ISOLATION OF LACTIC ACID BACTERIA FROM TRADITIONAL MOROCCAN PRODUCTS AND EVALUATION OF THEIR ANTIFUNGAL ACTIVITY ON GROWTH AND OCHRATOXIN A PRODUCTION BY ASPERGILLUS CARBONARIUS AND A. NIGER

and A. carbonarius, respectively; however, an 89.38% reduction for A. niger and 19.35 % A. carbonarius was achieved for the Leuconostoc mesenteroides ssp . mesenteroides (I) strain. These results revealed strain-dependent antifungal activity. Our study indicates that the use of these lactic acid bacteria can be applied as a biocontrol of fungal growth and mycotoxins production.


INTRODUCTION
Deterioration of food by filamentous fungi is a major preoccupation for farmers and the food industry due to their capacity to grow under harsh environmental conditions; they limit the shelf life of food products and cause a 5-10% loss in world crops (Pitt & Hocking, 2009).Molds produce compounds that have a negative impact on organoleptic properties, such as the molecules responsible for flavor defects, alteration of texture, or discoloration of food products (Muhialdin et al., 2013).In addition, some spoilage fungal species such as Aspergillus sp. can produce mycotoxins, including ochratoxin A (OTA).OTA is among the most studied mycotoxins because of its damaging effects on human and animal health, inducing nephrotoxic, carcinogenic possible effects, teratogenic, immunotoxic, and neurotoxic reactions (IARC, 1993; Pfohl-Leszkowicz & Manderville, 2007).In grapes and their derivatives, the most important species of Aspergillus sp.OTA producers are Aspergillus niger aggregate and Aspergillus carbonarius (Cabanes & Bragulat, 2018; Nielsen et al., 2009;Selouane et al., 2009;Leong, 2005).Several chemical and physical preservation methods are used to inhibit mold growth and mycotoxins production in food products (Farkas, 2001;Legan, 1993); however, the overuse of these chemicals is increasingly discouraged for economic reasons and because of growing concerns with environmental and food safety issues (Wagacha & Muthomi, 2008).As such, research for new methods to reduce the risk of fungal contamination is necessary, and the potential exploitation of the antifungal properties of lactic acid bacteria (LAB) for food bio-preservation has attracted the attention of several researchers (Muhialdin et al., 2013).LAB can be found naturally in different foodborne products (Carr et al., 2002;Gajbhiye & Kapadnis, 2016).They have been widely used in traditional fermentation processes since they are Generally Recognized As Safe (GRAS) and have important technological properties, such as inhibiting the growth of fungi by producing antifungal compounds (Gajbhiye and Kapadnis, 2016).In this work, the antifungal activity of lactic acid bacteria was evaluated.Lactic acid bacteria were isolated from different Moroccan biotopes, and the screening of strains with antifungal activity against A. carbonarius and A. niger was investigated.The effect of the selected isolate bacteria on OTA production by the Aspergillus sp. was also studied.

