some plants, them (Handelsman, 2004). At present, methods based on DNA analysing are more e. g. chestnut) is linked to higher conductivity we sorted samples to the three available for scientists. Metagenomic analysis, particularly 16S rRNA gene groups as follows (table 1): sequencing on high throughput sequencing platform Illumina became the most Low EC – honeys with EC to 0.29 mS.cm-1 (n = 13, EC: 0.11 - 0.29 common and accurate analyses. These techniques were previously used to asses mS.cm-1), gut microbiomes of bees. Bovo et al. (2018) analysed 2 honey samples (orange Middle EC - honeys with EC from 0.30 to 0.69 mS.cm-1 (n = 10, EC: tree blossom and eucalyptus honey from beekeepers in Sicily) by shotgun 0.32 - 0.69 mS.cm-1), metagenomics and surprisingly they noted that the largest number of reads Higher EC - honeys with EC from 0.70 mS.cm-1 (n = 7, EC: 0.72 - assigned to 5 organism group (Viruses, Bacteria, Plants, Fungi and Arthropods) 1.32 mS.cm-1). matched virus sequences in both honey samples (67.55 – 98.56%). Microbiology of honey is still full of secretes and it indicates, that honey is probably able to keep DNA or RNA of various organisms.
Table 1 Characterization of analysed samples Year of Sample code Botanical origin Geographical origin Obtaining Note production blossom (false acacia – commercial trade BloSCo1 Slovakia 2018 Robinia pseudoacacia) (Slovakia) blossom (rape – western Slovakia BloSBee1 directly from beekeeper 2017 creamed Brassica napus) (Male Krstenany) middle Slovakia BloSBee2 blossom directly from beekeeper 2018 (Dolny Pajer) blossom (with dominance of western Slovakia BloSBee3 directly from beekeeper 2018 1st extraction false acacia) (Dezerice) mix EU and non-EU commercial trade BloFCo1 blossom best before 2020 honey (Slovakia) western Slovakia BloSBee4 blossom (false acacia) directly from beekeeper 2018 1st extraction (Nitra-castle, Cerman) mix EU and non-EU commercial trade BloFCo2 blossom best before 2019 honey (Latvia) blossom (with dominance of eastern Slovakia BloSBee5 directly from beekeeper 2017 false acacia) (Sobrance) middle Slovakia BloSBee6 blossom directly from beekeeper 2018 1st extraction (Smrecany) middle Slovakia BloSBee7 blossom directly from beekeeper 2018 (Horna Orava) BloFCo3 blossom India (Himalaya) commercial trade (India) durability 2014 - 2017
BloSBee8 blossom middle Slovakia (Babin) directly from beekeeper 2018 middle Slovakia BloSBee9 blossom directly from beekeeper 2018 2nd extraction (Smrecany) blossom (sunflower – western Slovakia BloSBee10 directly from beekeeper 2018 Helianthus annus) (Dunajska Luzna) Japan (labelled as BloFCo4 blossom commercial trade (Japan) best before 2024 Mexican Orange honey) middle Slovakia BloHoSBee1 blended directly from beekeeper 2017 4th extracting (Smrecany) blossom (buckwheat – western Slovakia BloSBee11 directly from beekeeper 2018 Fagopyrum esculentum) (Risnovce)
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directly from beekeeper slightly BloFBee1 blossom Switzerland 2018 (farmers’ market) fermented eastern Slovakia directly from beekeeper BloSBee12 blossom (spring) 2018 (Nova Lubovna) (Switzerland) suspected of HoSBee1 honeydew eastern Slovakia (Kosice) directly from beekeeper 2017 adulteration western Slovakia directly from beekeeper BloSBee13 blossom 2018 2nd extraction (Dezerice) (farmers’ market) blossom (raspberry – Rubus BloSBee14 eastern Slovakia (Orlov) directly from beekeeper 2018 idaeus, linden – Tilia sp.) western Slovakia BloSBee15 blossom directly from beekeeper 2018 3rd extraction (Nitra-Zobor) BloSBee16 blossom (dandelion – Taraxacum eastern Slovakia (Orlov) directly from beekeeper 2018 officinale, willow – Salix sp.) slightly BloFBee2 blossom Switzerland directly from beekeeper 2018 fermented directly from beekeeper HoSBee2 honeydew eastern Slovakia (Orlov) 2018 (Switzerland) HoSBee3 honeydew middle Slovakia (Sutovo) directly from beekeeper 2017 eastern Slovakia HoSBee4 honeydew directly from beekeeper 2018 (Nova Lubovna) HoSBee5 honeydew western Slovakia (Nitra) directly from beekeeper 2018 5th extraction blossom BloFCo5 Tanzania (Kasalu) directly from beekeeper durability 2016 - 2019 (October blossom) sample code (according to botanical, geographical origin and obtaining): first part: Blo – blossom, Ho – honeydew, BloHo – blended (blossom-honeydew); second part: S – Slovakian, F – foreign; third part: Bee – from beekeeper, Co - from commercial trade; EU – European union, western Slovakia - Bratislava Region, Nitra Region, Trnava Region, Trencin Region; middle Slovakia – Banska Bystrica Region, Zilina Region, eastern Slovakia – Kosice Region, Presov Region
Microbiological analysis – dilution plating method (lactic acid bacteria) and MF (microscopic fungi, i. e. yeasts and MFF – microscopic filamentous fungi). The basic dilution (10-1) was performed by We used dilution plating method to quantify the microorganisms in honey. The homogenizing 5 g honey and 45 ml saline solution (0.85% NaCl, 0.10% peptone). target microbial groups were: TPC (total plate count), SAM (sporulating aerobic Specific conditions of microbiological analysis are listed in the table 2. microorganisms), bacteria from Enterobacteriaceae family, preliminary LAB
Table 2 Quantitative microbial examination of honey Cultivation Microbial group Medium Inoculation temperature time O2 requirement TPC GTY pouring 30 °C 2-3 days aerobic SAM NA 2 pouring* 25 °C 3 days aerobic Enterobacteriaceae VRBG pouring 30 °C 1-2 days aerobic preliminary LAB MRS pouring** 37 °C 3 days aerobic** MF DG 18 pouring 25 °C 5-7 days aerobic TPC – total plate count, cultivated on GTY – agar with glucose, tryptone and yeast extract (HiMedia®, India); SAM – sporulating aerobic microorganisms, inoculated by *pouring – after heat shock (at 80 °C for 10 min), cultivated on NA 2 – nutrient agar no. 2 (HiMedia®, India); Enterobacteriaceae family, cultivated on VRBG – violet red bile glucose agar (HiMedia®, India); LAB – lactic acid bacteria, inoculated by **double-pouring (decrease of oxygen in medium), cultivated on MRS – de Man, Rogosa and Sharpe agar (HiMedia®, India); MF – microscopic fungi, cultivated on DG 18 – Dichloran Glycerol agar - with chloramphenicol (HiMedia®, India)
DNA extraction were purified using a PCR purification kit (Jena Bioscience), quantified by qubit (Invitrogen), diluted to the same concentration and pooled together. Illumina We weighted 20 g of honey to sterile 50 ml tube and added sterile distilled water sequencing library was prepared by TruSeq LT PCR free kit (Illumina) with a to total volume of 45 ml. The solution was heated at 75 °C and rotated in modification involving omission of the DNA fragmentation and size selection. hybridization chamber to solve the honey. Then, samples were centrifuged at 8000 NebNext Quantification kit (New England Biolabs) was used for the library rpm for 10 min. Supernatant was removed and pellet was resuspended in 1 x PBS quantification then the library was diluted to 4 nM concentration, and denatured. solution (pH 7.4), which was added to total volume 30 ml. Then, samples were The sequencing reaction was performed on Illumina MiSeq using the MiSeq centrifuged at 8000 rpm for 5 min. Most of supernatant was removed. We left only Reagent Kit v3 (600-cycle). approximately 2 ml and transferred it to 2 ml tubes. These tubes were again Acquired sequencing data was processed in SEED environment (Větrovský et al., centrifuged at 13000 rpm for 5 min and supernatant was removed. Pellets were 2018). Forward and reverse reads were joined with minimum 100 base overlap. stored at -80 °C to next analysis. To pellet we added glass beads and 250 μl Only sequences with quality higher than Q30 were used in further analysis. PrepMan™ Ultra Sample Preparation Reagent (ThermoFisher Scientific) and then Sequences were assigned to samples according used barcodes and then barcoded homogenized by bead homogenizer BeadBug™ 3 (Benchmark Scientific) at primers were removed. Sequences were checked for chimeras and clustered to highest speed for 1 min. We heated the samples at 110 °C for 5 min. After heating, operational taxonomic unit (OTUs) using Vsearch (Rognes et al., 2016) at a the tubes were centrifuged and lysates were transferred to new tube and used for similarity level of 97%. From each cluster (OTU) the most abundant sequence was PCR reaction. found and identified using RDP classifier (Wang et al., 2007). Chloroplast sequences originated from pollen were removed. Metagenomic analysis Statistical analysis Barcoded primers 515F and 806R (Caporaso et al., 2011) which amplify V4 section of the 16S gene were used for PCR reaction. The composition of the PCR Data from microbiological analysis were calculated as log CFU.g-1. Significant mixture was as follows: 15 µl KAPA HIFI HotStart mix 2X (Kapa Biosystems), 8 difference was assessed if it was at least 1.00 log CFU.g-1. Data from cultivation µl of each primer with a concentration of 2.5 µM, and 1µl of isolated DNA. analysis as well as metagenomic data were descriptively processed in MS Excel Amplification was performed using SureCycler 8800 Thermal Cycler (Agilent) 2007. and thermal profile was following: Initial denaturation for 90 s at 98 °C followed Shannon and Chao1 diversity indices were calculated using ComEcolPaC (Drozd, by 35 cycles of denaturation for 15 s at 98 °C, annealing for 15 s at 62 °C and 2010). For analysis of microbial communities and their connection to physic- extension for 15 s at 72 °C. Final extension was 2 min at 72 °C. PCR products chemical parameters RDA was derived and significance were analysed by
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permutation test in package Vegan (Oksanen et al., 2013) in R statistical beekeepers and 18.6% in commercial samples. Water is a parameter, which is environment (Team R, 2013). OTUs with an only single member were removed important for evaluation of honey maturity and its low value relates directly with prior these analyses. long durability. According to Council Directive 2001/110/EC, honey has to Heatmaps were made using Heatmap3 (Zhao et al., 2014) in R. contain less than 20% of water. Only one sample (BloFBee1) exceeded this limit. The sample was gently fermented before analysis. According to Bogdanov et RESULTS AND DISCUSSION Martin (2002) the water content of honey can naturally be as low as 13.6% and as high as 23.0% depending on source of the honey, climatic conditions and other Physico-chemical and microbiological quality factors. Water in honey is related to honey preservation and storage, as high water content can lead to a growth of yeasts and microscopic filamentous fungi, causing In the table 3, there are results of basic physico-chemical parameters and microbial fermentation, flavour loss and low shelf life (Al-Farsi et al., 2018). Fermentation counts for each sample. Water content ranged from 15.0 to 20.7% with average does not usually a problem in honey with water content less than 18% (Bogdanov value 17.5 ± 1.3%. Similar results were published by Bušová et Kouřimská et Martin, 2002). (2018), who found average water content 18.3% in honey directly from Czech
Table 3 Physico-chemical parameters, microbial counts and diversity of microbial assemblage of tested honey samples Sample Physico-chemical quality Microbiological quality (log CFU.g-1) Diversity code Water FA EC Shannon’s pH TPC SAM E pLAB yeasts MFF (%) (meq.kg-1) (mS.cm-1) index BloSCo1 16.3 4.1 13.2 0.11 3.15 2.57 < 1.00 < 1.00 < 1.00 < 1.00 0.459 BloSBee1 16.1 3.8 19.1 0.13 1.85 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 6.659 BloSBee2 17.7 3.8 20.2 0.16 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 5.514 BloSBee3 18.5 3.8 18.2 0.17 2.28 2.30 < 1.00 < 1.00 1.00 < 1.00 5.198 BloFCo1 17.4 4.7 7.65 0.18 1.60 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 6.325 BloSBee4 17.6 3.8 18.8 0.20 < 1.00 1.60 < 1.00 < 1.00 1.30 1.00 4.662 BloFCo2 17.8 4.4 17.2 0.20 2.00 1.30 < 1.00 < 1.00 7.098 BloSBee5 16.9 4.1 28.5 0.22 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 7.077 BloSBee6 18.6 3.9 18.0 0.23 4.17 1.70 < 1.00 3.57 2.00 2.04 4.007 BloSBee7 19.9 3.5 48.0 0.26 1.00 1.00 < 1.00 < 1.00 1.48 1.00 4.760 BloFCo3 16.4 4.2 10.0 0.26 2.19 2.10 < 1.00 1.60 1.00 < 1.00 6.632 BloSBee8 18.6 3.3 31.3 0.28 1.48 < 1.00 < 1.00 < 1.00 2.00 1.00 3.719 BloSBee9 18.0 3.8 20.6 0.29 2.37 1.48 < 1.00 3.08 2.07 1.30 5.760 LOW EC n = 13 n = 13 n = 13 n = 13 n = 10 n = 8 n = 3 n = 7 n = 5 n = 13 ND mean ± SD 17.7 ± 1.1 3.9 ± 0.4 20.8 ± 10.4 0.21 ± 0.06 2.21 ± 0.90 1.76 ± 0.53 2.75 ± 1.03 1.55 ± 0.47 1.27 ± 0.45 5.22 ± 1.81 BloSBee10 15.0 3.7 29.4 0.32 < 1.00 1.48 < 1.00 < 1.00 < 1.00 < 1.00 4.261 BloFCo4 18.6 4.3 52.0 0.35 1.90 1.60 < 1.00 < 1.00 5.761 BloHoSBee1 18.3 3.8 54.5 0.36 3.58 1.00 < 1.00 3.54 1.30 < 1.00 6.956 BloSBee11 16.0 3.6 40.0 0.37 2.11 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 6.046 BloFBee1 20.7 4.0 25.2 0.42 1.48 1.48 < 1.00 < 1.00 1.00 < 1.00 2.127 BloSBee12 16.9 4.4 19.1 0.43 1.30 1.30 < 1.00 < 1.00 1.00 < 1.00 3.179 HoSBee1 16.2 3.8 42.7 0.49 1.00 1.00 < 1.00 < 1.00 < 1.00 < 1.00 6.989 BloSBee13 17.6 3.7 40.9 0.51 2.26 1.70 < 1.00 < 1.00 < 1.00 < 1.00 3.729 BloSBee14 18.2 3.7 42.3 0.64 1.95 < 1.00 < 1.00 < 1.00 1.70 < 1.00 1.774 BloSBee15 16.6 3.8 41.3 0.69 2.48 1.30 < 1.00 < 1.00 2.53 1.30 3.294 MIDDLE EC n = 10 n = 10 n = 10 n = 10 n = 9 n = 8 n = 1 n = 5 n = 1 n = 10 ND mean ± SD 17.4 ± 1.6 3.9 ± 0.3 38.7 ± 11.2 0.46 ± 0.13 2.01 ± 0.76 1.36 ± 0.26 3.54 1.51 ± 0.64 1.30 4.41 ± 1.92 BloSBee16 17.6 3.6 57.0 0.72 2.24 1.30 < 1.00 < 1.00 3.07 1.30 2.067 BloFBee2 18.2 3.8 74.3 0.80 1.60 2.21 < 1.00 < 1.00 1.00 < 1.00 3.355 HoSBee2 17.1 4.2 45.1 0.88 2.30 1.30 < 1.00 < 1.00 2.21 < 1.00 1.857 HoSBee3 15.2 4.7 16.0 1.00 1.30 1.60 < 1.00 < 1.00 1.00 < 1.00 3.357 HoSBee4 17.2 4.3 48.2 1.04 1.70 1.00 < 1.00 < 1.00 1.95 < 1.00 2.438 HoSBee5 16.4 4.5 68.0 1.20 2.10 1.70 < 1.00 < 1.00 2.00 < 1.00 4.838 BloFCo5 18.0 4.6 25.2 1.32 2.26 1.60 < 1.00 1.70 5.835 HIGHER EC n = 7 n = 7 n = 7 n = 7 n = 7 n = 7 n = 1 n = 6 n = 1 n = 7 ND mean ± SD 17.1 ± 1.0 4.2 ± 0.4 47.7 ± 21.3 0.99 ± 0.21 1.93 ± 0.39 1.53 ± 0.38 1.70 1.87 ± 0.79 1.30 3.39 ± 1.48 sample code (according to botanical, geographical origin and obtaining): first part: Blo – blossom, Ho – honeydew, BloHo – blended (blossom-honeydew); second part: S – Slovakian, F – foreign; third part: Bee – from beekeeper, Co - from commercial trade; FA – free acidity, EC – electrical conductivity; TPC – total plate count, SAM – sporulating aerobic microorganisms, E – bacteria from Enterobacteriaceae family, pLAB – preliminary lactic acid bacteria, MFF – microscopic filamentous fungi, SD – standard deviation, n – number of samples with detected value, ND – not detected
