Bioprotection of Fresh Food Products Against Blue Mold Using Lactic Acid Bacteria with Antifungal Properties

BIOPROTECTION OF FRESH FOOD PRODUCTS AGAINST BLUE MOLD USING LACTIC ACID BACTERIA WITH ANTIFUNGAL PROPERTIES

Received for publication, November 4, 2015 Accepted, January 20, 2015

G. M. MATEI1, S. MATEI1, A. MATEI2, C. P. CORNEA2, E. M. DRĂGHICI3, I. O. JERCA4 1National RD Institute for Soil Science, Agrochemistry and Environment Bd. Marasti 61, Sect.1, cod 011464, Bucharest, Romania 2UASVMB, Faculty of Biotechnologies/Center for Applied Biochemistry and Biotechnology BIOTEHNOL, Bd. Marasti 59, Sect.1, cod 011464, Bucharest, Romania 3UASVMB, Faculty of Horticulture, Bucharest, Romania 4UASVMB, Faculty of Land Reclamation and Environmental Engineering, Bucharest, Romania, Bd. Marasti 59, Sect.1, cod 7133, Bucharest, Romania *Corresponding author: [email protected]

Abstract The main goal of the present paper was to present the results of the research carried out on the effect of indigenous bacteria strains on development and fungal biofilm formation at Penicillium expansum and to investigate the possibilities to use selected lactic acid bacteria (LAB) strains in the biocontrol of apple spoilage caused by blue mold. Lactic acid bacteria are able to produce antagonistic compounds (organic acids, cyclic dipeptides, reuterin, biosurfactants) with an important role in controlling fungal development. Lactic acid bacteria strains selected from various plant materials or traditional Romanian foods were assayed for their antagonistic effect on the fungal biofilm in potato- dextrose broth and on the development of apples rot caused by blue mold in wounded and non-wounded fresh fruits. Strains LAB 13 and LAB 43 presented highly effective antifungal activity, comparably to the reference strain Lpa, against fungal growth and biofilm formation of spoilage fungus Penicillium expansum. Application of lactic acid bacteria demonstrated that the strains Lpl, LAB 43, LCM5 and Lpa presented bioprotection effect on apples from cultivar IONATAN of Voinești, experimentaly contaminated with Penicillium expansum, in both wounded and non-wounded fruits. Lactic acid bacteria with antifungal properties presented high potential to be utilized as bioprotectants.

Keywords: lactic acid bacteria, spoilage fungi, antifungal effect, biofilm, apples, biopreservation

1. Introduction Fungal species belonging to the genera Penicillium, Aspergillus and Fusarium, are known to play a role in damaging various fruits during storage and to have negative impact on human health, by the synthesis of exometabolites known as mycotoxins (TSITSIGIANNIS et al. [16]). Among these, Penicillium expansum can cause apples rot and synthesizes mycotoxins such as patulin and citrinin (ROUSE et al. [13]). In order to reduce the effects of the ingestion of foods contaminated with mycotoxins, different strategies involving a variety of physical, chemical, microbiological and biotechnological methods are applied in order to avoid

11201 Romanian Biotechnological Letters, Vol. 21, No. 1, 2016 GABI-MIRELA MATEI, S. MATEI, A. MATEI, CĂLINA PETRUȚA CORNEA, ELENA MARIA DRĂGHICI, IONUȚ OVIDIU JERCA contamination of mycotoxins, detoxify food products, and to inhibit mycotoxin absorption from gastrointestinal tract (SCHNÜRER & MAGNUSSON [14]; CUCIUREAN [3]) . In the last decades, a special attention received the control of mycotoxigenic fungal species by other microorganisms (MAGNUSSON et al. [11]; LAREF & GUESSAS [9]). Among lactic acid bacteria-LAB (known as natural biological antagonists of mycotoxigenic fungi) many species belong to intestinal microflora. They have practical applications in obtaining high quality fermented foods and in bio-preservation (CROWLEY et al., [2]), being generally recognized as safe. Recent research on the effect of lactic acid bacteria stated their capacity in producing antagonist compounds (weak acids, exopolysaccharides, biosurfactants, dipeptides, reuterin, hydrogen peroxide) and in competing for nutrients (TRIAS et al, [15]; DAHLIE et al., [4]). These properties lead to increased interest in using lactic acid bacteria in food preservation as a response to consumers demand for reducing the application of chemical preservatives able to control pathogenic fungi (MAFTEI et al. [10]). The aim of the present study, was to investigate the effect of indigenous strains of lactic acid bacteria with antifungal properties on biofilm formation at Penicillium expansum and to use them in bioprotection of apples against spoilage caused by blue mold as an alternative to chemical preservatives.