Isolation of lactic acid bacteria
The strains of lactic bacteria were isolated from different Moroccan biotopes and food products such as milk and its traditional derivatives (Jben, Lben, and Smen) as well as brine from fermented olives and fermented capers.Serial decimal dilutions were made in a physiological water solution, then 0.1 mL from dilutions Different agricultural products, including grapes, are susceptible to contamination and damage by several microorganisms, one being fungi and their mycotoxins such as ochratoxin A. These alterations are a source of food waste and lead to significant economic losses.Thus, new alternatives have been sought in parallel with existing methods.In this work, after isolation, identification by 16S rDNA sequencing and differentiation by Pulsed-Field Gel Electrophoresis (PFGE) of four lactic acid bacteria from different traditional Moroccan food products, antifungal activity was studied against Aspergillus carbonarius and A. niger isolated from Moroccan grapes.Their effect on ochratoxin A production was also assessed.A strain of Lactoccocus lactis ssp.lactis (F) and three strains of Leuconostoc mesenteroides ssp.mesenteroides (I, C, and D) selected from 16 isolated strains were tested.Their antifungal activities against Aspergillus spp.were quite significant, with growth inhibition rates ranging from 28.69% to 53.31%.Likewise, ochratoxin A production by Aspergillus strains was significantly reduced.Indeed, in the presence of the Lactoccocus lactis ssp.lactis (F) strain, this reduction was about 63.90% and 65.12% for A. niger and A. carbonarius, respectively; however, an 89.38% reduction for A. niger and 19.35 % A. carbonarius was achieved for the Leuconostoc mesenteroides ssp.mesenteroides (I) strain.These results revealed strain-dependent antifungal activity.Our study indicates that the use of these lactic acid bacteria can be applied as a biocontrol of fungal growth and mycotoxins production.
of 10 -4 , 10 -5 , and 10 -6 were inoculated on the surface of Petri plates containing MRS (De Man, Rogosa and Sharpe, 1960) agar medium.The plates were incubated at 35 °C for 24 to 48 h.The colonies were selected, purified, and preliminarily identified.

Screening of the antifungal activity of lactic acid bacteria
The antifungal activity of lactic acid bacteria was tested using the double layer method described by Cheong et al. (2014) with some modifications.The bacteria were inoculated on two streaks on Petri plates.Two culture media, MRS (Tryptic digest of casein, 10 g ; beef extract, 8 g ; yeast extract, 4 g ; glucose, 20 g ; Tween 80, 1 g ; di-potassium hydrogen orthophosphate, 2 g ; magnesium sulphate 7H20, 0.2 g ; manganese (II) sulphate 4H20, 0.05 g ; ammonium citrate, 2 g ; sodium acetate 3H20, 5 g ; agar, 15g ; distilled water, 1 litre) and M17 (Peptone, 5.0 g ; soya peptone, 5.0 g ; yeast extract, 2.5 g ; beef extract, 5.0 g ; lactose, 5.0 g ; sodium glycerophosphate, 19.0 g ; magnesium sulfate, 0.25 g ; ascorbic acid, 0.5 g ; agar, 15 g ; distilled water, 1 liter; Terzaghi & sandine, 1975) were used to compare the influence of culture media on antifungal activity.The plates were subsequently incubated at different temperatures (25, 30 and 37 °C) and for different times (18, 24 and 48 h).After incubation, the Petri plates were covered by 10 mL of CYA containing 10 5 spores/mL and incubated at 25 °C for 72 h.Control plates of MRS agar medium were used to inoculate the two strains of Aspergillus sp. to eliminate the effect of sodium acetate, as an antifungal agent.The following scale was used for the selection of lactic acid bacteria strains with potential antifungal activity: (-), no inhibition (the bacteria were totally covered by the fungal strain); (+), weak inhibition (the bacteria were not covered by the fungi); (++), inhibition (formation of a small zones inhibition around the bacterial culture); and (+++), strong inhibition (a large zones of inhibition has been observed around the bacterial culture).The antifungal activity tests were conducted in triplicate.

Identification of selected lactic acid bacteria by 16S rDNA sequencing
The selected lactic acid isolates were inoculated in MRS broth medium and then incubated at 30 °C for 24 h.For each isolate studied, the DNA was extracted using a Qiagen Blood and Tissue kit (Qiagen, Germany).The concentration of extracted DNA was then measured using Nanodrop® (Thermo Fisher Scientific, USA).First, the extracted DNA was amplified by polymerase chain reaction (PCR) using universal primers W001 and W002 (Godon et al., 1997).Then, it was sequenced according to the Sanger method (Sanger & Coulson, 1975).The sequencing results were analyzed using Invitrogen-Vector NTI software, and the sequence obtained was compared with the sequences in the NCBI (Nucleotide Blast) database (https://www.ncbi.nlm.nih.gov/).Finally, the strains were subjected to species-specific PCR in order to differentiate the subspecies.