Values of pH ranged from 3.3 to 4.7 with average value 4.0 ± 0.4. Similarly, Amir Spain). Al-Farsi et al. (2018) tested 58 Omani honey samples and found wider et al. (2010) found pH of Algerian honey from 3.7 to 4.7, with average pH 4.0. All range of pH – from 3.46 to 7.51, while they stated that in Oman there are two main honeys are acidic with a pH-value generally lying between 3.5 and 5.5, due to the sources for honey – summer plant Acacia tortilis (from Fabaceae family) and presence of organic acids that contribute to honey flavour and stability against winter plant Ziziphus spina-Christi, L., called Sidr (from Rhamnaceae family). microbial spoilage (Bogdanov et al., 2004). Organic acids constitute 0.5% of Value of pH as well as total acidity and free acidity have some classification power honey and include gluconic acid which is bye product of enzymatic break down of for the discrimination between unifloral, while lactones, due to their strong glucose (Olaitan et al., 2007). According to Escuredo et al. (2012) pH and variability, do not provide useful information (Bogdanov et al., 2004). Overall, electrical conductivity are two parameters widely used to distinguish between free acidity of tested samples ranged from 7.7 to 74.3 meq.kg-1. According to nectar and honeydew honeys, but they found pH from 3.5 to 5.0 without significant Council Directive 2001/110/EC, honey has to contain not more than 50 meq.kg-1. differences between blossom and honeydew honeys from Galicia (Nortwest Five samples (2 from middle EC group and 3 from higher EC group) exceeded the
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limit. Average values of free acidity were different depending on electrical yeasts or MFF. Sporulating bacteria, MFF and yeasts are typically found in honey, conductivity (EC) dividing: 20.8 ± 10.4 meq.kg-1 in honeys with low EC, 38.7 ± often at low numbers, while spores can persist indefinitely (Snowdon et Cliver, 11.2 meq.kg-1 in honeys with middle EC and 47.7 ± 21.3 meq.kg-1. In general, 1996). blossom honey posses lower free acidity comparing with honeydew honey, except lime, buckwheat, eucalyptus honey or some other blossom honey. Zielińska et al. Diversity of bacteria (2014) found free acidity 14.7 ± 4.6 meq.kg-1 in rape (Brassica napus), 15.4 ± 6.6 meq.kg-1 in multifloral, 30.7 ± 17.4 meq.kg-1 in lime (Tilia sp.), 29.8 ± 11.8 meq.kg- Totally 155310 of high quality and chimera-free sequences were obtained 1 in goldenrod, 35.6 ± 8.5 meq.kg-1 in honeydew and 45.0 ± 6.4 meq.kg-1 in representing average 5177 sequences per sample. Sequences were clustered into buckwheat (Fagopyrum esculentum) Polish honey. 11926 clusters and 1743 of them had at least 5 members and while 5630 OTUs Electrical conductivity (EC) in tested honey ranged from 0.11 to 1.32 mS.cm-1. were presented by single sequence. A part of sequences, 323 OTUs comprising Low EC was 0.21 ± 0.06 mS.cm-1 on average and these honeys were mainly from 11517 (7.5%) sequences were identified as chloroplast 16S rRNA genes. false acacia, rape or blossom – multifloral honey. Middle EC was 0.46 ± 0.13 Chloroplast co-amplification is common issue when plant tissue samples are mS.cm-1 on average and these honeys were from sunflower, buckwheat, raspberry, analysed (Medo et al., 2018). In our samples chloroplasts sequences were linked linden, blossom - multifloral or blended. Sample HoSBee1 was labelled as to pollen grains naturally occurring in honey. honeydew honey. We found EC 0.49 mS.cm-1, what is not sufficient for EC of There were found occurrence of microbial species from 18 phyla (figure 1). The honeydew honey. According to Council Directive 2001/110/EC, EC of honeydew most common phylum was Firmicutes followed by Proteobacteria and honey has to be not less than 0.8 mS.cm-1. EC correlates well with the mineral Bacteroidetes and Actinobacteria. Other phyla were significantly less common. content of honey (Bogdanov et al., 2004). Minerals are present in honey in very There was apparent difference between samples in occurrence of Bacteroidetes. small quantities (0.17%) with potassium as the most abundant (Olaitan et al., Differences between samples were most apparent on genus level which is the 2007). Minerals in honeydew as well as chestnut honey are higher comparing with lowest taxonomic level with fully reliable identification for the used method. blossom honey. Escuredo et al. (2012) determined average EC of blossom honey Diversity of bacteria expressed as values of Shannon’s index shoved values in very 0.540 ± 0.200 mS.cm-1 (with range: 0.224 - 0.920 mS.cm-1) and total minerals of wide range 0.46 to 7.10 (Table 3). Dominance of single or few species in a sample blossom honey 148.6 ± 55.9 mg.kg-1 (with range: 47.9 - 280.3 mg.kg-1); and resulted to very low diversity indices. Analysis of sequences identified 492 genera average EC of honeydew honey 0.830 ± 0.200 mS.cm-1 (with range 0.482 - 1.168 however only 52 had more than 2% share in any sample (Figure 2). mS.cm-1) and total minerals 235.4 ± 80.8 mg.kg-1 (with range 95.2 - 387.4 mg.kg- According to Bovo et al. (2018), bacteria from honey could be group combining 1). Amir et al. (2010) found of Algerian honey - ash content 0.13-1.02%, with their main role/action or prevalent ecological niche and their putative origin: average 0.4% and electrical conductivity 0.3 - 1.2 mS.cm-1, with average 0.6 Predominant group consists of bacteria typical for the hive micro-environment mS.cm-1 and indicated the honeys as blossom (63%), honeydew (16%) and blended (also common microbiota of the bee gut), e. g. Lactobacillus kunkeei (obligate (21%). The electrical conductivity is good criterion related to botanical origin of fructophilic LAB), Parasaccharibacter apium, Gilliamella apicola, Frischella honey and thus is very often used in routine honey control instead of the ash content perrara. Bee pathogens, e. g. Melissococcus plutonius or Paenibacillus larvae (Gulfraz et al., 2011). represent the second group. The third abundant group of microbes in honey are We detected viable microbial counts as follows: TPC (87% of samples) > SAM plant associated species, e. g. Pseudomonas syringae, Erwinia amylovora, (77% of samples) > yeasts (60% of samples) > MFF (23% of samples) > pLAB Spiroplasma citri. Ubiquitous and specialized species, e. g. Escherichia coli, (17% of samples) > bacteria from Enterobacteriaceae family (0% of samples). Bacillus cereus as well as antagonistic bacteria like Pseudomonas agglomerans Target microbial groups ranged from ND (not detected, i. e. less than 1.00 log (with action against E. amylovora) are also common in honey. CFU.g-1) to 4.17 log CFU.g-1 (table 3). Average values of tested microbial groups were at level 1 – 2 log CFU.g-1. Tolba et al. (2007) demonstrated total viable counts from ˂100 to 1700 CFU.g-1 (˂2.00 to 3.23 log CFU.g-1) and did not find 100% Armatimonadetes
90% Cyanobacteria/Chloroplast Nitrospirae 80% Planctomycetes 70% Spirochaetes Chlamydiae 60% Chloroflexi 50% Synergistetes Acidobacteria 40%
Proportion ofreads Proportion Verrucomicrobia 30% Fusobacteria
20% candidate division WPS-1 Candidatus Saccharibacteria 10% Tenericutes 0% Actinobacteria Bacteroidetes
Proteobacteria
BloSCo1 BloFCo1 BloFCo2 BloFCo3 BloFCo4 BloFCo5
HoSBee1 HoSBee2 HoSBee3 HoSBee4 HoSBee5
BloSBee1 BloSBee2 BloSBee3 BloSBee4 BloSBee5 BloSBee6 BloSBee7 BloSBee8 BloSBee9 BloFBee1 BloFBee2
BloSBee10 BloSBee11 BloSBee12 BloSBee13 BloSBee14 BloSBee15 BloSBee16
BloHoSBee1 Firmicutes Honey samples
Figure 1 Proportion of reads assigned to bacterial phylum in honey samples sample code (according to botanical, geographical origin and obtaining): first part: Blo – blossom, Ho – honeydew, BloHo – blended (blossom- honeydew); second part: S – Slovakian, F – foreign; third part: Bee – from beekeeper, Co - from commercial trade
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Figure 2 Bacterial genera with at least 2% share in any sample of honey and grouping of samples according bacteria occurrence sample code (according to botanical, geographical origin and obtaining): first part: Blo – blossom, Ho – honeydew, BloHo – blended (blossom- honeydew); second part: S – Slovakian, F – foreign; third part: Bee – from beekeeper, Co - from commercial trade
At presented heatmap (figure 2), honey samples were clustered to two big groups (clusters) – A and B - according to most common bacterial genera in samples. The main factor of dividing into the group A and group B seems to be the freshness Fructobacillus were very abundant while Paenibacillus were apparent in some of the sample, because the first 19 samples are blossom/honeydew honeys from samples. However, in sample BloSCo1, genus Lactobacillus was not detected, only beekeepers and commercial trade from the year 2018, except HoSBee3, which is genus Paenibacillus, which was dominant and in detailed analysis, it was identified from 2017 and BloSCo1, which is commercial. Next 11 samples originated from as Paenibacillus alvei (from honey DNA followed by confirmation of culture DNA beekeepers from the year 2017 (except BloSBee 2, 11) and from commercial trade. from the NA2 plate). Paenibacillus sp. were separated from the bacilli, they Production year of commercial samples can be uncertain, because only packaging originated mainly from the soil and are common in the honey, but P. alvei is one and/or expiration date are present at the label and in fact honey could be produced of the secondary invaders of European Foulbrood (EFB) and P. larvae is the several years before packaging. causative agent of American Foulbrood (AFB) (Generch, 2010). In group A consist of 19 honey samples, genera Lactobacillus was dominant Composition of microbial assemblage in group B (11 honey samples) samples was followed by Bombella. In detailed analysis lactobacilli OTUs highest sequence significantly different. Besides genera Leuconostoc and Melissococcus which were similarity was found with Lactobacillus apinorum. Lactobacillus kunkeei is reported in honey, few other detected genera are not often in honeys or bees. dominant LAB in honey stomach (crop) and fresh honey (Olofsson et Vásquez, Bacteria Melissococcus pluton causes the brood disease EFB, while some other 2008). L. apinorum, originated from the honey bee gut, is the second fructophilic organisms, like Paenibacillus alvei, P. apiaries, Brevibacillus laterosporus, LAB within the genus Lactobacillus (Maeno et al., 2017). Bombella apis was Enterococcus faecalis or Bacterium eurydice are present as secondary invaders of detected in midgut, crop and hive of honey bees and bumble bees (Bonilla-Rosso dead brood (Shimanuki et Knox, 2000). Presence of genera like Prevotella, et Engel, 2018). Preliminary LAB were found in samples BloSBee 6, 9; Staphylococcus or Porphyromonas suggests contamination of these samples, BloHoSBee 1 and BloFCo 3, 5 by dilution plating method, but presence of LAB during the extracting, processing and/or storage of the samples. On the other side, by lactobacilli detection was only in samples BloSBee 6 and 9. Although LAB do last time, presence of Neisseria sp., which mostly known as human pathogen, were not survive in stored honey, it is likely that humans have been consuming viable confirmed in gut of bees and bumblebees (Kwong et Moran, 2013). LAB in fresh honey during and directly after honey hunts throughout human history (Olofsson et Vásquez, 2008). In 3 member subgroup (BloSBee 10, 13, 15)
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Figure 3 RDA scatterplot from analysis of physico-chemical properties and microbial assemblage of honey sample code (according to botanical, geographical origin and obtaining): first part: Blo – blossom, Ho – honeydew, BloHo – blended (blossom-honeydew); second part: S – Slovakian, F – foreign; third part: Bee – from beekeeper, Co - from commercial trade; FA – free acidity, EC – electrical conductivity
