Antifungal activity of Lysinibacillus macroides against toxigenic Aspergillus flavus and Fusarium proliferatum and analysis of its mycotoxin minimization potential
BMC Microbiology volume 23, Article number: 269 (2023)
Toxigenic fungi (Aspergillus and Fusarium) and their metabolites represent the major cause of corn and corn-based products contamination and consequently lead to severe economic and health issues.
Our current study aimed to investigate the efficacy of using L. macroides Bac6 as a biological control agent against the toxigenic fungi; A. flavus f10 and F. proliferatum f30 and their mycotoxins.
The results illustrated that A. flavus f10 produced the aflatoxins AFB1 and AFG2 with concentrations of 21.239 and 13.593 ppb, respectively. While F. proliferatum f30 produced fumonisin B1 (9600 ppb). Furthermore, L. macroides showed a high potential for inhibition of toxigenic fungal growth using a dual culture method. F. proliferatum f30 and A. flavus f10 were found to be inhibited by a percentage of 80 and 62.5%, respectively. The results were confirmed using the scanning electron microscope. The antagonistic bacteria, L. macroides, showed chitinase productivity and activity of 26.45 U/L and 0.12 U/mL/min, respectively, which illustrates its potential application as a biocontrol agent. The GC-MS analysis revealed an abundance of Pyrrolo[1,2-a] pyrazine-1,4-dione, Hexahydro in the bacterial supernatant that exhibited antifungal characteristics. L. macroides had a significant reduction of AFB1 and AFG2 produced by A. flavus f10, recording 99.25% and 99% inhibition, respectively. It also showed strong inhibition of fumonisin B1 (90% inhibition) produced by F. proliferatum f30. Conclusion: Thus, the current study is a prospective study evaluating for the first time the potential impact of L. macroides Bac6 against the toxigenic fungi and their toxins.
The world’s population is estimated to be 8 billion by 2025 and 9.8 billion by 2050 . As a result, increasing global agricultural productivity is required to meet food demand [2, 3]. Grains, pulses, and oil seeds continue to play an important part in both human and animal nutrition around the world. Aspergillus flavus is typically found in decaying vegetation and soil . This fungus is well-known for producing aflatoxin, a strong carcinogen that is dangerous to both people and animals [5, 6]. A. flavus may infect a diverse range of hosts, such as maize, cotton, and peanuts. . Infections that develop during growth or storage might cause significant monetary losses . Due to its capacity to create secondary metabolites such as aflatoxins, cyclopiazonic acid, and kojic acid, A. flavus is fatal . In the host organism, these metabolites may result in cell damage and death . A. flavus’s aflatoxins have the potential to harm people either immediately after exposure or over time, leading to conditions such as liver cancer, immune system suppression, and growth retardation [11, 12]. A filamentous fungus called Fusarium proliferatum infects maize and causes illnesses in plants. It creates mycotoxins, which, when consumed by people or animals eating tainted feed or crops, can have major negative effects on their health . F. proliferatum infects maize plants, causing root, stalk, and ear rot as well as producing fumonisins, a mycotoxin that can taint maize kernels and pose a major risk to human and animal health, results in a serious health risk to consumers. . Additionally, grains, including corn (maize), are susceptible to mycotoxins before and after harvest . When specific fungal species are exposed to environmental conditions (e.g., water activity, temperature, pH, and intergranular gas composition), mycotoxins are produced. Mycotoxins from toxigenic fungal growth are produced in food and animal feeds via the secondary metabolism process . Mycotoxins, in contrast to many bacterial toxins, are typically extremely stable when exposed to the conventional heating method used in the preparation of foods for human consumption . These fungi and mycotoxins have serious consequences for human and animal growth and health [18,19,20]. Aflatoxin and fumonisin B1 are two mycotoxins that have been linked to cancer in humans and other mammals as well as renal and neurological disorders. [21, 22]. One of the primary issues in managing mycotoxigenic fungi and related mycotoxin, which is a source of contamination is that fungicides are ineffective at treating agricultural commodities, that are usually applied in the agricultural sector to treat diseases caused by fungi. [23, 24]. Biological control