Differentiation of lactic acid bacteria strains selected by Pulsed-Field Gel Electrophoresis (PFGE)
Clonal differentiation of the selected strains by PFGE was conducted.This technique is largely used for the analysis of genomic diversity of lactic acid bacteria (Adesulu-Dahunsi et al., 2017).For this, the strains were cultivated in MRS medium at 30 °C for 24 h and analyzed.After migration, profiles were analyzed using Bionumerics software (AppliedMath, Belgium).The gel was first normalized using a molecular weight marker.The results were compiled in the form of a dendogram for comparing the samples with each other by considering the number of bands and their position.Two samples with the same number of bands at the same positions were considered identical (duplicates of the same strain).

Determination of the antifungal activity of bacterial supernatants
The isolates of lactic acid bacteria were inoculated in 10 mL of MRS broth medium for 18 h at 30 °C.An aliquot (200 µL) of each culture was then inoculated into MRS broth medium (20 mL) and incubated at 30 °C for 48 h.After centrifugation (7200 × g, 10 min) (Sigma 1-14, Sigma Laborzentrifugen GmbH, Germany), the supernatants obtained were sterilized by filtration (Millipore 0.45 µm) and immediately tested for their antifungal activities according to Cortés-Zavaleta et al. (2014) with some modifications.The bacterial supernatant was mixed with CYA agar medium at 45 °C to reach a final concentration of 5% (v/v) and poured into Petri plates (15 mL per plate).Next, 10 µL of the spore suspension was placed in the center of the plates.Control plates containing only CYA agar medium mixed with MRS broth medium in the same proportions (5%, v/v) were used.During incubation at 25 °C, the diameters of the mold growth were measured daily for 7 days, and the percentage of growth inhibition (I) % was calculated according to the following formula: Where AC and AT are the diameters of mycelial growth in treated and control plates, respectively.All analyses were performed in triplicate.

Ochratoxin A extraction and analysis by High-performance liquid chromatography with fluorescence detection (HPLC-FLD)
Ochratoxin A (OTA) was extracted using the method described by Bragulat et al. (2001).Three agar plugs (diameter = 7 mm) were taken from the inner, central, and outer areas of each colony.After the plugs were weighed and distributed in flasks, 1 mL of methanol (99.9 %) was added, then the mixture was shaken for 5 s and incubated at 25 °C for 60 min.The extracts were centrifuged (HERMLE Z 230 MA, Labnet International, USA) three times for 10 min at 13,000 rpm.The supernatant was filtered on a hydrophilic PVDF filter (0.22 μm), then analyzed by HPLC (Agilent Technologies, USA) with fluorescence detection (FLD) (excitation at 333 nm, emission at 460 nm; calibration with OTA standard (Sigma Aldrich, Steinheim, Germany).The metabolites were separated on a C18 reverse phase column (Zorbax SB, particle size 4.6 × 250 mm × 5 µm).OTA was analyzed in isocratic mode.The mobile phase (acetonitrile-water-acetic acid; 99: 99: 2, v/v/v) was pumped at 0.7 mL/min with an injection volume of 20 µL.OTA was detected at around 11 min.By comparing the retention time of the peaks of the extract with the OTA standard and with co-injection, the final concentration (expressed in ng/g of CYA) was determined based on the calibration curve established for each series.All analyses were performed in triplicate.

Isolation of lactic acid bacteria
As previously described, lactic acid bacteria were isolated from various natural products of different Moroccan origin, including raw milk (cow, sheep, goats and camel), Smen, Jben, Lben, as well as brine of fermented olives and capers.Among 66 isolates, only 16 isolates were considered lactic acid bacteria since they were Gram-positive, catalase and oxidase negative, and immobile (Table 1).These isolates were tested for antifungal activity against A. carbonarius and A. niger.