Microbial assemblage of honey is connected mainly to the microbiome of bees. Probably, most of them are not in viable state as honey represents a suitable Honey seems to be a suitable indicator of surrounding during the honey production, environment for keeping of DNA and RNA intact. Subsequently, data from processing and storage. Relation of physic-chemical attributes and microbial metagenomic analysis can provide us various information about surrounding assemblage using redundancy analysis showed electrical conductivity as main during honey production, processing and storage. factor related to variation in honey microbiome (figure 3). First RDA factor explained majority of variance (82.7%) in microbial assemblage. This factor was Acknowledgments: The work was supported by the grant KEGA 025SPU-4/2019 in strong correlation (R2 = 0.69) with EC. Permutation test approved EC as the and VEGA 1/0661/19. Authors would thank to prof. Radoslav Omelka from only one significant physico-chemical attribute (P = 0.003) among tested. University of Constantine the Philosopher in Nitra for access to Next Generation According to Bogdanov et Martin (2002), EC is suitable parameter for evaluation Sequencer. differentiation between blossom and honeydew honeys as well as for unifloral honeys. As EC is strongly depended on source of honey, probably the source is REFERENCES important determinant of microbial assemblage at least the same that bees themselves. AL-FARSI, M., AL-BELUSHI, S., AL-AMRI, A., AL-HADHRAMI, A., AL- RUSHEIDI, M., AL-ALAWI, A. 2018. Quality evaluation of Omani honey. Food CONCLUSION Chemistry, 262(2018), 162-167. http://doi.org/10.1016/j.foodchem.2018.04.104 AMIR, Y., YESLI, A., BENGANA, M., SADOUDI, R., AMROUCHE, T. 2010. Overall, electrical conductivity was the main parameter, which was connected with Physico-chemical and microbiological assessment of honey from Algeria. the botanical origin. Viable counts of microorganisms could be influenced by the Electronic Journal of Environmental, Agricultural and Food Chemistry, 9(9), water content as well as freshness of the sample. We found sporulating aerobic 1485-1494. microorganisms and yeasts as the most often occurring viable microbes. However, BOGDANOV, S., MARTIN, P. 2002. Honey Authenticity. Mitteilungen aus metagenomic analysis gave us interesting view into the presence of bacterial DNA Lebensmitteluntersuchung und Hygiene, 93(3), 232-254. in the honey. Bacterial diversity in the honey samples indicated main differences BOGDANOV, S., RUOFF, K., PERSANO ODDO, L. 2004. Physico-chemical between the Slovak and Swiss fresh samples (produced in 2018) and the older ones methods for the characterisation of unifloral honeys: a review. Apidologie, 35(1), (produced in 2017) together with commercial samples from Slovakia and foreign S4-S17. http://doi.org/10.1051/apido:2004047 countries. In general, lactobacilli were dominant in Slovakian and Swiss fresh BONILLA-ROSSO, G., ENGEL, P. 2018. Functional roles and metabolic niches honeys. In older samples, Prevotella sp. and other representatives probably in the honey bee gut microbiota. Current Opinion in Microbiology, 43(2018), 69- originating in human contamination during the honey extracting and processing 76. http://doi.org/10.1016/j.mib.2017.12.009 were dominant. Surprisingly, the spectrum of identified bacterial genera was broad.
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MICROBIOLOGY SAFETY ASSESSMENT OF BRINE AFTER WET SALTING OF MEAT
Miroslav Kročko1*, Viera Ducková1, Ondřej Bučko2, Marek Bobko1, Jana Tkáčová1
Address(es): 1Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Technology and Quality Animal Products, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia. 2Slovak University of Agriculture, Faculty of Agrobiology and food resources, Department of Animal Husbandry, 949 76 Nitra, Nitra, Tr. A. Hlinku 2, Slovakia.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2019.9.special.431-433
ARTICLE INFO ABSTRACT
Received 30. 7. 2019 The aim of the study was to determine the microbial safety of brine and the effect of bacterial starter culture containing CNS strains of Revised 15. 10. 2019 Staphylococcus carnosus and Staphylococcus xylosus (Indasia, Germany) on the microbiological quality of the brine during wet salting Accepted 16. 10. 2019 of the meat. In the first experiment was determined the microbial safety of brine with different concentrations of curing salt and with Published 8. 11. 2019 addition of bay leaf, whole black pepper; whole juniper and whole new pepper water extracts. In the second experiment were prepared brine samples with 9.5% of curing salt, brine samples with the same concentration of curing salt but with the addition of a starter culture and brines with the concentration 7.5% of curing salt, starter culture and spices water extracts same as in previous experiment. The Regular article commercial starter culture contains strains of Staphylococcus carnosus and Staphylococcus xylosus (CNS) was added in amount of 0.2g. Safety assessment of the brine was established according to results of pH value, salinity, color and count of coliform bacteria, psychrotrophic bacteria, Lactobacillus sp. and Streptococcus sp. The results show that the very presence of 9.5% salt and nitrite does not prevent the decline of microorganisms in brine samples used for salting meat. Protective culture helped reduce the number of unwanted microorganisms and promoted lactobacilli. However, the demonstrably higher antibacterial effect of the protective culture, determined on the basis of the number of psychotropic microorganisms, was found to be combined with the spice extract.
Keywords: brine, wet salting, bacteria, CNS, color
INTRODUCTION Over time, it has been found that only some types of salt have helped to develop the desired color and characteristic taste of the meat. The nineteenth-century Salting is one of the oldest processes of meat preservation. Sodium chloride investigation revealed that sodium nitrate, present as an impurity in these salts, is solution was commonly used to treat meat pieces or the meat was covered with a responsible for the development of the characteristic color and taste of salted dry salt layer. The salt itself caused a decrease of the water activity value in the meat. Later it was found that the desired effect occurs through microbial meat, thus preventing microbial damage and unwanted organoleptic changes. The reduction of nitrates to nitrites. From the subsequent experiments, regulations ancient Sumerians consider themselves the first civilization to use meat and fish were introduced for the direct addition of sodium nitrite in order to prolong the preserving salt around 3000 BC. Dead Sea salt was first used in ancient Palestine shelf life of the meat while maintaining the color and desired flavor. Current as early as 1600 BC. Chinese and Greeks also used stone salt, which was later practice includes the addition of sodium nitrite, ascorbates or erythorbates, used by ancient Romans (Pereira and Vicente, 2013; Wojnowski et al., 2017). additives such as e.g. sweeteners, phosphates, spices and smoke flavor Salting methods can be divided into two basic categories: dry and wet (saline, (Skovgaard, 1992). brine). Dry salting is the oldest traditional technique in which salt with nitrites or The production of fermented meat products is based primarily on the metabolic even nitrates is applied to the surface of the meat. In brine salting processes, the activities of lactic acid bacteria and subsequently catalysis of positive cocci, in individual components are dissolved in water to form brine, in which the meat is particular the coagulase-negative staphylococci (CNS). On the other hand, in the naturally located for a period of time or injected directly into the meat (Shahidi production of salted meat, the CNS has been monitored primarily for a long time. and Samaranayka, 2004). These two salting methods can also be combined with Their use usually leads to the appropriate formation of meat color based on their each other. Combined method has obtained popularity in the fish industry. The nitrate reductase activity, while their catalase activity reduces oxidative damage. method that is generally used is as follows: the fish is immersed for 1–4 days in a In addition, CNS metabolism contributes to taste, although the exact effects are solution of salt, with salt concentration is 17% NaCl; removal from the brine; the difficult to estimate. There are reasons to believe that these basic technological fillets are placed with thin layers of salt, into stacks or in plastic tubs; the fish is features of the CNS can be further enhanced by examining their overall metabolic then kept stacked for 10–14 days for dry salting after which it is packed potential. Rational selection of CNS strains can lead to the development of new (Aberoumand and Nejad, 2015). cultures with enhanced functionality. Their ability to contribute to the formation Wet curing (saline, brine) has been applied to a wide range of food, such as of meat color could be optimized to reduce the amount of saline needed, either by vegetables, cheese, fish, and meat products. The curing of meat is important, as selecting effective nitrate reducing CNS strains or by exploring a potential meat is a valuable source of protein which decomposes rapidly when not alternative based on nitric oxide synthase activity. Finally, bacteriocin-producing preserved (Pereira and Vicente, 2013; Wojnowski et al., 2017). The rate of CNS strains may offer solutions for bioprotection against meat pathogens such as diffusion of salt and functional ingredients into the feedstock depends on the salt Clostridium botulinum and Staphylococcus aureus (Sanchez Mainar et al., concentration, the size of the individual pieces of raw material, tumbling process 2017). and on the content of fibrous and fat portions that act as a diffusion barrier The aim of the study was to determine the microbial safety of brine with different (blending for protein activation) (Feiner et al., 2006). The antimicrobial activity amount of salt and spices extract and the effect of bacterial starter culture of NaCl is reviewed in light of currrent calls for a reduction of Na+ in the human containing CNS strains of Staphylococcus carnosus and Staphylococcus xylosus diet due to health reasons, and the possible replacement of NaCl in processed (Indasia, Germany) on the microbiological quality of the brine during wet salting foods with chloride salts of other ions (i.e. KCl, MgCl2, CaCl2) (Sofos, 1984). of the meat.
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MATERIAL AND METHODS
Brines models preparation and experimental set-up
First experiment
It is well known that the concentration of salt as well as nitrites in the wet salting process in the brine gradually decreases; thereby the antibacterial effect of this brine is reduced. For this reason, the first experiment was established to determine the microbial safety of brine with different concentrations of curing salt additions. Samples of brines were prepared according to traditional procedures used in a small and large fully functional curing facilities located in the Slovak republic. Pork samples (musculus longissimus dorsi) were cut to a weight of 100g. The ratio of raw material and brine set to 1: 3. Preparation of 300 ml samples of the brine consisted of a 9.5%; 8.5%; 7.5%; and 6.5% curing salt (NaCl 99.5% and NaNO2 0.5%) addition. For last experimental sample of brine, was first added to Figure 1 Values of pH in brines after 10 days of wet salting phase 500 ml of water; 0.5 g of bay leaf, 0.5g whole black pepper; 0.5g of whole juniper and 0.5g of whole new pepper. In a sealed vessel, the solution was The process of salting and subsequent drying of meat is the most common way of brought to boiling for 10 minutes (this time was sufficient to reduce the volume treating meat to obtain a safe product intended also for direct consumption by from 500 ml to 300 ml.) and, after cooling the spices were removed. The curing consumers. Therefore, it is important to ensure sufficient bactericidal effect of salt (7.5%) was added to 300 ml of this solution. Meat samples were stored at 4 ± brine, especially against coliform bacteria and streptococci (Cardoso-Toset et 1 °C for 10 days after loading in saline. al., 2017).
Second experiment
In the second part of the experiment, were prepared brine samples with 9.5% of curing salt (NaCl 99.5% and NaNO2 0.5%) (K), brine samples with the same concentration of curing salt but with the addition of a starter culture (P1). Only brines of P2 were prepared with the concentration 7.5% of curing salt and spices water extracts same as in previous experiment. Starter culture was also added to these samples in amount of 0.2g. The commercial starter culture contains strains of Staphylococcus carnosus and Staphylococcus xylosus (Indasia, Germany) in the amount of log 11 KTJ.g-1. Samples of meat were not investigated in this study. pH and salinity measurement
After sampling, the pH was measured directly in the brine with a Gryf 209L pH meter (Gryf, Czech Republic) equipped with an insertion pH probe. Salinities of each brine solutions were measured using a refractometer. Figure 2 Number of microorganisms in brines after 10 days of wet salting phase
Color measurement The number of coliform bacteria, Streptococcus sp. and TPC bacteria was found
to be highest in all of brine with concentration of curing salt 6.5%. These findings Color spaces of L*, a*, b* were determined by CM 2600D spectrophotometer are in accordance with study of Durack et al. (2013) who reported that salt (Konica Minolta, Germany). Color of brine in second experiment was measured within brine was introduced to reduce the rate of deterioration of meat by with SCE (Specular Component Excluded). reducing the growth of microorganisms. High concentration of salt in brine
results in greatly reduced loads of Psychrotrophic bacteria in brine and inhibits Enumeration of microorganisms microbial spoilage of meat.