2. Material and methods Test organisms used in experiments LAB strains (LAB43, LAB58, LAB35, LAB15, LCM5, and LAB13) have been isolated from various plant materials or traditional Romanian foods (fermented wheat bran, pickles, sauerkraut) by dr. Medana Zamfir, from the Institute of Biology and dr. A. Matei from UASVM, Bucharest. As reference strains, two collection strains were used: Lactobacillus plantarum ATCC8014 (Lpl) and Lactobacillus paracasei IC13239 (Lpa). All strains were grown in MRS broth at 360C. Potential mycotoxigenic fungal species Penicillium expansum, from infected apples was isolated in pure culture on PDA medium. Assay of antifungal activity of LAB strains on fungal growth and biofilm formation The antifungal effect of lactic acid bacteria was assayed on 48 hours cultures of each strain (LAB43, LCM58, Lpa, LAB35, LAB15, and LAB13) inoculated in 100ml flasks over a fungal culture of Penicillium expansum (106 spores ml-1), in PD broth and monitored for a period of 14 days for biofilm formation and development. Dry weight measurements of fungal mycelia biomass were used for direct quantification of influence of lactic acid bacteria on the fungal biofilms. The percent inhibition of growth rate was calculated using the formula (R1-R2)/R1, where R1 represents the dry weight of control and R2 represent the dry weight of indicator fungus grown in the presence of LAB strains (ADEBAYO & ADERIYE [1]). The values obtained represent the mean of three replicate for each variant. Results have been statistical interpreted by variance analysis (ANOVA monofactorial) for p<0.05, according to Student test. The evolution of fungal biofilm was monitored for morphological appearance and sporulation dynamics. Ex vivo experiments for antifungal activity of LAB against P.expansum on apples Two ex vivo experiments using wounded and non-wounded apples from cv. IONATAN of Voinesti have been done to assess the efficiency of LAB strains in bioprotection against the blue mold Penicillium expansum. All apples were surface disinfected with sodium 11202 Romanian Biotechnological Letters, Vol. 21, No. 1, 2016

BIOPROTECTION OF FRESH FOOD PRODUCTS AGAINST BLUE MOLD USING LACTIC ACID BACTERIA WITH ANTIFUNGAL PROPERTIES hypochlorite in concentration of 5%. In the first experiment, the apples rested non-wounded and in the other, their surface was wounded with a sterile scalpel. Apples from both experiments sprayed uniformly with the LAB strains Lpa, Lpl, LAB43, LAB58, LAB35, LAB15, LCM5 and LAB13, grown on MRS broth for 48 hours. After 30 minutes, the apples were infected by spraying with a suspension containing the indicator fungus Penicillium expansum with a concentration of 106 spores ml-1. Apples experimentally infected with Penicillium expansum and without lactic acid bacteria were utilized as control in both experiments. The development of infections with Penicillium expansum was monitored for evolution of infection spot diameters. The percent of inhibition of spot development under the influence of treatment with LAB strains was calculated by reporting to the diameter of spots measured to the control infected with indicator fungus and non-treated with lactic acid bacteria (TRIAS et al., [15]). All assays were carried out in triplicate. Results have been statistical interpreted by variance analysis (ANOVA) for p<0.05, according to Student test.

3. Results and discussions Antifungal activity of LAB on fungal growth and biofilm formation Effect of LAB strains producing extracellular compounds on the fungal biofilm developed by Penicillium expansum was analyzed by way of quantitative changes occurring in the biomass accumulation processes, in conditions of simultaneous growth of both species. Biomass accumulations analysis revealed that the strains influenced the processes involved in fungal cells aggregation or the perforation of fungal biofilm and the inhibition of growth of fungal mycelia in the indicator fungus species. Also, the influence of antifungal metabolites on spore development was assessed. Penicillium expansum biofilm formation was most strongly inhibited by the strains LAB 13 and Lpa. Under the influence of LAB 43 several points of initiation of non-sporulated mycelium appeared and with LAB 58, the points were more frequent, sporulated and presented subsequent confluence trend (Fig. 1).

Figure 1. The influence of LAB strains on fungal biofilm formation of Penicillium expansum (right image – control )

LAB strains LAB15 and LAB35 caused the formation of lighter colored mycelia conflu in a complete biofilm with sporulation started on the edges.

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As a general remark, all LAB strains induced various inhibition degree on Penicillium expansum biofilm formation comparatively with the control. Penicillium expansum biofilm was complete, thick and heavily sporulated on its entire surface (Fig. 1). As a consequence, significantly lower mycelium dry weight registered in variants with LAB strains comparatively with Penicillium expansum alone (control). The highest growth inhibition of mycelia biomass was induced by the strain LAB13. Another class of inhibition was represented by the strains LAB43 and Lpa that led to a decline of over 80% of fungal growth. The lowest growth inhibition (8.82%) was induced by the strain LAB35 (Table 1).