Screening of the antifungal activity of lactic acid bacteria and molecular identification
According to the results of the 16 isolates studied, only four, named (F), (I), (C), and (D), were found to inhibit the growth of the two Aspergillus sp.tested.They were incubated at temperature of 30 °C for 48 h (Table 2, Figure 1); however, no inhibition was observed for the 16 isolates incubated at 25 °C and 37 °C for incubation periods of 18 h and 24 h, respectively.In addition, the MRS agar medium appeared to promote the production of antifungal substances by the isolates (F), (I), (C), and (D) because the zones of inhibition that appeared on the MRS agar medium were larger than those on the M17 agar medium (Figure 2).Isolates (F), (I), (C), and (D) were retained for the following studies.
Table 2 Antifungal activity of lactic acid bacteria selected isolates using double layer method after incubation at 30 °C for 48 h

Antifungal activity of bacterial supernatants
The antifungal activity results of the four prepared supernatants are shown in Figure 4. A. carbonarius, Lactococcus lactis ssp.lactis (F) and Leuconostoc mesenteroides ssp.mesenteroides (I) were found to inhibit fungal radial growth with significant percentages of 53.31% and 50.02%, respectively.The percentage of inhibition by the two strains of Leuconostoc mesenteroides ssp.mesenteroides (C and D) was moderate, with 33.02% and 28.89% growth inhibition, respectively.Likewise, the growth inhibition of A. niger was about 51.39% by Lactococcus lactis ssp.lactis F, with moderate growth inhibition of 37.45%, 36.61% and 28.69% by the three strains of Leuconostoc mesenteroides ssp.mesenteroides (I, C, and D) supernatants, respectively.

The effect of lactic acid bacteria supernatants on OTA production
The concentrations of OTA in the control culture of both Aspergillus species are similar, with 96.36 and 97.96 ng/g for A. niger and A. carbonarius, respectively (Table 3).The effect of the supernatants obtained from Lactococcus lactis ssp.lactis (F) and Leuconostoc mesenteroides ssp.mesenteroides (I), which appeared significant percentages of growth inhibition on OTA production by A. niger was very important.Indeed, this reduction was around 63.90% and 89.38% for the Lactococcus lactis ssp.lactis (F) and Leuconostoc mesenteroides ssp.mesenteroides (I) supernatants, respectively.Concerning A. carbonarius, while the reduction percentage of OTA production in the presence of the Lactococcus lactis ssp.lactis (F) supernatant (65.12%) was similar to that of A. niger (63.90 %), it was only 19.35% with the Leuconostoc mesenteroides ssp.mesenteroides (I) supernatant.

DISCUSSION
Currently, the control of black mold contamination in vineyards is mainly based on the use of synthetic fungicides; however, the appearance of the secondary effects of these fungicides has called into question their use.Indeed, the appearance of resistant strains, the problems of residues at different stages of product life, and the negative brand image of the products induced by consumer dissatisfaction also justify the search for alternative methods to control, prevent, inactivate, retard, or inhibit growth of these spoilage fungi (Arfaoui, 2019; Garnier, 2017).In this study, the results obtained by qualitative methodology showed that the three Leuconostoc mesenteroides ssp.mesenteroides strains (I, C and D) and Lactococcus lactis ssp.lactis (F) inhibited the growth of A. carbonarius and A. niger.This inhibition can be attributed to the antifungal compounds produced by lactic acid bacteria, such as organic acids, fatty acids, bacteriocins, reuterin, diacetyl, and hydrogen peroxide  2011) observed that the maximum antifungal activity for Lactobacillus plantarum NB and Lactobacillus plantarum DC2 was obtained at temperatures of 25 and 37°C.Regarding incubation period, antifungal activity was not detected until after 48 h.This is in accordance with the results of Dalié (2010), which showed that antifungal activity improved with increasing incubation time.This could be related to antifungal compounds, likely secondary metabolites, which are released only during cell lysis when lactic acid bacteria are in a phase of declining growth (Dalié, 2010).We have also shown that antifungal activity of the four strains (F), (I), (C), and (D) was most important in MRS medium compared to M17 medium.According to Salas et al.
(2017), the composition of MRS medium can have a significant impact on the expression of antifungal activity of lactic acid bacteria.This impact could be related to the presence of sodium acetate in MRS medium, which appears to reinforce antifungal activity.In addition, no effect was observed by using MRS medium as a control to inoculate both Aspergillus sp.tested.This shows that sodium acetate alone has no antifungal effect.2019), which showed that the antifungal activity of lactic acid bacteria is linked to the nature and quantity of the inhibitory compounds they produce.
Our study indicates that the selected strains of lactic acid bacteria can be applied as a biocontrol of fungi growth and mycotoxins production in food product.In fact, several studies showed that strains with in vitro antifungal activity were found to be either less or more active in food products.In general, this can be achieved either by adding the bacterial supernatant, purified antifungal compounds, or whole bacteria as active ingredients, or by spraying them into the surface of an intact fruit or wound, prior to inoculation of the fungal target (Le Lay et al., 2016;Ben Taheur et al., 2019).