The amount of Lactobacillus bacteria were not detected in brine solutions of 6.5 One mililiter of brine were aseptically transferred into a glass tubes and and 7.5%. This group of microorganisms was probably suppressed by other appropriate decimal dilutions in saline were prepared and spread on selective species of microorganisms as they occurred in brines with higher salt agar. concentrations. The determination of coliform bacteria was carried out on VRB agar (after The total number of coliform microorganisms in the saline samples at the end of cultivation for 24 hours at 30 ° C ± 1 ° C (ISO 4832). the wet salting time ranged from 0.3 to 4.68 log CFU / ml. The lowest numbers The total number of psychrotorphic microorganisms (TPM) was determined on of coliform microorganisms were recorded in samples with addition of spices the PCA agar (HiMedia, India) after 10 days of culture at 4 ± 1 ° C. extract and with a reduced salt concentration of 7.5%. Lactobacillus bacteria were assayed on MRS agar by culture at 30 ° C ± 1 ° C. The results show that curing salt is effective against undesirable microorganisms After 5 days of culture, their numbers were read (ISO 15214, 2002). in the wet salting process only if its concentration is maintained at 9.5% or more The total number of streptococci was determined on the M17 agar (HiMedia, throughout the salting time. This also ensures a sufficient amount of nitrites that India) by incubation at 30 ± 1°C for 3 days (ISO 15214). act as antimicrobials.
Nitrate itself has little antimicrobial effect and in most applications could be RESULTS AND DISCUSSION replaced by lower concentrations of nitrite. Further, improved hygiene diminishes
the need for nitrite. The antimicrobial activity and technological needs for nitrate Regarding pH, statistically differences among the samples were observed. Value and nitrite are reviewed. It is concluded that the technological needs for nitrite in of pH was compare to other brines significantly higher in 6.5% brine and the meat products stored at < 10°C could be met by added nitrite concentrations of lowest in 9.5% brine after 10 days of salting. Statistically no differences were 50 mg/kg. In such products, Vibrio parahaemolyticus does not grow at salt observed among the samples of 7.5%; 8.5 and 7.5 + spices brines. The pH value concentrations of 10% and food poisoning by this organism is not related to the of brine can be used as an indicator of degree of freshness or spoilage of salted absence of nitrate or nitrite; growth of Clostridium botulinum Type E (the meat. In fresh meat pH is close to neutral, first it decreases due to lactic acid predominant cause of botulism from fish products) is arrested by salt arising from death, however then it increases because of deterioration. The reason concentrations of 3–4%. Listeria monocytogenes in seafood cannot be controlled for this increase is the disruption to the oxidation-reduction balance along with by nitrite (Skovgaard, 1992). the effect of enzymes and bacteria, and the changes in the concentration of free hydrogen and hydroxyl ions (Varlık et al., 1993). The increase in pH indicates the loss of quality (Latifa et al., 2014).
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based on the number of psychotrophic microorganisms was found to be a* b* combined with the extract from the spices.
5 Acknowledgments: This work has been supported by a grant of KEGA 025SPU- 4/2019. 4 3 REFERENCES
2 ABEROUMANDAND, A., NEJAD, S. Z. 2015. Effects of brining process on nutrient composition of fish species (kharo, govazim and kijar) from Iran. 1 International Journal of Agricultural Research, Innovation and Technology 5, 0 36-39, https://doi.org/10.3329/ijarit.v5i1.24585 K P1 P2 BINICI, A., KAYA, G. K.. 2017. Effect of brine and dry salting methods on the physicochemical and microbial quality of chub (Squalius cephalus Linnaeus, Figure 3 Intensity of redness (a*) and yellowness (b*) of brine after 10 day of 1758). Food Science and Technology 38, 66-70. http://dx.doi.org/10.1590/1678- salting period 457x.15717. CARDOSO-TOSET, F., LUQUE, I., MORELES-PARTERA, A. et al. 2017. Microbiologically unstable meat brines are characterized by a color change. Survival of Streptococcus suis, Streptococcus dysgalactiae and Trueperella Microbiologically degraded solutions may have a reddish or dark brown color pyogenes in dry-cured Iberian pork shoulders and loins. Food Microbiology 61, and unpleasant smell. Intensity of brine lightness after 10 days of meat salting 66-71. https://doi.org/10.1016/j.fm.2016.09.002 period was highest in samples of P2 (26.52), followed samples of P1 (25.57) and CASSENS, R. G. 2008. Meat preservation:Preventing Losses and Assuring K (25.51). Intensity of red color (a*) during the whole period of salting was Safety. Hoboken : John Wiley & Sons : 133 p. ISBN 0-917678-34-6. significantly highest in brine samples of control group. https://doi.org/10.1002/9780470385029 The higher yellowness and discoloration in the meat products might be related to DURACK, E., ALONSO‐GOMEZ, M., WILKINSON, M. G. 2013. The Effect of a higher lipid oxidation caused by the microbial lipases produced by the starter Salt Reduction on the Growth of Food Spoilage Bacteria in Model Broth Systems cultures. The lipid oxidation in meat products can reduce myoglobin stability, and Salt‐Adjusted Ready Meals. Journal of Food Safety 33, 302-312. making it more susceptible to oxidation, which decreases the red color and https://doi.org/10.1111/jfs.12053 increases the discoloration of red meat (Kennedy et al., 2004). The increased FEINER, G. 2006. Meat Products Handbook. Cambridge : Woodhead Publishing, intensity of yellow color in brine samples of P2 group is probably caused due to 672 p. ISBN 9781845690502 the addition of spices extract. KENNEDY, C., BUCKLEY, D. J., KERRY, J. P. (2004). Display life of sheep meats retail packaged under atmospheres of various volumes and compositions. Meat Science, 68, 649-658. https://doi.org/10.1016/j.meatsci.2004.05.018 LATIFA, G. A., CHAKRABORTY, S. C., BEGÜM, M., NAHID, M. N., FARID, F. B. 2014. Nutritional quality analysis of bangladeshi fish species, M. tengra (Hamilton-Buchanan, 1822) preserved with different salt curing methods in laboratory condition. American Journal of Food and Nurition 2(6), 100-107. https://doi.org/10.3329/bjz.v42i2.23369 PEREIRA, P. M., AND VICENTE, A. F. 2013. Meat nutritional composition and nutritive role in the human diet. Meat Science 93, 586-592. https://doi.org/10.1016/j.meatsci.2012.09.018. SKOVGAARD, N. 1992. Microbiological aspects and technological need: Technological needs for nitrates and nitrites. Food Additives and Contaminants 9 (5), 391-397, https://doi.org/10.1080/02652039209374089. WOJNOWSKI, W.,MAJCHRZAK, T., DYMERSKI, T., GEBICKI, J., AND NAMIESNIK, J. 2017. Electronic noses: powerful tools in meat quality assessment. Meat Science, 131, 119-131. https://doi.org/10.1016/j.meatsci.2017.04.240 SÁNCHEZ MAINAR, M., STAVROPOULOU, D.A., LEROY, F., 2017.
Figure 4 Number of microorganisms in brines with starter cultures after 10 days Exploring the metabolic potential of coagulase-negative staphylococci to improve of wet salting phase the quality and safety of fermented meats: a review. Int. J. Food Microbiol. roč. 247, 24–37. https://doi.org/10.1016/j.ijfoodmicro.2016.05.021. In the second experiment were among count of coliform bacteria in brine samples SOFOS, J. N. 1984. Antimicrobial effects of sodium and other ions in foods: not detected significant differences and their count were below 1 log CFU/ml in a review. Journal of Food Safety 6, 45-78. https://doi.org/10.1111/j.1745- all tested brine samples (figure 3). The total number of streptococci in brine 4565.1984.tb00478.x samples ranged from 1.0 to 2.65 log CFU/ml. The lowest numbers of this group VARLIK, C., UĞUR, M., GÖKOĞLU, N., GÜN, H. (1993). Quality control of microorganisms were recorded in both samples with the addition of protective methods and principles in aquaculture (174 p., No. 17). AnkaraI : Ankara culture and also with protective culture and spices. Üniversitesi Ziraat Fakültesi Gıda Böl. 174 p. In the brine samples prepared with the spice extract and also bacterial culture, the number of psychrotrophic microorganisms was found to be significantly lower by 1.1 log CFU/ml than that of the other brine samples. In addition to taste, spices can also provide antiseptic and preservative properties due to the presence of essential oils. Antimicrobial activity depends on the type of spices, the type of food to which it is added, the composition and content of the extracts. Thus, in addition to providing strong flavors, they have antioxidant properties that inhibit lipid oxidation. The antioxidant properties of spices are caused by the presence of flavonoids, terpenoids, lignans or polyphenols. Thanks to these properties, they can help maintain the stability of the saline solution (Cassens, 2008). Growth of psychrotrophic bacteria in the control group may be related to an increase of the accompanying microflora characteristic for brine and meat, but may also be associated with the development of CNS strains that have been directly added to the brine samples of P1 and P2. Our findings are consistent with the findings of Binici and Kaya (2017), who also noted an increase in the number of psychotrophic microorganisms during 15 days of wet fish salting to 8 log CFU/g.
CONCLUSION
The results indicate that the presence of 10% salt and nitrites alone will not reduce the microorganisms in saline solutions used for salting meat. Starter culture helped to reduce the number of undesirable microorganisms and promoted lactobacilli growth. However, a significantly higher antibacterial effect
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EFFECT OF PEPTIDOGLYCAN OF STAPHYLOCOCCUS AUREUS ON APOPTOSIS OF BOVINE MAMMARY GLAND LYMPHOCYTES
Petr Slama*,1, Terezie Zavadilova1, Lucie Kratochvilova1, Kristina Kharkevich1, Michal Uhrincat2, Vladimir Tancin2,3
Address(es): Ing. Petr Slama, Ph.D. 1Mendel University, Faculty of AgriSciences, Department of Animal Morphology, Physiology and Genetics, Zemedelska 1, 613 00 Brno, Czech Republic, phone number: +420 545 133 146. 2NPPC-Research Institute for Animal Production, Hlohovecka 2, 951 41 Luzianky, Slovak Republic. 3Slovak University of Agriculture in Nitra, Faculty of Agrobiology and Food Resources, Department of Veterinary Sciences, Trieda A. Hlinku 2, 949 76 Nitra, Slovak Republic.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2019.9.special.445-446
ARTICLE INFO ABSTRACT
Received 31. 7. 2019 The goal of this stud was to analyze whether apoptosis of lymphocytes is modulated by experimental infection caused by peptidoglycan Revised 29. 9. 2019 of Staphylococcus aureus. Lymphocytes were obtained by lavage of the mammary glands at four intervals (24, 48, 72 and 168 hours) Accepted 7. 10. 2019 following experimental stimulation of mammary glands. The portion of apoptotic lymphocytes peaked at 48 hours following infection Published 8. 11. 2019 with peptidoglycan of Staphylococcus aureus. The results show that during the mentioned infection the apoptosis of lymphocytes is induced.
Regular article Keywords: peptidoglycan, lymphocyte, apoptosis, Staphylococcus aureus, mammary gland
INTRODUCTION quarter of the udder was injected with 20 ml phosphate buffered saline (PBS) with 50 μg of peptidoglycan of S. aureus (Sigma, USA). Before experimental Staphylococcus aureus is one of the most important pathogen which is able to infection, the mammary glands were used for preparation of control samples cause bovine mastitis (Petersson-Wolfe et al. 2010). These bacteria contain through treatment by PBS as previously described (Sladek et al., 2005). peptidoglycan in their cell wall. Peptidoglycan is also found in the cell wall of The lymphocytes obtained by lavages of mammary glands were analysed by flow Gram-negative bacteria but in different structure than in Gram-positive bacteria cytometry (FACS Calibur Apparatus, Becton Dickinson, CA, USA) and (Vollmer et al., 2008). This natural molecule of bacteria is able to induce subsequently by software WinMDI 2.8 (Trotter, 2000) as in previous studies (for inflammatory response of bovine mammary gland (Furukava et al., 2018). example Slama et al., 2006). Proportion of apoptotic lymphocytes was Sulabh et al. (2019) have confirmed the stimulant effect of peptidoglycan of S. enumerated by staining with Annexin-V (FITC) and propidium iodide (PE) aureus on peripheral blood mononuclear cells obtained from blood samples of previously described by Vermes et al. (1995). crossbred cattle, Tharparkar cattle, and Murrah buffaloes. Peptidoglycan For statistical analysis, there were used statistical software STATISTICA 8.0 stimulates leukocytes and also epithelial cells during mastitis (Im et al., 2014). (StatSoft, Czech Republic). Arithmetic means and standard deviations were used S. aureus is also able to produce enterotoxins. These enterotoxins (staphylococcal to describe apoptosis of lymphocytes. Statistically significant differences in the enterotoxin C, α-toxin) induce activation and apoptosis of T cells (Damle et al., proportion of apoptotic lymphocytes were determined by paired t-test. 1993; Webb and Gascoigne, 1994; Boshell et al., 1996) and apoptosis of peripheral blood mononuclear cells (Haslinger et al., 2003). RESULTS AND DISCUSSION In context of previous information, changes of programmed cell death can be caused by different inducers in frame work of bacteria – by components of cell The aim of this study was to evaluate the effect of peptidoglycan on apoptosis of wall or product of bacteria mainly toxins. We previously studied the effect of S. bovine mammary gland lymphocyte during experimentally induced aureus and Streptococcus uberis on lymphocyte apoptosis of bovine mammary inflammation. gland (Slama et al., 2009a). We have found that apoptosis of lymphocytes was In this study, the apoptosis of lymphocytes was changed during the inflammatory delayed by those bacterial species during experimental infection and in in vitro response. Experimental infusion of peptidoglycan into the mammary gland led to study, too. Therefore, we would like to know whether peptidoglycan as the increase of lymphocyte apoptosis with the maximum in 48 hours following molecule naturally found in the cell wall of S. aureus can also modulate stimulation of the mammary glands compare to the control (Figure 1). lymphocyte apoptosis in the same manner or in different way. The aim of this study was to determine whether apoptosis of bovine mammary gland lymphocytes is modulated during an inflammatory response of bovine mammary gland induced by peptidoglycan.
