Inhibitory Properties of Lactic Acid Bacteria against Moulds Associated with Spoilage of Bakery Products

Aim: To evaluate the potentiality of LAB strains isolated from different fermented products to inhibit moulds associated with spoilage of bakery products. Methodology: Lactic acid bacterial (LAB) strains obtained from fermented products (“burukutu”, “pito”, yoghurt, and “iru”) were screened for antifungal activity against moulds (Aspergillus flavus, Aspergillus fumigatus, Aspergillus repens and Penicillium sp.) isolated from spoilt bakery products. Inhibitory activities of the lactic acid bacterial isolates were determined by the dual agar overlay method and well diffusion method. Results: Eleven isolates (79%) out of the fourteen lactic acid bacterial strains screened showed antifungal activity against one or more of the moulds when the method used was dual agar overlay method. When agar well diffusion assay was used to check the antifungal activity of the cell-free culture supernatants of these eleven LAB isolates after excluding inhibition due to organic acids and hydrogen peroxide, only four LAB strains (BE1, IO1, PO4 and PO9) continued to show antifungal activity against the moulds. The selected bacteriocin-producing LAB strains (BE1, IO1, PO4, and Original Research Article Adesina et al.; JAMB, 4(3): 1-8, 2017; Article no.JAMB.31386 2 PO9) were identified as Lactobacillus cellobiosus, Pediococcus pentosaceus, Lactobacillus rhamnosus, and Tetragenococcus halophilus respectively using API 50 CHL system. The cell-free culture supernatant of P. pentosaceus IO1, T. halophilus PO9, and L. cellobiosus BE1 sprayed on bread surface inhibited the growing of moulds during 14 days of storage in polythene bags. Conclusion: These LAB isolates from fermented products capable of inhibiting the moulds have potential that could be used as biopreservative agents in bakery products.


INTRODUCTION
Bakery products are the important staple foods in most countries and cultures. Bakery products such as breads and cakes are essential food items of the vast majority of the world population [1]. Mould spoilage is the main cause of substantial economic loss in bakery industry and might also cause public health problems due to the production of mycotoxins. The reduction of mould growth in bakery products is thus, of crucial importance and there is great interest to develop safe and efficient strategies for this purpose [2].
Lactic acid bacteria (LAB) have a long history of safe use in food and are generally recognized as safe. LAB are known to produce a wide variety of antagonistic compounds, including lactic acid, hydrogen peroxide, and bacteriocins [3,4]. The use of LAB to control moulds is a good alternative approach to physical and chemical preservation methods normally applied in food products. Careful selection of specific strains of lactic acid bacteria with antifungal properties can allow the reduction of moulds and can, therefore, improve the shelf-life of many fermented products and reduce the presence of mycotoxins [5].
The objective of this study was to evaluate the potentiality of LAB strains isolated from different fermented products to inhibit moulds associated with spoilage of bakery products.

Lactic Acid Bacterial Strains
Lactic acid bacterial (LAB) strains from fermented products ("burukutu", "pito", yoghurt, "wara", and "iru") procured from sellers were isolated using de Man Rogosa Sharpe (MRS; Oxoid Ltd) agar and incubated at 30°C for 48 h under anaerobic conditions [6]. The LAB strains were then stored at 4°C in MRS broth and MRS agar slants.

Isolation and Identification of Fungal (mould) Strains
Loaves of bread and cakes were stored at room temperature (28 ± 2°C) for 4 -7 days to allow mould growth on them. Moulds in pure culture were then isolated from mouldy breads and cakes using streak plate method and inoculated onto the surface of malt extract agar medium, which were incubated afterward for the growth of mould colony at 28 ± 2°C for 5 days [7]. The mould cultures were preserved at 4°C and identification was done on the basis of morphological and microscopic characterization [8].

Fungal inocula preparation
Moulds isolated from loaves of bread and cakes were grown on malt extract agar medium at 28 ± 2°C for 5 days. A sterilized cork borer was used to punch out agar disk containing the spores from agar plate into 5 ml sterile distilled water to disperse the spores. The mould suspension was adjusted to give approximately 10 7 spores/ ml using sterile distilled water [7].

Initial screening for antifungal activity by overlay method
Inhibitory activity of the lactic acid bacteria isolates was determined by the overlay method as described by Muhialdin and Hassan [9] with a slight modification. Lactic acid bacteria were inoculated in 2 cm lines on MRS agar plates and incubated anaerobically at 30°C for 24 h. The plates were then overlaid with 10 ml of malt extract soft agar containing 10 7 spores/ml of each mould and incubated aerobically. After 48 h of aerobic incubation at 28 ± 2°C, zone of inhibition was observed. Inhibitory activity was scored as follow: -, no inhibition; +, very weak inhibition; ++, low inhibition with a little clear zone "near the edge of the colony"; +++, strong inhibition with a large clear zone.

Detection of antifungal activity of cellfree culture supernatant by welldiffusion method
The anti-mould activity of the cell-free supernatant from LAB isolates which showed inhibitory activity was determined by welldiffusion assay as described by Muhialdin and Hassan [9]. The LAB isolates were grown in de Man Rogosa Sharpe (MRS) broth separately for 48 h at 30°C. The broth culture was centrifuged at 4000 x g for 20 min to get the cell-free supernatant. Fungi 10 7 spore/ml was mixed with molten malt extract agar and allowed to solidify. Then, wells of size 6 mm were made using cork borer and 20 µl of malt extract agar was pipette to cover the base of the well to avoid leaking of the supernatant. Eighty (80) microlitres of LAB supernatants (pH-neutralized cell-free culture supernatant with addition of catalase enzyme) were added to different wells and the plates were incubated at room temperature (28 ± 2°C) for 48 h. The zone of inhibition of mycelia growth was measured with a ruler and recorded in millimetres.