CONCLUSION
This study demonstrated that four selected strains of lactic acid bacteria isolated from different fermented Moroccan products, are able to inhibit the growth of A. niger and A. carbonarius, with Lactococcus lactis ssp.lactis (F) and Leuconostoc mesenteroides ssp.mesenteroides (I) being the most active.Indeed, these two strains of lactic acid bacteria also reduced the production of OTA as indicated by high percentages.Nevertheless, Lactococcus lactis ssp.lactis (F) was able to reduce OTA production by A. carbonarius strain (65.12%).In contrast, Leuconostoc mesenteroides ssp.mesenteroides (I) was proved less efficient to inhibit OTA production from the same fungal strain (19.35%).In this regard, we suggest a resistance mechanism of fungal isolate upon bacterial antagonism.As such, the use of lactic acid bacteria as an alternative against fungal alterations would be more effective in the presence of a mixture of strains in order to avoid resistance mechanisms.

Figure 2
Growth of Aspergillus niger on CYA agar medium (control, A), inhibitory zone of A. niger due to the growth of isolate (F) after incubation at 30 °C/48 h (B) and growth of A. niger on MRS agar medium (control, C) A B Comparison of the inhibition zones observed due to the growth of isolate (I) on M17 (A) and MRS (B) culture media Molecular identification and differentiation of lactic isolates selected by PFGE (Pulsed-Field Gel Electrophoresis) The four retained isolates were molecularly identified as Leuconostoc mesenteroides ssp.mesenteroides for (I), (C), and (D) and as Lactococcus lactis ssp.lactis for (F).After differentiation by PFGE of the three species of Leuconostoc (I, C and D) and after analysis of the dendrogram (Figure 3), it appeared that the three isolates are different but genetically similar with high percentages of similarity; (C) and (D) displayed 93.8% similarity, (I) and (D) displayed 97% similarity, and (I) and (C) displayed 95.3% similarity.

Figure 3
Figure 3 Dendrograms of differentiation between three isolates of Leuconostoc mesenteroides ssp.mesenteroides C, D and I, obtained after analysis by Bionumerics

Figure 4
Figure 4 Growth inhibition (%) of Aspergillus niger and A. carbonarius, in presence of supernatants of Lactococcus lactis ssp.lactis (LL-F) and three strains of Lenconostoc mesenteroides ssp.mesenteroides (LM-I, LM-C and LM-D)

Table 1
Origin and source of lactic acid bacterial isolates used for the study of antifungal activity

Table 3
Quantification of OTA produced by the Aspergillus sp.treated with the supernatants of Lactococcus lactis ssp.lactis F and Lenconostoc mesenteroides ssp.mesenteroides I (LOD = 0.2 ppb)