MATERIAL AND METHODS
For our experiments, we used eight clinically healthy virgin heifers (Holstein x Bohemian Red Pied crossbred) in age 16 to 18 months. All animals were free of infection of the mammary glands. Bacteriological examination of mammary gland lavages was executed by culturing on blood agar with aerobic incubation at 37 °C for 24 hours. For the experimental infection, there were used urethral catheter (AC5306CH06, Porges SA, France) to insert into the teat canal after disinfection of the teat orifice (Sladek et al., 2005; Slama et al., 2009a). Each mammary gland in each
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CD11a/CD18-dependent mechanism. European Journal of Immunology, 23, 12 1513-1522. PBS 10 ** FURUKAVA, M., YONEYAMA, H., HATA, E., IWANO, H., HIGUCHI, H., peptidoglycan ANDO, T., SATO, M., HAYASHI, T., KIKU, Y., NAGASAWA Y., NIIMI, K., 8 USAMI, K., ITO, K., WATANABE, K., NOCHI, K., ASO, H. 2018. Identification of a novel mechanism of action of bovine IgG antibodies specific 6 for Staphylococcus aureus. Veterinary Research, 49, 22. http://dx.doi.org/10.1186/s13567-018-0517-y 4 HASLINGER, B., STRANGFELD, K., PETERS, G., SCHULZE-OSTHOFF, K.,
SINHA, B. 2003. Staphylococcus aureus α-toxin induces apoptosis in peripheral Apoptosis Apoptosis (%) 2 blood mononuclear cells: role of endogenous tumour necrosis factor-α and the mitochondrial death pathway. Cellular Microbiology, 5, 729-741. 0 IM, J., LEE, T., JEON, J.H., BAIK, J.E., KIM, K.W., KANG, S.S., YUN, C.H., 24 h 48 h 72 h 168 h KIM, H., HAN, S.H. 2014 Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from
Figure 1 Apoptosis of mammary gland lymphocytes (%) following stimulation Staphylococcus aureus. International Immunopharmacology, 21, 231-240. by peptidoglycan in four timepoints (24, 48, 72, 168 hours). PBS – phosphate http://dx.doi.org/10.1016/j.intimp.2014.05.002 buffered saline; ** P˂0.01 PARK, Y.H., LEE, S.U., FERENS, W.A., SAMUELS, S., DAVIS, W.C., FOX, L.K., AHN, J.S., SEO, K.S., CHANG, B.S., HWANG, S.Y., BOHACH, G.A. These results suggest that peptidoglycan can induce apoptosis of lymphocytes. 2006. Unique features of bovine lymphocytes exposed to a staphylococcal Contrary to that, our previous results shown that S. aureus or S. uberis can delay enterotoxin. Journal of Veterianry Science, 7, 233-239. apoptosis of lymphocytes (Slama et al., 2009a). Slight increase in apoptosis of T http://dx.doi.org/10.4142/jvs.2006.7.3.233 lymphocytes was detected by Park et al. (2006). Those authors cultivated PETERSSON-WOLFE, C.S., MULLARKY, I.K., JONES, G.M. 2010. lymphocytes with staphylococcal enterotoxin C in vitro. Those mentioned Staphylococcus aureus mastitis: cause, detection, and control. Virginia information suggest that components of bacterial cell wall could be more Cooperative Extension, 404-229. effective in induction of lymphocyte apoptosis in the initial stage of RATH, P.C., AGGARWAL, B.B. 1999. TNF-induced signaling in apoptosis. inflammation. Later, bacteria produce toxins which can be effective apoptotic Journal of Clinical Immunology, 19, 350-364. inducer but in the late stage of inflammation. Apoptosis of cells is very important SLADEK, Z., RYSANEK, D. RYZNAROVA, H., FALDYNA, M. 2005. to protect mammary gland tissue damage during mastitis. Apoptotic cells have Neutrophil apoptosis during experimentally induced Staphylococcus aureus stable cell membrane and the content of the cell is not spread into surrounding mastitis. Veterinary Research, 36, 243-262. tissue. On the other hand, necrosis of cells is dangerous to tissue because the cell http://dx.doi.org/10.1051/vetres:2005023 membrane of these cells is damaged. If the necrotic cells are neutrophils, they can SLAMA, P., SLADEK, Z., RYSANEK, D. 2006. Effect of isolation techniques harm the mammary tissue by releasing reactive oxygen intermediates and on viability of bovine blood neutrophils. Acta Veterinaria Brno, 75, 343-353. proteolytic enzymes (Zhao and Lacasse, 2008). http://dx.doi.org/10.2754/avb200675030343 In our study, we used peptidoglycan of S. aureus as Gram-positive bacteria. SLAMA, P., SLADEK, Z., RYSANEK, D., LANGROVA, T. 2009a. Effect of Question remains, if Gram-negative bacteria or the components of their cell wall Staphylococcus aureus and Streptococcus uberis on apoptosis of bovine are able to affect the lymphocyte apoptosis in the same way. Our preliminary mammary gland lymphocytes. Research in Veterinary Sciences, 87, 233-238. results (unpublished data) indicate that lipopolysaccharide of Escherichia coli has http://dx.doi.org/10.1016/j.rvsc.2009.03.005 similar effect on apoptosis of lymphocyte in vivo. Yokochi et al. (1996) also SLAMA, P., SLADEK, Z., RYSANEK, D. 2009b. Lipopolysaccharide delays shown that lipopolysaccharide induced apoptosis of B lymphocytes. In contrary apoptosis of bovine lymphocytes. FEBS Journal, 276, 223. to that, we reported that lipopolysaccharide can delay apoptois of lymphocytes in SLAMA, P., SLADEK, Z., RYSANEK, D. 2009c. Effect of muramyl dipeptide in vitro conditions (Slama et al., 2009b). We also used muramyl dipeptide to on apoptosis of bovine mammary gland lymphocytes in vitro. Slovak Journal of stimulate lymphocytes in vitro. Muramyl dipeptide is the minimal structural unit Animal Science, 42, 90-93. of peptidoglycan. We found out that apoptosis of lymphocytes was also delayed SOHN, E.J., PAAPE, M.J., CONNOR, E.E., BANNERMAN, D.D., FETTERER, using muramyl dipeptide (Slama et al., 2009c). Contrast in results of in vitro and R.H., PETERS, R.R. 2007. Bacterial lipopolysaccharide stimulates bovine in vivo studies can be possible. In vitro study only says what cells are able to do neutrophil production of TNF-alpha, IL-1beta, IL-12 and IFN-gamma. Veterinary but in in vivo conditions there are more variables which can play important role Research, 38,809-818. http://dx.doi.org/10.1051/vetres:2007033 in final results of experiments. Moreover, there are produced different cytokine SULABH, S., PANIGRAHI, M., AHMAD, S.F., VARSHNEY, R., VERMA, A., during mastitis by immune cells. These cytokines are able to modulate apoptosis BABA, N.A., KUMAR, S., KUMARI, S., CHAUHAN, A., KUMAR, P., of cells. In the initial stage of mastitis, there is produced tumor necrosis factor BHUSHAN, B. 2019. Peptidoglycan and Lipoteichoic Acid Induces Differential alpha by neutrophils (Sohn et al., 2007). That cytokine is able to induce mRNA Response of Immune-Related Genes in PBMC of Crossbred, Tharparkar apoptosis of cells (Rath and Aggarwal, 1999) and therefore the highest Cattle and Murrah Buffalo. Animal Biotechnology, 30, 166-174. percentage of lymphocyte apoptosis is found out in 48 hours following the start http://dx.doi.org/10.1080/10495398.2018.1461633 of inflammation. TROTTER, J.: WinMDI Version 2.8. 2000. http://facs.scripps.edu/. VERMES, I, HAANEN, C., STEFFENS-NAKKEN, H., CONCLUSION REUTELINGSPERGER, C. 1995. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using The results suggest that the cell wall components as peptidoglycan are able to fluorescein labelled Annexin V. Journal of Immunological Methods, 184, 39-51. modulate lymphocyte apoptosis during the process of inflammation of the VOLLMER, W., BLANOT, D., DE PEDRO, M.A. 2008. Peptidoglycan structure mammary gland. We studied whole population of lymphocytes and therefor it is and architecture. FEMS Microbiology Reviews, 32, 149-167. necessary to continue to investigate different subpopulation of lymphocytes. http://dx.doi.org/10.1111/j.1574-6976.2007.00094.x Different subpopulation of lymphocytes could have different resistance to WEBB, S.R., GASCOIGNE, N.R. 1994. T-cell activation by superantigens. bacterial compounds and toxins. Current Opinion in Immunology, 6, 467-475. YOKOCHI, T., KATO, Y., SUGIYAMA, T., KOIDE, N., MORIKAWA, A., Acknowledgments: The authors wish to express their thanks for financial JIANG, G.Z. KAWAI, M., YOSHIDA, T., FUKADA, M., TAKAHASHI, K. support to the projects of IGA AF MENDELU No. AF-IGA-2018-tym002 and 1996. Lipopolysaccharide induces apoptotic cell death of B memory cells and the Ministry of Education Science Research and Sports of the Slovak regulates B cells in antigen non-specific manner. FEMS Immunology and Republic/the Slovak Research and Development Agency (Project No. APVV-15- Medical Microbiology, 15, 1-8. 0072). ZHAO, X., LACASSE, P. 2008. Mammary tissue damage during bovine mastitis: causes and control. Journal of Animal Science, 86, 57-65. REFERENCES http://dx.doi.org/10.2527/jas.2007-0302
BOSHELL, M., MCLEOD, J., WALKER, L., GALL, N., PATEL, Y., SANSOM, D. 1996. Effects of antigen presentation on superantigen-induced apoptosis mediated by Fas/Fas ligand interactions in human T cells. Immunology, 87, 586- 592. DAMLE, N.K., LEYTZE, G., KLUSSMAN, K., LEDBETTER, J.A. 1993. Activation with superantigens induced programmed cell death in antigen primed CD4+ class II+ major histocompatibility complex T lymphocytes via a
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EFFECT OF PEPTIDOGLYCAN ON EXPRESSION OF CD44 ON BOVINE MAMMARY GLAND LYMPHOCYTES
Petr Slama*, Terezie Zavadilova, Lucie Kratochvilova, Kristina Kharkevich
Address(es): Ing. Petr Slama, Ph.D. Mendel University, Faculty of AgriSciences, Department of Animal Morphology, Physiology and Genetics, Zemedelska 1, 613 00 Brno, Czech Republic, phone number: +420 545 133 146.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2019.9.special.447-448
ARTICLE INFO ABSTRACT
Received 2. 8. 2019 The aim of this study was to analyze changes in the expression of CD44 on bovine mammary gland lymphocytes during experimental Revised 12. 9. 2019 infection caused by peptidoglycan of Staphylococcus aureus. Lymphocytes were obtained by lavage of the mammary glands at four Accepted 13. 9. 2019 intervals (24, 48, 72 and 168 hours) following experimental intramammary injection of peptidoglycan. The percentage of CD44 positive Published 8. 11. 2019 lymphocytes peaked at 24 and 48 hours following infection with peptidoglycan of Staphylococcus aureus. The results show that during the experimental infection the expression of CD44 on lymphocytes is induced in early stage of mastitis (till 48 hours from the stimulation of inflammation). In the resolution of mastitis, CD44 expression is dramatically decreased. Regular article
Keywords: peptidoglycan, lymphocyte, CD44, Staphylococcus aureus, mammary gland, mastitis
INTRODUCTION peptidoglycan of S. aureus (Sigma, USA). The first sample of cells was obtained by PBS lavage of right-front quarter in 24 hours, right-rear quarter in 48 hours, Staphylococcus aureus is very important pathogen causing bovine mastitis left-front quarter in 72 hours, and left-rear quarter in 168 hours following (Petersson-Wolfe et al. 2010) with high economic loss for dairy farmers peptidoglycan challenge. Before experimental infection, the mammary glands (Heikkila et al., 2018). These bacteria contain peptidoglycan in the cell wall. were used for preparation of control samples through treatment by PBS which Peptidoglycan is also found in the cell wall of Gram-negative bacteria but in was previously described by Sladek et al., (2005). different structure than in Gram-positive bacteria (Vollmer et al., 2008). The lymphocytes obtained by lavages of mammary glands were processed to Peptidoglycan is able to induce inflammatory response of bovine mammary gland detect CD44 positive lymphocytes by flow cytometry (FACS Calibur Apparatus, (Furukava et al., 2018). Becton Dickinson, CA, USA) and subsequently by software WinMDI 2.8 CD44 is an adhesion receptor that is associated with different biological (Trotter, 2000) as in previous study. Mouse anti-ovine antibody CD44 BAG40A processes including inflammation. Increased amount of CD44 remains on the (VMRD Inc. Pullman, Washington, USA) and IgG3 surface of memory T lymphocytes mediate protection against re-infection (SouthernBiotech,Birmingham, Alabama, USA) were used as the primary and the (Baaten et al., 2010). This glycoprotein is multifunctional and it is involved in secondary antibody (Langrova et al., 2008). aggregation, migration, and activation of cells (Heider et al., 1993; Senbanjo For statistical analysis, there were used statistical software STATISTICA 8.0 and Chellaiah, 2017). Bacteria or their components or toxins are able to (StatSoft, Czech Republic). Arithmetic means and standard deviations were used influence expression of CD44 receptor on the cell membrane of different immune to describe CD44 positive lymphocytes. Statistically significant differences in the cells. There were analyzed the effect of muramyl dipeptide and proportion of CD44 positive lymphocytes were determined by paired t-test. lipopolysaccharide on the expression of CD44 on neutrophils (Langrova et al., 2008) and macrophages (Sladek and Rysanek, 2009). The effect of those RESULTS AND DISCUSSION bacterial components on CD44 expression was primarily evident during initial stage of the inflammation. High expression of CD44 in the resolution of The aim of this study was to evaluate the effect of peptidoglycan on the inflammation can be related to macrophages involvement in the processes of lymphocyte CD44 expression during experimentally induced inflammation. renewing of injured tissues (Sladek and Rysanek, 2009). In the early stage of In our experiments, the expression of CD44 receptor on the cell membrane of the inflammation, there was a gradual increase in the portion of CD44 positive lymphocytes was changed during the inflammatory response. Experimental neutrophils. The percentage of CD44 positive neutrophils is low in the start of infusion of peptidoglycan into the mammary gland led to increase of CD44 inflammation and therefore CD44 is not marker of activation of neutrophils positive lymphocytes with the maximum in 24 and 48 hours following infection (Langrova et al., 2008). of the mammary glands compare to the control (Figure 1). The goal of this study was to analyze whether expression of CD44 receptor on bovine mammary gland lymphocytes is modulated during an inflammatory response induced by peptidoglycan.