Identification of LAB Isolates
LAB that possessed inhibitory activity after excluding inhibition due to organic acids and H 2 O 2 from their supernatants were selected for identification using API 50CH Kit (Bio Merieux, France).

Screening for Anti-mould Activity of LAB on Bread Surface
Three LAB strains were tested for anti-mould activities on the bread surface using the method described by Cizeikiene et al. [

RESULTS
The colonial and morphological features of the fungal isolates from spoilt bakery products are shown in Table 1. The fungal isolates B1, B2, B4, and C1 were identified as Aspergillus fumigatus, Aspergillus repens, Penicillium sp. and Aspergillus flavus. Table 2 shows the inhibitory activities of lactic acid bacterial (LAB) isolates against spoilage moulds as determined by the overlay method. Eleven (11) isolates out of the fourteen (14) LAB strains screened showed antifungal activity against one or more of the moulds. Although the inhibition zones varied with the lactic acid bacteria as well as with the mould strain tested. Aspergillus flavus, which was the most sensitive indicator strain, was inhibited by most of the LAB strains. Fig. 1 shows the clear zones of inhibition of growth of A. flavus formed around colonies of LAB strains (IO1, BE1, and PO9) on MRS agar.
When agar well diffusion assay was used to check the antifungal activity of the cell-free culture supernatants of the eleven LAB isolates after excluding inhibition due to organic acids and hydrogen peroxide, only four LAB strains (BE1, IO1, PO4, and PO9) continued to show antifungal activity against the moulds (Fig. 2).

Fig. 2. Inhibition of moulds by bacteriocin-containing cell-free culture supernatants of four selected lactic acid bacteria
The LAB supernatants and crude bacteriocins of P. pentosaceus IO1, T. halophilus PO9, and L. cellobiosus BE1, sprayed on bread surface, inhibited the growing of moulds during 14 days of storage (Table 4); whereas on the control bread sample, large mould growths were detected after 14 days of storage.

DISCUSSION
The four most common fungal isolates that were obtained from spoilt bread and cake samples in this study were identified as Aspergillus fumigatus, A. repens, A. flavus and Penicillium sp. Similarly, Mishra et al. [11] isolated Aspergillus flavus and Penicillium oxalicum from spoiled bread and cake samples. Abellana et al. [12] had reported Aspergillus and Penicillium isolates which spoiled sponge cake. Filamentous fungi (moulds) involved in spoilage of bakery products include Aspergillus sp., Penicillium sp., Rhizopus sp., Monilia sp., Mucor sp., and Eurotium sp. [1]. In addition to the economic losses associated with the spoilage of bakery products by moulds, another concern is the possibility of mycotoxins production [1]. The occurrence of mycotoxinogenic moulds such as Aspergillus and Penicillium in foods is potentially dangerous for public health and also constitutes a major economic problem [13]. The screening of some selected lactic acid bacteria strains for antifungal activity against the spoilage moulds using agar-well diffusion method demonstrated that only four LAB strains; Lactobacillus cellobiosus BE1, Pediococcus pentosaceus IO1, L. rhamnosus PO4, Tetragenococcus halophilus PO9 were able to produce bacteriocins with antifungal activities. Pediococcus pentosaceus IO1 was most effective in the inhibition of Aspergillus repens when compared with the other LAB isolates.
Studies concerning antibacterial proteinaceous compounds, such as bacteriocins, are extensive in comparison to proteins with antifungal properties, although during the last decade various LAB-derived proteinaceous compounds with anti-yeast and anti-mould abilities have been identified [14][15][16]. Adebayo and Aderiye [15] reported that only 17 of the 25 bacteriocinproducing LAB isolates from some fermented foods ("eko", "fufu", "iru", and "ogi") possessed antifungal activity against Penicillium citrinum, Aspergillus niger and A. flavus. Lactobacillus rhamnosus VT1 was reported to inhibit the growth of spoilage and toxigenic fungi species in the genera Aspergillus, Penicillium, and Fusarium [17]. Muhialdin and Hassan [9] found that Lactobacillus brevis G004, Lactobacillus fermentum Te007 and Pediococcus pentosaceus Te010 isolated from fermented guava juice and tempeh possessed strong antifungal activity against Aspergillus oryzae. Kim [18] also reported that the five Lactobacillus strains isolated from Kimchi exhibited antifungal activity against several fungi and concluded that some different compounds produced by the bacteria caused the inhibitory activity.
Lactic acid bacteria are well known for their antifungal activity, which is related to the production of a variety of compounds including organic acids, diacetyl, reuterin, hydrogen peroxide, phenyllactic acid, bacteriocins and cycle peptides [19][20][21][22][23]. The organic acids are active at low pH and the activity relies on the undissociated form of the acids. Recently, interest has dramatically increased in the use of bioactive peptides produced by LAB as an antifungal agent. The use of protein-like compounds such as bacteriocins are preferred over the use of acids because their activity is present over a wide range of pH and they are heat stable compounds which are ideal for use in heat processed foods [24].
The findings in this study using sprayed LAB supernatant on bread surface is in agreement with the report of Cizeikiene et al. [10] which indicated that effective inhibition of undersirable microorganisms could be achieved by LAB for preventing bread moulding.

CONCLUSION
Based on the findings of this study, it can be concluded that the selected LAB strains from different fermented products possess inhibitory activity against the spoilage moulds and could be used as biopreservatives in bakery products.