(Salas et al., 2017; Crowley et al., 2013; Gerez et al., 2009; Prema et al., 2008; Magnusson et al., 2003; Lavermicocca et al., 2000).
(Dalié et al., 2010)iciency of the antifungal activity of lactic acid bacteria depends on several factors, including temperature, incubation period, and culture medium used(Dalié et al., 2010).In this work, the antifungal activity observed in four tested strains was detected at a temperature of 30 °C and an incubation period of 48 h.Our results are in agreement with those obtained by different authors who showed that 30 °C was the most adequate temperature for the antifungal activity of different bacterial strains, such as Lactococcus lactis ssp.

and Magnusson (2005), Stiles et al. (2002), and Delavenne et al. (2012), confirming
Our results are in agreement with Schnürer that sodium acetate present in MRS medium participates, in synergy with some antifungal compounds produced by lactic acid bacteria, in increasing the inhibitory effect.Regarding the results of our quantitative methods, the supernatants of four selected isolated strains inhibited growth of both Aspergillus sp.studied, with percentages between 28.69% and 51.39%.These results confirm the presence of antifungal compounds in the supernatants.These percentages are similar to those obtained by Ben Taheur et al. (2019), which was 37.78% when using the supernatant of Lactobacillus kefiri (FR7) against A. carbonarius.Conversely, the results obtained by Le Lay et al.(2016)showed that Leuconostoc mesenteroides and Lactococccus lactis ssp.lactis had no effect on the growth of A. niger.isolatestrains(thosewithsignificant percentages of growth inhibition) were selected.To our knowledge, this article is one of the first to study the effect of Lactococcus lactis ssp.lactis and Leuconsotoc mesenteroides ssp.mesenteroides on OTA production by A. niger and A. carbonarius.In the case of A. niger, OTA production was reduced by roughly 89.38% with the use of the Leuconsotoc mesenteroides ssp.mesenteroides (I) supernatant and 63.90% in the case of Lactococcus lactis ssp.lactis (F).This reduction may be due to reduced mycelial growth after using bacterial supernatants.Indeed,Gerbaldoet al. (2012) and Dallagnol et al. (2018) noted a correlation between fungal growth and production of mycotoxins when using lactic acid bacteria.Thus, in our case, the growth inhibition observed may have contributed to this weak synthesis of mycotoxins; however, this correlation is not always effective (Dallagnol et al., 2018).Furthermore, this reduction of OTA production may also be linked to antifungal compounds produced by the lactic acid bacteria studied.Indeed, it is known that some antifungal compounds, especially organic acids, can diffuse through the membrane of target organisms in their undissociated hydrophobic form.In the cytoplasm, due to its neutrality, acid will dissociate and generate an accumulation of protons, thereby reducing intracellular pH.This generally causes inhibition of key enzymes of metabolism, inducing a loss of viability and cell destruction ( (Varga et al., 2005)ioration of the nutritional and organoleptic quality of food, contamination by fungi often induces the formation of mycotoxins.Their presence in food constitutes a danger to human and animal health(Dalié et al., 2010).The most effective way to prevent contamination of food with mycotoxins is to prevent growth of mycotoxigenic fungi(Varga et al., 2005).In this study, we evaluated the ability of lactic acid bacteria to not only inhibit growth of A. niger and A. carbonarius but also their effects on OTA production.For this, (F) and (I)

Dalié et al., 2010; Le Lay, 2015; Lappa et al., 2018).
By using the supernatant of Leuconsotoc mesenteroides ssp.mesenteroides (I), we found the percentage reduction of OTA production by A. carbonarius is only 19%.It is possible that A. carbonarius has developed resistant bacterial antagonism; however, this resistance is only observed in the presence of Leuconsotoc mesenteroides ssp.mesenteroides (I) and not with Lactococcus lactis ssp.lactis (F).This suggests the existence of other antifungal compounds involved in the reduction of OTA that are produced only by the Lactococcus lactis ssp.lactis (F) strain.This agrees with the work of Sadeghi et al.(