MATERIAL AND METHODS
Experimental design: Eight clinically healthy virgin heifers (Holstein x Bohemian Red Pied crossbred) aged 16 to 18 months were used for our experiments. All these animals were free of infection of the udder. Bacteriological examination of mammary gland lavages was executed by culturing on blood agar with aerobic incubation at 37 °C for 24 hours. For the experimental infection, there were used urethral catheter (AC5306CH06, Porges SA, France) to inject into the teat canal after disinfection of the teat orifice (Sladek et al., 2005; Slama et al., 2009). Each mammary gland of the udder was injected with 20 ml phosphate buffered saline (PBS) with 50 μg of
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SENBANJO, L.T., CHELLAIAH, M.A. 2017. CD44: A Multifunctional Cell 70 ** Surface Adhesion Receptor Is a Regulator of Progression and Metastasis of 60 PBS ** Cancer Cells. Frontiers in Cell and Developmental Biology, 5,18. http://dx.doi.org/10.3389/fcell.2017.00018 50 SLADEK, Z., RYSANEK, D. RYZNAROVA, H., FALDYNA, M. 2005. ** Neutrophil apoptosis during experimentally induced Staphylococcus aureus 40 mastitis. Veterinary Research, 36, 243-262. http://dx.doi.org/10.1051/vetres:2005023 30 SLADEK, Z., RYSANEK, D. 2009. Expression of macrophage CD44 receptor in 20 the course of experimental inflammatory response of bovine mammary gland induced by lipopolysaccharide and muramyl dipeptide. Research in Veterinary 10 Science, 86, 235-240. http://dx.doi.org/10.1016/j.rvsc.2008.07.016
SLAMA, P., SLADEK, Z., RYSANEK, D., LANGROVA, T. 2009. Effect of CD44+ lymphocytes CD44+ lymphocytes (%) 0 Staphylococcus aureus and Streptococcus uberis on apoptosis of bovine 24 h 48 h 72 h 168 h mammary gland lymphocytes. Research in Veterinary Sciences, 87, 233-238. http://dx.doi.org/10.10 16/j.rvsc.2009.03.005 Figure 1 CD44 positive lymphocytes (%) following stimulation by TROTTER, J.: WinMDI Version 2.8. 2000. http://facs.scripps.edu/. peptidoglycan in four timepoints (24, 48, 72, 168 hours). PBS – phosphate VOLLMER, W., BLANOT, D., DE PEDRO, M.A. 2008. Peptidoglycan structure buffered saline; ** P˂0.01 and architecture. FEMS Microbiology Reviews, 32, 149-167. http://dx.doi.org/10.1111/j.1574-6976.2007.00094.x These results suggest that peptidoglycan can induce expression of CD44 on lymphocytes in the initial phase of inflammation. Contrary to that, the previous study showed gradual increase of CD44 expression on neutrophils in experimental inflammation of the udder using lipopolysaccharide and muramyl dipeptide (Langrova et al., 2008). This comparison indicates the different role of this receptor on lymphocytes and neutrophils. In the population of macrophages, there is different expression between two subpopulation of macrophages (non- vacuolised x vacuolised) (Sladek and Rysanek, 2009). Vacuolised macrophages has similar trend of CD44 expression as neutrophils. Baaten et al. (2010) refer about the importance of this adhesion receptor to immune cell regulation and protection against intracellular bacteria. S. aureus is also able to internalize and survive within host immune cells (Hamza et al., 2013) and therefore we suggest that component of the cell wall of that bacteria as peptidoglycan is involved in the process of activation of immune cells, internalization of the bacteria into host cells and expression of CD44 of host cells including lymphocytes. The expression of CD44 on lymphocytes probably correlates with apoptosis of lymphocytes in the first stage of mastitis (unpublished preliminary results).
CONCLUSION
The results suggest that peptidoglycan can modulate expression of CD44 receptor on the cell membrane of lymphocytes during the process of inflammation of the mammary gland. We studied whole population of lymphocytes and therefor it is necessary to continue to investigate different subpopulation of lymphocytes. Different subpopulation of lymphocytes could have different expression of CD44.
Acknowledgments: The authors wish to express their thanks for financial support to the projects of IGA AF MENDELU No. AF-IGA-2018-tym002.
REFERENCES
BAATEN, B.J.G., LI, C.R., BRADLEY, L.M. 2010. Multifaceted regulation of T cells by CD44. Communicative and Integrative Biology, 3, 508-512. http://dx.doi.org/10.4161/cib.3.6.13495 FURUKAVA, M., YONEYAMA, H., HATA, E., IWANO, H., HIGUCHI, H., ANDO, T., SATO, M., HAYASHI, T., KIKU, Y., NAGASAWA Y., NIIMI, K., USAMI, K., ITO, K., WATANABE, K., NOCHI, K., ASO, H. 2018. Identification of a novel mechanism of action of bovine IgG antibodies specific for Staphylococcus aureus. Veterinary Research, 49, 22. http://dx.doi.org/10.1186/s13567-018-0517-y HAMZA, T., DIEZT, M., PHAM, D., CLOVIS, N., DANLEY, S., LI, B., 2013. Intra-cellular Staphylococcus aureus alone causes infection in vivo. European Cells and Materials, 25, 341-350. http://dx.doi.org/10.22203/eCM.v025a24 HEIDER, K.H., HOFMANN, M., HORS, E., VAN DEN BERG, F., PONTA, H., HERRLICH, P., PALS, S.T. 1993. A human homologue of the rat metastasis- associated variant of CD44 is expressed in colorectal carcinomas and adenomatous polyps. Journal of Cell Biology, 120, 227-233. http://dx.doi.org/10.1083/jcb.120.1.227 HEIKKILA, A.M., LISKI, E., PYORALA, S., TAPONEN, S. 2018. Pathogen- specific production losses in bovine mastitis. Journal of Dairy Science, 101, 9493-9504. http://dx.doi.org/10.3168/jds.2018-14824 LANGROVA, T., SLADEK, Z., RYSANEK, D. 2008. Expression of CD14 and CD44 on bovine polymorphonuclear leukocytes during resolution of mammary inflammatory response induced by muramyldipeptide and lipopolysaccharide. Veterinarni medicina, 53, 1-11. http://dx.doi.org/10.17221/1935-VETMED PETERSSON-WOLFE, C.S., MULLARKY, I.K., JONES, G.M. 2010. Staphylococcus aureus mastitis: cause, detection, and control. Virginia Cooperative Extension, 404-229.
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MICROFUNGI OF GRAPES FROM SMALL CARPATHIAN REGION IN SLOVAKIA
Soňa Felšöciová*1, Miroslava Kačániová1
Address(es): doc. Ing. Soňa Felšöciová,PhD. 1Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Microbiology, Tr. A. Hlinku 2, 949 76 Nitra, Slovak republic, phone number: + 4210376415813.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2019.9.special.478-482
ARTICLE INFO ABSTRACT
Received 10. 7. 2019 A total of 10 samples of grapes (bunches) without apparent fungal contamination were analyzed. The samples were collected during the Revised 10. 10. 2019 2017 and 2018 harvest from Suchá nad Parnou and Vrbové village in Small Carpathian region of Slovakia. Samples were sent to Accepted 15. 10. 2019 mycological laboratory, where they were stored at 4°C until their analysis. The objectives of this study were to gain more knowledge Published 8. 11. 2019 about mycobiota on grapes originating from Slovakia, with a focus on genus Penicillium and its ability to produce mycotoxins in in vitro conditions by thin layer chromatography method. For the isolation of fungi were used the direct plating technique on DRBC plates while surface sterilized grapes were used for endogenous mycobiota analysis. The plates were then incubated aerobically at 25 ±1 °C for one Regular article week in the dark. Overall, we isolated 818 strains belonging to 14 genera of filamentous microscopic fungi from surface mycobiota of grapes. The most frequent were genera Alternaria, Penicillium, Rhizopus, Sordaria, Aspergillus, Botrytis, Cladosporium and Epicoccum. The main occurring Penicillium species of the samples were P. expansum (60% Isolation frequency, 93% Relative density). A total of 388 isolates belonging to 12 genera were obtained from endogenous mycobiota. The most frequent and the most abundant genera were Alternaria, Cladosporium and Penicillium. From 3 different Penicillium species the most common was again P. expansum (30% IF, 92.5% RD). The selected isolates – P. citrinum, P. expansum, and P. chrysogenum were tested for their toxigenic ability. Out of 69 strains, 74% produced at least one mycotoxin as revealed by the method used here.
Keywords: grape, mycobiota, Penicillium sp., mycotoxins, TLC method
INTRODUCTION The most important mycotoxin in wine is ochratoxin A (OTA) (Amézqueta et al., 2009; Varga and Kozakiewicz, 2006). The role of OTA producing penicillia The incidence of filamentous fungi and toxin levels in grapes and wines varies in contaminating wine is of interest, although isolation from grapes is considered depending on the variety of grapes, the wine region, agricultural practices, infrequent. However, Mikušová et al. (2010) isolated OTA-producing P. weather conditions, the harvest and the winemaking process (Freire et al., 2017). verrucosum from Slovakian grapes. Rousseaux et al. (2014) reported OTA- The mycobiota frequently isolated from grapes includes the genera Aspergillus, producing Penicillium species from grapes in northern Italy and France, Penicillium, Mucor, Rhizopus, Alternaria, Cladosporium, Botrytis and Fusarium suggesting they could be involved in OTA contamination. The production of (Trinidad et al., 2015). OTA from Chinese penicillia (Zhang et al., 2016) requires confirmation Penicillium is a diverse fungal genus of ascomycetous fungi and contains more (Perrone et al., 2017). In general, isolating OTA fungi from grapes should not than 350 species (Visagie et al., 2014) playing various roles in natural exclude penicillia. The aim of our study was to monitor the mycobiota of grapes ecosystems, agriculture, and biotechnology. Species of Penicillium are ubiquitous and determine the characteristic mycotoxin production profiles of Penicillium soil fungi, preferring cool and moderate climates commonly present in organic strains (patulin, citrinin, and roquefortin C) isolated from grapes for wine materials. Most of the species are saprophytes and live mainly on organic production in the Small Carpathian region of Slovakia. biodegradable substances (Kirk et al., 2008). Penicilli have a large economic impact on human life. They have two sides – a good and beneficial one and a bad MATERIAL AND METHODS and economically destructive one. Many species of Penicillium are of proven importance because of their widespread occurrence and ability to produce a wide Study area range of bioactive metabolites, including antibacterial, antifungal, immune suppressants, cholesterol-lowering agents etc. (Petit et al., 2009). Some species Grape samples were collected in moderate region of Slovakia, as it can be produce toxins and may render food inedible or even dangerous (Visagie et al., common in the Middle Europe areas, from Terra Parna winery, Suchá nad Parnou 2013). Penicillum expansum can cause rot in grapes, but does not usually attack and from Sabo winery, Vrbové in Small Carpathian wine region during the years grapes before harvest. Aside from losses in fruit, this species is regarded as the 2017 and 2018 (Table 1). Slovak republic has 6 distinct wine-growing zones (the major producer of patulin, although this species produces many other toxic Small Carpathians, the Southern Slovak, the Nitra, the Central Slovak, the metabolites such as citrinin, roquefortine C or chaetoglobosins among others Eastern Slovak and the Tokaj wine regions). They spread from the west to the (Andersen et al., 2004). east of the country along its southern and south-western borders. The largest in Mycotoxins are secondary metabolities produced by filamentous fungi either pre- size and the most important over the centuries has been the Small Carpathian area or postharvest and which can contaminate agricultural food and feed products (4260 ha of vineyards) spreads in the western of Slovakia (ÚKSÚP, 2019a). The and have detrimental effects on human and animal health. Much of the research Small Carpathian wine region is divided into 12 subregions. The subregion is the on mycotoxins, including recent research on the effects of climate change on area with the same soil and climate conditions. Wine-growing zones are defined mycotoxins, has focused on Aspergillus, Fusarium, and Penicillium species, as as geographic regions with distinct climatic conditions for grape cultivation they are the major mycotoxin-producing fungi in field crops and stored products (ÚKSÚP, 2019b). The Small Carpathian wine-growing region has medium in the world (Paris et al., 2015). climates and abundant moisture.