may be a long-term solution, safe for human and environmentally healthy, self-sustaining treatment strategy to manage mycotoxins, resulting in reduced agri-operational costs [25, 26]. Biological control is effective, and many new approaches are being developed, most of them are based on microbiological research and the usage of microbial organisms that inhibit fungal growth and detoxify mycotoxins . Bio-acceptable approaches should not only be beneficial to the environment and the crop, but also to the producers and the consumers . One of the approaches that are most important for ensuring humanity’s health and sustaining eco-friendly food production will unquestionably be the introduction and administration of biological and natural protective agents against fungal contamination. [28, 29]. Biocontrol methods include an antibiosis, mycoparasitism, competition, development of resistance in the host plant, and competition . As a result, mycotoxigenic biocontrol by antagonistic agents is regarded as a promising strategy for minimizing and reducing toxin production by these fungal species, and thus reducing the hazards of these toxins on human health when consuming food products . Flavobacterium aurantiacum B-184 had been effectively evaluated for aflatoxins destruction and was effective in eradicating Aspergillus toxins from liquids irreversibly [32, 33]. Lactobacillus plantarum CECT 749 CFS had a strong antifungal impact on maize kernels and maize ears against A. flavus and F. verticillioides, and FB1 and AFB1 levels were extremely dropped . Bacillus velezensis RC 218 and Streptomyces albidoflavus RC 87B successfully reduced Fusarium Head Blight by up to 30%, its severity by up to 25%, and deoxynivalenol accumulation by up to 51% on durum wheat under field conditions [35, 36]. Interestingly, Lysinibacillus macroides, isolated from damaged Waste materials from fruits and vegetables were found to possess an inhibiting effect on food-borne microbes . It has been proven that sugarcane bagasse can be applied for producing extracellular laccase on a large scale using Lysinibacillus macroides at a low cost and with ease . Additionally, Lysinibacillus macroides produces some antimicrobial compounds and chitinase, glucanase, and protease, which are enzymes that have the ability to degrade the cell wall, are well-known to be used as biocontrol techniques. . Lysinibacillus has the ability to down regulate the fungal-hyphal growth . Additionally, by lowering the prevalence of Salmonella, which is well-known for producing significant morbidity in poultry, people, and other animals such as cattle and pigs, as well as in several plants, Lysinibacillus macroides was working as a natural control agent . The most common way for these illnesses to spread is through the intake of contaminated food and drink . It is proven that Lysinibacillus macroides shows a significant concentration reduction ability for chromium (VI), which is known as an accumulated pollutant in lakes in Mexico . Lysinibacillus has been proven to be effective at pest management , improve crop production and clean up heavy metal-contaminated ecosystems . Zinc-tolerant Lysinibacillus spp. are additionally known to boost maize production in Zinc-contaminated soil [43, 44].
All across the world, mycotoxins pose a serious threat to the safety of food. Farmers and consumers suffer financial losses in poor nations such as Egypt when food lots contain high toxin levels. Extremely polluted lots are either totally discarded or sold for an undesirable price. In years and locations with extremely high levels of mycotoxins, leading to significant food loss and waste. In low- and middle-income countries, where mycotoxin limits may not even exist or may not be routinely implemented, human health consequences can be substantially get worse . For the bio-management of toxic fungus and their potential to manufacture mycotoxins, the use of bacterial strains is recommended as a non-chemical, successful, environmentally friendly, and affordable biological control strategy . There are no published papers that demonstrate the potential effect of using L. macroides as a natural deterrent towards toxigenic fungi that are related to corn and its products; therefore, we aim for the first time to investigate the efficacy of L. macroides Bac6 as a natural barrier to toxigenic fungi (A. flavus f10 and F. proliferatum f30) and minimize their mycotoxins to reduce the loss of crops and increase the productivity and quality of the corn and its products, which will reflect on the consumers’ health.