Table 1. Dry weight and percent of growth inhibition of Penicillium expansum Variant Dry weight (mg) Growth inhibition (%)

P.expansum 0.34a* - P.expansum +LAB 13 0.03f 91.18a P.expansum +LAB 15 0.25c 27.45d P.expansum +LAB 35 0.31b 8.82e P.expansum +LAB 43 0.06e 83.33b P.expansum +LAB 58 0.15d 54.90c P.expansum +Lpa 0.07e 80.39b DL 0.05 = 0.021 DL 0.05 = 6.87 DL 0.01 = 0.030 DL 0.01 = 9.63 DL 0.001 = 0.041 DL 0.001 = 13.61 *Values followed by the same letter are not significantly different for p<0.05 (Student test).

EL-DEEB et al. [7] reported similar results for extracellular extracts from different LAB strains that showed different pathways of inhibition on bacterial biofilm development. For example, one strain belonging to Lactobacillus acidophilus was able to inhibit with over 59% the biofilm formation in Shigella and one Klebsiella strain but not in two other strains of Klebsiella. ADEBAYO & ADERIYE [1] assayed the antifungal activity of LAB strains from genus Lactobacillus against Penicillium citrinum and obtained similar patterns of fungal sensitivity to antifungal agents released by Lactobacillus strains in liquid and solid media. However, a higher percentage of growth inhibition in liquid media than on agar plates was explained by the greater diffusion of antifungal compounds in liquid media and a higher contact with fungal spores. Inhibition levels of over 25% observed for 10 of 17 strains was atributed to the inhibitory effect of bacteriocins. DESAI et al. [5] studied the inhibition of the mechanisms of biofilm formation using Aspergillus fumigatus and Fusarium sp. as indicator fungi species. Inhibition of fungal biofilm in Aspergillus niger and Penicillium chrysogenum by lipopeptides produced by lactic acid bacteria, with fungal hyphae and cell surface deformations were registered by JADHAV et al. [8]. In our present experiments, antifungal agents produced by LAB strains were able to inhibit the fungal growth and biofilm formation leading to the hypothesis that extracellular metabolites may interfere with factors influencing the mechanisms of quorum sensing in fungi. Research on fungal biofilm stated that these factors represented by various alcohols, peptides, lipids, acetaldehyde or volatile compounds can act as molecules with role in morphogenesis, mycelia formation, as well as in pathogenesis or other fungal functions (MAJUMDAR & MONDAL [12]). DUSANE et al. [6] suggested that potential biofilm disruptors in Candida species may be represented by surface- active agents of chemical and biological origin.

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BIOPROTECTION OF FRESH FOOD PRODUCTS AGAINST BLUE MOLD USING LACTIC ACID BACTERIA WITH ANTIFUNGAL PROPERTIES

Antifungal activity of LAB against P. expansum (ex vivo experiments on apples) Eight LAB strains (LAB 58, LAB 15 LAB 13, LCM5, LAB 35, LAB 43, Lpl and Lpa) were selected for ex vivo experiments on model systems represented by fresh fruits. Experiments on fresh apples from cultivar IONATAN of Voinești assumed the treatment by spraying with bacterial suspensions of LAB strains on the non-wounded fruits (surface intact) and fruits injured by the tip of a sterile scalpel (Fig. 2). Application of the target fungus, Penicillium expansum was achieved after bacterial treatment, in the same manner.

Figure 2. Antifungal activity of LAB strains against Penicillium expansum in wounded and non-wounded apples experiments at 7 days (a) and 14 days (b), and with non-wounded apples at 21 days (c). 1-LAB 58, 2-LAB 15, 3-LAB 13, 4- LCM5, 5-LAB 35, 6-Lpl, 7-Lpa, 8-LAB 43, 9- control+P.expansum, 10- nontreated, noninfected control)

The administration of LAB strains for the bioprotection against the fungal species Penicillium expansum, the agent of apple rot, resulted in different effects in the two experiments conducted, depending on the presence or absence of wounds on the surface of apples. The inhibition percent of rot spots produced by P.expansum in the presence of each LAB strain was calculated by measuring the diameter of spots and reference to the diameter of the untreated control. In the experiments with non-wounded apples, better protective effects were obtained than in the experiments with injured apples. The strains LAB 43, Lpl, LAB 58, LAB 13 and LCM5 conferred complete protection at 5 and 7 days, and the first four strains showed a percentage of 100% inhibition of the fungus to 14 days, significantly higher than the other strains (Fig.4).

*Values followed by the same letter are not significantly different for p<0.05 (Student test).