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Table 1 Wine grape varieties used in the study from the Small Carpathian region No of samVillage Subregion Grape variety Date of harvest Date of analyses 1. Suchá nad Parnou Orešanský Alibernet 27.09.2017 28.09.2017 2. Suchá nad Parnou Orešanský Blue Portugal 27.09.2017 28.09.2017 3. Suchá nad Parnou Orešanský Rheinriesling 27.09.2017 28.09.2017 4. Vrbové Vrbovský Pálava 28.09.2017 29.09.2017 5. Vrbové Vrbovský Dornfelder 28.09.2017 29.09.2017 6. Vrbové Vrbovský Alibernet 04.09.2018 05.09.2018 7. Vrbové Vrbovský Cabernet Sauvignon 04.09.2018 05.09.2018 8. Vrbové Vrbovský Dornfelder 04.09.2018 05.09.2018 9. Vrbové Vrbovský Pálava 04.09.2018 05.09.2018 10. Vrbové Vrbovský Chardonnay 04.09.2018 05.09.2018
Sampling once. The relative density (%) is defined as the percentage of isolates of the species or genus, occurring in the analyzed sample (Guatam et al., 2009). These Two samples of red grapes (Alibernet, Blue Portugal) and one sample of white values were calculated according to González et al. (1999) as follows: grape (Rheinriesling) were collected from Suchá nad Parnou and one sample of IF (%) = (ns / N) x 100 ; RD (%) = (ni / Ni) x 100 white grape (Pálava) and one sample of red grape (Dornfelder) were collected ns – number of samples with a species or genus; N – total number of samples; ni from Vrbové in the final stages of maturation of the berries (harvest season), at – number of isolates of a species or genus; Ni – total number of isolated fungi. the end of September, 2017. Three samples of red grapes (Alibernet, Cabernet Sauvignon and Dornfelder) and Toxinogenity analysis two samples of white grapes (Pálava and Chardonnay) were collected from Vrbové at the beginning of September, 2018. The sample comprised 3 bunches of Toxinogenity of selected isolates was screened in in vitro conditions by means of grapes collected across two diagonal transects. Grape samples were put directly thin layer chromatography (TLC) according to Samson et al. (2002b), modified each into a sterile plastic bag. Samples were brought into the laboratory and kept by Labuda and Tančinová (2006). Extracellular metabolites – citrinin and at 5 °C till fungal analysis. patulin were carried out on YES agar and intracellular roquefortin C on CYA agar. A few plugs of mycelium were removed from different points of the colony Mycological analysis of grapes in an Eppendorf tube with 500 µL of chloroform:methanol – 2:1 (Reachem, Slovak Republic). The content of the tubes was stirred for 5 min by Vortex Genie A total of 50 berries (7 - 8 berries per bunch) from each sample were plated in ® 2 (MO BIO Laboratories, Inc. – Carlsbad, CA, USA). The volume 30 µL of Dichloran Rose Bengal Chloramphenicol agar medium (DRBC) and incubated at liquid phase of extracts along with 10 µL of standards (Sigma, Germany) was 25±1 °C in the dark for one week. In this way was determined an exogenous applied on TLC plate (Alugram ® SIL G, Macherey – Nagel, Germany). The mycobiota. Another 50 grapes were surface-disinfected in 1% NaClO for 1 min plate was put into TEF solvent (toluene:ethyl acetate:formic acid – 5:4:1, toluene according methods of Magnoli et al. (2003) and 3 times rinsed by submersion in – Mikrochem, Slovak Republic; ethyl acetate and formic acid – Slavus, Slovak sterile distilled water (total amount 1L) to remove incidental surface Republic). After elution the plate was air-dried. Identification of the metabolites contaminants, dried, plated in the same medium and incubated at 25 °C in the was done by comparison with metabolite standards. Roquefortin C was visible dark for 7 days. In this way was determined an endogenous mycobiota. The after spraying with Ce(SO4)2 x 4 H2O as an orange spot. Patulin detection was identification of fungal taxa was based on macroscopic and microscopic features, achieved by spraying with 0.5% methylbenzothiazolone hydrochloride (MBTH) with guidelines by Pitt and Hocking (2009). Penicillium strains were isolated (Merck, Germany) in methanol and heating at 130 °C for 8 min and then detected and cultivated on MEA (Malt extract agar) (Samson et al., 2010), CYA (Czapek as a yellow-orange spot under visible light. Citrinin was detected directly as an yeast agar) (Samson et al., 2010), Creatine-Sucrose agar (CREA) (Samson et al., intense yellow‐green streak under ultraviolet light (365 nm). 2010) and Yeast Extract agar (YES) (Samson et al., 2010). From the pure cultures, genus Penicillium was identified to species level based on macroscopic RESULTS AND DISCUSSION and microscopic characteristics according to the manuals of Pitt and Hocking (2009), Samson and Frisvad (2004) and Samson et al. (2002a, 2010). The filamentous fungi identified in 4 white and 6 red grape varieties from surface mycobiota during the years 2017 and 2018 are indicated in table 2. A total of 818 Results evaluation strains belonging to 14 genera were identified. From varieties Alibernet (1), Blue Portugal (2) and Rheinriesling (3) from Suchá nad Parnou in 2017, the highest The obtained results were evaluated and expressed according to isolation number of isolates (from 89 to 102) with 8 or 9 genera were found. All samples frequency (IF) and relative density (RD). The isolation frequency (%) is defined were colonised by genera Alternaria, Aspergillus, Cladosporium, Epicoccum and as the percentage of samples within which the species or genus occurred at least Penicillium, concretely P. expansum.
Table 2 Fungi identified in Slovak wine grapes from exogenous mycobiota from 2017 to 2018 by the direct plating method Fungal taxa 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Alternaria 36 16 7 35 9 93 90 70 54 73 Arthrinium 3 - 2 2 ------Aspergillus 3 4 1 1 - 1 1 - - - Aureobasidium ------1 - Botrytis 2 6 - 1 1 - - 2 - 12 Cladosporium 19 13 31 2 - - - - - 2 Epicoccum 8 12 20 - - 1 2 - - - Eurotium 6 ------Fusarium 6 - - 2 2 - - - - - Mucor - 3 - 8 ------Penicillium 19 29 29 2 4 1 3 - - 1 P. aurantiogriseum - - - - 1 - - - - - P. brevicompactum - - - - 1 - - - - - P. citrinum ------3 - - - P. expansum 19 29 29 2 2 - - - - 1 P. glabrum - - - - - 1 - - - - Rhizopus - 4 7 5 - 4 4 7 5 2 Sordaria - 2 4 5 - 9 4 1 2 Trichoderma - - - 1 - 1 ∑ 102 89 101 64 16 109 104 79 61 93 Legend: 1, 6 - Alibernet, 2 - Blue Portugal, 3 – Rheinriesling, 4, 9 – Pálava, 5, 8 – Dornfelder, 7 - Cabernet Sauvignon, 10 - Chardonnay
The lower number of isolates (64) were isolated from the white variety Pálava (4) grape varieties Alibernet (6), Cabernet Sauvignon (7) and the white variety from Vrbové in 2017 but on the other hand with the highest number of genera Chardonnay (10) reached approximately the same quantitative and qualitative (11). The least isolates and genera of filamentous microscopic fungi were isolated representation of micromycetes from Vrbové with the dominance of the genus from the red grape variety Dornfelder (5) from Vrbové in 2017. In 2018, the red Alternaria. The lowest quantitative and qualitative representation of
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micromycetes was isolated from the Pálava (9) and Dornfelder (8) varieties again Epicoccum and Rhizopus (5%, each) of all the fungi found. The remaining genera with the dominance of the genus Alternaria. were detected in less than 5% of all the isolates. From 5 different Penicillium Alternaria spp. are ubiquitous plant pathogens that may invade fruit (Asam et al., species the most frequent and most abundant was P. expansum. Alternaria is the 2010). Fungal contamination of grapes could occur before or during harvest and main component of wine grape mycobiota from different winemaking regions processing (Prendes et al., 2016). Globally, Alternaria has been isolated from worldwide (Magnoli et al., 2003; Rousseaux et al., 2014; Prendes et al., 2015; grapes, musts, wines, and raisins. Alternaria cause rot bunch of damaged berries, Tančinová et al., 2015). Alternaria growth on grapes has been reported in scrape and pedicels (Kakalíková et al., 2009; Steel et al., 2013). The exposure to several countries such as Argentina, Brazil, Spain, Italy, Portugal, USA, Alternaria toxins has been linked to a great variety of adverse effects to both Slovakia, Hungary and Czech Republic. The incidence of Alternaria in grapes human and animal health (Dall'Asta et al., 2014). There are not specific has been reported in different percentages with respect to total mycobiota: 5–23% international regulations of Alternaria toxins in food (Trinidad et al., 2015). in USA, 25% in Italy, 80% in Argentina, 3–18%, 24%, 17% in different studies Without surface disinfection, a total of 388 strains belonging to 12 genera were in Portugal, 75%, 3–58%, 13% in Spain, and A. alternata and A. tenuissima were identified during the years 2017 and 2018, while Alternaria colonised all the reported in 16–19% in Slovakia (Prendes et al., 2016). samples examined (Table 3). In 2018, we isolated a lower number of genera from From the twelve vineyards in the Small Carpathian area were collected 14 our tested samples (from 2 to 4) than in 2017 (from 3 to 8). Species Penicillium samples of wine grapes (white 6, red 8) during harvesting 2011, 2012 and 2013 expansum were dominated from the samples of Terra Parna (1-3) in 2017. In (Felšöciová et al., 2015c). In these samples were identified 22 genera and 79% of 2018, P. expansum was not detected. The occurrence of Penicillium spp. in other samples were colonized by the genus Aspergillus. During the survey, 37 isolates samples was generally low. The highest number of isolates was detected in the belonging to 7 Aspergillus species (A. clavatus, A. flavus, A. section Nigri, A. genus Alternaria (53) in the grape variety Pálava (4), however in most samples it ostianus, A. parasiticus, A. versicolor and A. westerdijkiae) were isolated and was dominated. identified from exogenous mycobiota. The main occurring aspergillus species of the samples were A. section Nigri (64%). Black aspergilli have been reported as Table 3 Fungi identified in Slovak wine grapes from endogenous mycobiota the predominant fungi from Spanish wine and liqueur grapes at harvesting time, from 2017 to 2018 by the direct plating method constituting from 88.7% to 98.5% of the total Aspergillus isolates (Bau et al., Fungal taxa 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 2005, 2006; Gómez et al., 2006). In our research Aspergillus isolates were found Alternaria 7 15 8 53 10 37 41 45 11 25 in grape samples but with low relative density (1%). Arthrinium 6 2 3 1 ------By the endogenous (surface-disinfected) plating method were identified 12 Aspergillus - - - - 1 - - - - - different genera from the 388 fungal strains (Table 4): Alternaria, Arthrinium, Botrytis - 1 2 9 - - - - - 1 Aspergillus, Botrytis, Cladosporium, Epicoccum, Fusarium, Penicillium, Cladosporium 9 11 18 - 1 2 2 5 2 - Rhizopus, Sordaria, Talaromyces and Trichoderma. Data in the same table Epicoccum 3 1 - - - - 1 - - - showed, that Alternaria was the most frequently occurring genus (100%), Fusarium - 1 ------followed by Cladosporium (80%) and Penicillium (50%). The 3 most abundant Rhizopus - - - 1 - 3 1 - genera found by descending order and with the highest relative density were Penicillium 10 2 25 - - 1 4 - - - Alternaria (65%), Cladosporium (13%) and Penicillium (11%). From 3 different P. citrinum ------3 - - - Penicillium species the most common in grape samples was again P. expansum. P. expansum 10 2 25 ------Felšöciová et al. (2015a) investigated an endogenous mycobiota of grapes from P. chrysogenum - - - - - 1 1 - - - Eastern wine region of Slovakia. A total of 582 isolates were obtained that Sordaria 1 1 - 1 ------belonged to 10 genera: Alternaria, Aspergillus, Botrytis, Cladosporium, Talaromyces - - - 2 ------Epicoccum, Fusarium, Mucor, Penicillium, Rhizopus, and Trichoderma. The Trichoderma - - - 2 ------most frequent were 4 genera Alternaria, Aspergillus, Botrytis and Penicillium ∑ 36 34 56 69 12 40 48 53 14 26 with 100% frequention. The relative density of genera Alternaria, Cladosporium Legend: 1, 6 - Alibernet, 2 - Blue Portugal, 3 – Rheinriesling, 4, 9 – Pálava, 5, 8 – and Penicillium, were the highest (42%, 19.6% and 15.8%, respectively) as in our Dornfelder, 7 - Cabernet Sauvignon, 10 - Chardonnay results. Frequency of isolation of Penicillium spp. in Spanish vineyards was low (Bau et al., 2005, 2006; Bellí et al., 2006; Gómez et al., 2006). In other surveys Data in table 4 show that, fourteen fungal genera namely Alternaria, Arthrinium, made in European vineyards with different climatic conditions, Penicillium was Aspergillus, Aureobasidium, Botrytis, Cladosporium, Epicoccum, Eurotium, the predominant genus (Abrunhosa et al., 2001; Serra et al., 2005, 2006). Fusarium, Mucor, Penicillium, Rhizopus, Sordaria and Trichoderma were Penicillium expansum, the responsible agent of blue mould on fruits, has been identified from fresh grape samples. Alternaria was the most frequently reported in grapes in different studies (Abrunhosa et al., 2001; Serra et al., occurring genus (100%), followed by Penicillium, Rhizopus (80%, each), 2005, 2006; Felšöciová and Kačániová, 2019). Sordaria (70%), Aspergillus, Botrytis (60%, each), Cladosporium and Epicoccum (50%, each). The most abundant genus from exogenous mycobiota was Alternaria (59%), followed by Penicillium (11%), Cladosporium (8%),
Table 4 The occurrence, isolation frequency and relative density of filamentous microscopic fungi in exogenous and endogenous mycobiota of grapes (n=10) harvested in Small Carpathian wine region Fungal taxa No of isolates Isolation Relative No of isolates Isolation Relative exo frequency (%) density (%) endo frequency (%) density (%) Alternaria 483 100 59 252 100 65 Arthrinium 7 30 0.9 12 40 3 Aspergillus 11 60 1 1 10 0.3 Aureobasidium 1 10 0.1 - - - Botrytis 24 60 3 13 40 3 Cladosporium 67 50 8 50 80 13 Epicoccum 43 50 5 5 30 1 Eurotium 6 10 0.7 - - - Fusarium 10 30 1 1 10 0.3 Mucor 11 20 1 - - - Penicillium 88 80 11 42 50 11 P. aurantiogriseum 1 10 - - - - P. brevicompactum 1 10 - - - - P. citrinum 3 10 - 3 10 - P. expansum 82 60 - 37 30 - P. glabrum 1 10 - - - - P. chrysogenum - - - 2 20 0.5 Rhizopus 38 80 5 5 30 1 Sordaria 27 70 3 3 30 0.8 Talaromyces - - - 2 10 0.5 Trichoderma 2 20 0.2 2 10 0.5 Total 818 388 Legend: No - number of isolated micromycetes from exogenous mycobiota/ from endogenous mycobiota
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The toxigenic profile of the 69 Penicillium isolates representing P. citrinum, P. from the region. The present work showed the toxigenic potential of the expansum and P. chrysogenum from the Slovak grapes is shown in Table 5. Penicillium species isolated from wine grapes, which indicates the potential risk Patulin, citrinin and roquefortin C production was tested by thin layer of mycotoxin accumulation in the fruits. Extracts of fungal strains were analysed chromatography method in in vitro condition. Almost all the isolates were able to for patulin, citrinin and roquefortin C by thin-layer chromatography. Patulin was produce mycotoxins. From the exogenous mycobiota 44 strains were tested, produced by 73% (45/62), citrinin was produced by 100% (68/68), roquefortin C namely P. citrinum and P. expansum. Positive toxigenity was detected for citrinin production was observed in 98% (62/63). Our results showed that the Penicillium by P. citrinum and P. expansum, for roquefortin C by P. expansum and 35 strains species commonly isolated from grapes are a source of the mycotoxins patulin, out of 41 screened produced patulin. From the endogenous mycobiota 25 strains citrinin and roquefortin C. Altogether, these results indicate that the higher were tested, namely P. citrinum, P. expansum and P. chrysogenum. Positive presence of potentially toxigenic genera Alternaria and Penicillium in wine toxigenity was detected for citrinin and roquefortin C by P. expansum (100%, grapes could represent a risk in the health of wine consumers. each) and 10 strains out of 21 produced patulin. All tested strains of P. citrinum were positive for citrinin and one strain of P. chrysogenum did not produce Acknowledgments: This work has been supported by grants of the European roquefortin C. Out of 69 strains, 74% produced at least one mycotoxin as Community of project No. 26220220180: Building Research Centre revealed by the method used here. “AgroBioTech” and of Slovak Research and Development Agency VEGA No. 1/0411/17 and KEGA 015SPU-4/2018. Table 5 Toxinogenity of selected Penicillium strains, isolated from exogenous and endogenous mycobiota of wine grapes REFERENCES Penicillium species patulin citrinin roquefortin C Toxinogenity of exogenous mycobiota ABRUNHOSA, L., PATERSON, R. R. M., KOZAKIEWICZ, Z., LIMA, N., P. citrinum nt 3*/3** nt VENÂNCIO, A. 2001. Mycotoxin production from fungi isolated from grapes. P. expansum 35/41 41/41 41/41 Lett Appl Microbiol, 32(4), 240–242. https://doi.org/10.1046/j.1472- Toxinogenity of endogenous mycobiota 765x.2001.00897.x P. citrinum nt 3/3 nt AMÉZQUETA, S., GONZÁLEZ-PEÑAS, E., MURILLO-ARBIZU, M., LÓPEZ P. expansum 10/21 21/21 21/21 DE CERAIN, A. 2009. Ochratoxin A decontamination: A review. Food Control, P. chrysogenum nt nt 0/1 20(4), 326-333. https://doi.org/10.1016/j.foodcont.2008.05.017 Legend: * - number of isolates with ability to produce mycotoxin, ** - number of tested ANDERSEN, B., SMEDSGAARD, J., FRISVAD, J. C. 2004. Penicillium isolates, nt – not tested expansum: consistent production of patulin, chaetoglobosins and other secondary metabolites in culture and their natural occurrence in fruit products. J. Agric. Patulin is a tetraketide lactone produced by a variety of moulds, in particular, Food Chem., 52(8), 2421-8. https://doi.org/10.1021/jf035406k Aspergillus, Penicillium, and Byssochlamys species (Puel et al., 2010). The main ASAM, S., KONITZER, K., RYCHLIK, M. 2010. Precise determination of the producer of patulin is Penicillium expansum, which contaminates mainly apple Alternaria mycotoxins alternariol and alternariol monomethyl ether in cereal, and apple products, and also other fruits like cherries, blueberries, plums, fruit and vegetable products using stable isotope dilution assays. Mycotoxin bananas, strawberries, and grapes, processed grape juice and fermenting wine Research, 27(1), 23-28. https://doi.org/10.1007/s12550-010-0071-6 (Abrunhosa et al., 2001). Penicillium expansum is found frequently in botrytized BAU, M., BRAGULAT, M., ABARCA, M., MINGUEZ, S., CABAÑES, F. grapes (Russell et al., 2018). The occurrence of patulin in wine is low because it 2005. Ochratoxigenic species from Spanish wine grapes. Int J Food Microbiol., is well-known to be degraded partially by the fermentation process (Moss and 98(2), 125–130. https://doi.org/10.1016/j.ijfoodmicro.2004.05.015 Long, 2002). Patulin is thermal resistant, causes gastrointestinal problems, BAU, M., BRAGULAT, M. R., ABARCA, M. L., MINGUEZ, S., CABAÑES, including ulceration, distension and bleeding, skin rashes, and is known to be F. J. 2006. Ochratoxin A producing fungi from Spanish vineyards. In HOCKING, mutagenic, immunotoxic, and neurotoxic mycotoxin (Medina et al., 2005). A. D., PITT, J. I., SAMSON, R. A., THRANE, U. Advances in Food Mycology. Patulin is known to be antibacterial, cytotoxic and perhaps even anticarcinogenic Advances in Experimental Medicine and Biology. New York : Springer, 173– (Kumar et al., 2018). It is classified in group 3 as not classifiable as to its 179. https://doi.org/10.1007/0-387-28391-9_10 carcinogenicity to human by IARC (Varga et al., 2015). Out of 62 screened BELLÍ, N., BAU, M., MARÍN, S., ABARCA, M., RAMOS, A., BRAGULAT, strains in our research, 73% produced patulin. The predominant Penicillium M. 2006. Mycobiota and ochratoxin A producing fungi from Spanish wine species was not only Penicillium expansum as in our study, but also Penicillium grapes. Int J Food Microbiol., 111, 40–45. chrysogenum (100 %) from Tokaj wine region (Felšöciová et al., 2015b). https://doi.org/10.1016/j.ijfoodmicro.2006.03.011 Penicillium expansum produced patulin (15 out of 18 strains screened), citrinin COLE, R. J., COX, R. H. 1981. Handbook of Toxic Fungal Metabolites. New (13 out of 18 strains screened) and all of them produced roquefortin C. York : Academic Press, 937 p. ISBN : 0121797600. Citrinin is a pentaketide derivate produced principally by species of the genera https://doi.org/10.1016/c2009-0-03073-6 Penicillium and Aspergillus (A. terreus, A. niveus). Penicillium citrinum, the DALL'ASTA, C., CIRLINI, M., FALAVIGNA, C. 2014. Mycotoxins from main producer of citrinin, has been isolated from grapes, P. expansum could Alternaria: toxicological implications. In FISHBEIN, C., HEILMAN, J. M. ed. produce this mycotoxin, too. Citrinin, a hepato-nephrotoxic compound, is not Advances in Molecular Toxicology. Amsterdam : Elsevier B.V., 107– 121. degraded during alcoholic fermentation and may be present in very small https://doi.org/10.1016/b978-0-444-63406-1.00003-9 amounts in wine (Samson et al., 2011). All 68 tested strains were positive on FELŠÖCIOVÁ S., KAČÁNIOVÁ, M. 2019. Mycobiota in traditional grapes and citrinin in our study. The metabolite citrinin, a characteristic yellow-lemon grape for ice wine production cultivated in Slovakia. Scientific Papers: Animal pigment, was also produced by all strains of P. expansum from grapes and must Science and Biotechnologies, 52(1), 70-77. under laboratory conditions in Small Carpathian wine growing region of Slovakia FELŠÖCIOVÁ, S., MAŠKOVÁ, Z., KAČÁNIOVÁ, M. 2018. Fungal diversity in the year 2017 (Felšöciová et al., 2018). in the grapes-to-wines chain with emphasis on Penicillium species. Roquefortin C is the most widespread mycotoxin produced by various fungi, Potravinarstvo, 12(1), 379-386. https://doi.org/10.5219/882 particularly species from the Penicillium genus, and is known as an important FELŠÖCIOVÁ, S., RYBÁRIK, Ľ., TANČINOVÁ, D., MAŠKOVÁ, Z., neurotoxic (paralytic) secondary metabolite (Cole and Cox, 1981). Out of 63 KAČÁNIOVÁ, M. 2014. Penicillium strains isolated from grapes grown in the tested strains, 98% produced roquefortin C. A total of 10 wine producing grapes Central Slovak wine region. Journal of Microbiology, Biotechnology and Food were collected from the Central Slovak region in 2011 and 2012, which involved Sciences, 3(1), 210-214. 7 vineyards (Felšöciová et al., 2014). Twenty five potentially toxigenic species FELŠÖCIOVÁ, S., RYBÁRIK, Ľ., TANČINOVÁ, D., MAŠKOVÁ, Z., were tested for their toxigenic ability on roquefortin C, namely P. crustosum, P. KAČÁNIOVÁ, M. 2015a. Microfungi and mycotoxins of grapes from Eastern expansum, P. griseofulvum, P. chrysogenum and P. hordei. Positive toxinogenity Slovak wine region. Journal of Microbiology, Biotechnology and Food Sciences, was also high, 92 % strains produced mentioned mycotoxin. Felšöciová et al. 4(1), 12-15. https://doi.org/10.15414/jmbfs.2015.4.special1.12-15 (2015c) also tested 68 Penicillium strains on roquefortine C from Small FELŠÖCIOVÁ, S., RYBÁRIK, Ľ., TANČINOVÁ, D., MAŠKOVÁ, Z., Carpathian winemaking region from exogenous mycobiota of grapes which all KAČÁNIOVÁ, M. 2015b. Microfungi and mycotoxins of grapes from Tokaj were positive. wine region. Journal of Microbiology, Biotechnology and Food Sciences, 4(1), 16-18. https://doi.org/10.15414/jmbfs.2015.4.special1.16-18 CONCLUSION FELŠÖCIOVÁ, S., TANČINOVÁ, D., RYBÁRIK, Ľ., MAŠKOVÁ, Z., KAČÁNIOVÁ, M. 2015c. Mycobiota of Slovak wine grapes with emphasis on From 10 samples of wine grapes from exogenous and endogenous mycobiota Aspergillus and Penicillium species in the Small carpathian area. Potravinarstvo, were isolated 1206 strains belonging to 15 genera. Alternaria, Cladosporium and 9(1), 501-508. https://doi.org/10.5219/529 Penicillium were the most common genera within the surface and endogenous FREIRE, L., PASSAMANI, F. R. F., THOMAS, A. B., NASSUR, R. D. M. R., colonisation. The most abundant genus was Alternaria almost in all samples. SILVA, L. M., PASCHOAL, F. N., PEREIRA, G. E., PRADO, G., BATISTA, L. During the survey six Penicillium species were isolated: P. aurantiogriseum, P. R. 2017. Influence of physical and chemical characteristics of wine grapes on the brevicompactum, P. citrinum, P. expansum, P. glabrum and P. chrysogenum. Of incidence of Penicillium and Aspergillus fungi in grapes and ochratoxin A in the 130 fungi of the Penicillium genus isolated from grapes, 119 (91%) were P. wines. International journal of food microbiology, 241, 181-190. expansum, which shows a higher risk of the toxin in grapes and their derivatives https://doi.org/10.1016/j.ijfoodmicro.2016.10.027
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