Figure 3. Percentage of inhibition of Penicillium expansum infection in experiments with non-wounded apples, treated with lactic acid bacteria, at 5 and 7 days

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Strains that have ensured the highest percentage of inhibition in non-wounded apples to 5 days were Lpl, LAB 43, LCM5, LAB 13 and LAB 58 (Fig. 3). The protective effect of these bacteria to the non-wounded apples against P.expansum infection maintained for the whole duration of the experiment, up to 14 days of incubation, no blowout infection (Fig. 4).

*Values followed by the same letter are not significantly different for p<0.05 (Student test).

Figure 4. Percentage of inhibition of Penicillium expansum infection in experiments with non- wounded apples, treated with lactic acid bacteria, at 14 days

When evaluating the protective effects of lactic acid bacteria against fungal infection of apples injured it was found that it was lower than in non-wounded apples. The percentage of growth inhibition of fungus P.expansum was lower in the case of injured apples due to easier penetration of propagules into the apple. However, delaying the development of fungal contamination and reduced lesion size during the 14 days of incubation was constantly observed in the treatment with strains Lpl, LAB 43 LCM5 and Lpa (Fig. 5). The strains LAB 58, LAB13, LAB 35 and LAB15 induced to 5 days the lower percentage of inhibition. By day 14 of monitoring, the percent inhibition has not exceeded 10%, with the exception of LAB 58 strain whose protective effect increased over time to more than 20% (Fig. 6).

*Values followed by the same letter are not significantly different for p<0.05 (Student test).

Figure 5. Percentage of inhibition on Penicillium expansum infection in experiments with wounded apples, treated with lactic acid bacteria at 5 and 7 days.

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BIOPROTECTION OF FRESH FOOD PRODUCTS AGAINST BLUE MOLD USING LACTIC ACID BACTERIA WITH ANTIFUNGAL PROPERTIES

*Values followed by the same letter are not significantly different for p<0.05 (Student test).

Figure 6. Percentage of inhibition on Penicillium expansum infection in experiments with wounded apples, treated with lactic acid bacteria at 14 days.

The percentage of inhibition for LAB strains tested against Penicillium expansum on the apples variety IONATAN are consistent with the literature. TRIAS et al.[15] obtained a percentage of inhibition of infection generated by P.expansum in apples variety Golden Delicious injured of 20% in four strains of lactic acid bacteria and 50% for strain TM128 of Weissella cibaria. The production of dextran, and the accumulation of exopolysaccharides synthesized by the lactic acid bacteria are related, in some cases to the formation of biofilms, which contribute to the colonization of the surface and the prevention of fungal attack, but the accumulation has to appear in combination with other mechanisms of antagonism, since not all exopolysaccharides producing bacteria inhibited production of P.expansum infections. In the present experiments, conducted with IONATAN of Voineşti apple variety, both injured and non-wounded, the LAB strains which induced the highest percentage of growth inhibition of P.expansum infection spots are those that inhibited fungal growth and biofilm formation. ROUSE et al.[13] assigned the antifungal effect of lactic acid bacteria against P.expansum, to the production of antifungal peptides, in the antagonism tests performed ex vivo on culture medium supplemented with apple extract. The inhibition of spoilage fungus induced by these antifungal compounds was produced by a LAB strain of Pediococcus pentosaceus (with inhibition zones over 10mm diameter). The results of present experiments showed that LAB strains Lpl, LAB 43, LCM5 and Lpa produced antifungal agents with strongly antagonistic effect against the blue mold Penicillium expansum showing potential for protection against apple rot, as an alternative to chemicals.

4. Conclusions LAB strains LAB 13 and LAB 43 presented highly effective antifungal activity, comparably to the reference strain Lpa, against fungal growth and biofilm formation of spoilage fungus Penicillium expansum. Application of antifungal lactic acid bacteria demonstrated that the strains Lpl, LAB 43, LCM5 and Lpa presented bioprotection effect against Penicillium expansum on both wounded and non-wounded apples from cultivar IONATAN of Voinești. Data obtained from in vitro and ex vivo assays were similar, confirming the reliability of results and the antifungal activity of exometabolites produced by the LAB strains. Lactic acid bacteria with antifungal properties presented high potential to control the blue mold in apples and may be further utilized as bioprotectants, an environmental friendly alternative to chemical fungicides. Romanian Biotechnological Letters, Vol. 21, No. 1, 2016 11207

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Acknowledgements This work was supported by a grant of the Romanian National Authority for Scientific Research, CNDI – UEFISCDI, financed from project number PN-II-PT-PCCA-2011-3.2- 1351 - Contract No.68/2012 (acronym FRIENDLYHORT) and partly from PN-II-PT-PCCA- 2011-3.1-0621 nr.105/2012, (acronym PLANTLAB).

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