Molecular studies for the most predominant toxigenic fungi isolated from yellow-corn and cornflakes ( A. flavus f10 and F. proliferatum f30) strains
From the previous screening study performed by , A. flavus f10 and F. proliferatum f30 showed the highest productivity of mycotoxins, so they are representative producers of aflatoxins and fumonisin B1, respectively. A. flavus f10 and F. proliferatum f30 isolates were identified at the species level using macro- and microscopic characteristics. Moreover, A. flavus f10 and F. proliferatum f30 isolates were subjected to confirmation of their identification using molecular techniques. The molecular typing resulted in partial 18 S rRNA gene sequences of 582 and 521 bp for A. flavus f10 and F. proliferatum f30, respectively. The partial 18 S rRNA gene sequence of 582 bp of the representative A. flavus f10 isolate had a sequence with 99% similarity to Aspergillus flavus strain PUXX-FS06 (KR296888.1), Aspergillus flavus IFM 42,126 (LC602022.1), Aspergillus flavus strain TN533D12 (MH271095) and Aspergillus flavus strain USMG09 (KF434090.1) available in the GenBank database (S 1 A). The candidate isolate was identified as Aspergillus flavus f10, which belongs to the family Aspergillaceae, order Eurotiales, class Eurotiomycetes. Similarly, the partial 18 S rRNA gene sequence of 521 bp of F. proliferatum f30 isolate had a sequence with 100% similarity to Fusarium proliferatum strain HC01-1 (MT560215.1), Fusarium proliferatum strain TH12-5 (MT560218.1) and Fusarium proliferatum strain CF2 (MN658457.1), which are available in the Genbank database (S 1B). The chosen isolate was identified as Fusarium proliferatum, which belongs to the family Nectriaceae, order Hypocreales, class Sordariomycetes. The phylogenetic trees were built independently from various sequence alignments of 18 S rRNA genetic sequences. The obtained sequences of toxigenic fungal strains A. flavus f10 and F. proliferatum f30 were deposited in the genebank database under accession numbers OQ087136 and OQ087105, respectively.
Quantification of mycotoxins production by A. flavus f10 and F. proliferatum f30 strains using the HPLC technique
High-performance liquid chromatography analysis was employed to figure out the concentrations of aflatoxins and fumonisin B1 produced by A. flavus f10 and F. proliferatum f30 strains, respectively. HPLC analyses continued for 15–25 min of retention time, but all the produced mycotoxins were recovered at the first 6 min of retention time (Fig. 1) and the tested fungal strains showed a high ability to produce mycotoxins. A. flavus f10 strain showed the ability to produce two types of aflatoxins namely, AFB1 and AFG2 in considerable concentrations of 21.239 ppb and 13.593 ppb, respectively. Additionally, F. proliferatum f30 strain showed a high degree of toxigenicity since it produces 9600 ppb of fumonisin B1 (Fig. 2).
The antagonistic effect of bacterial isolates against the toxigenic A. flavus f10 and F. proliferatum f30 strains
Four bacterial isolates demonstrated various antagonistic activities against the two highest mycotoxin-producing fungal strains, A. flavus f10 and F. proliferatum f30. Bacterial isolate Lysinibacillus macrolides Bac6 exhibited the highest efficiency for inhibition of the growth of toxigenic fungi A. flavus f10 and F. proliferatum f30 recording a 13 mm and 8 mm inhibition zone, respectively. Whereas, bacterial isolates Bacillus subtilis, Pseudomonas sp. and Bacillus cereus showed inhibition zone of 8 mm, 4.5 mm and 2 mm, respectively against (A) flavus f10, as well as inhibition zone of 0.9 mm, 4.5 mm and 3 mm, respectively, against F. proliferatum f30 as shown in Table 1; Fig. 3A. Additionally, Lysinibacillus macroides Bac6 due to its highest efficacy among the four isolates as demonstrated in Fig. 3B. The accumulated data from three plates for each fungal strain are expressed as the mean ± SEM in L. macroides Bac6 (open bars), Bacillus subtilis (hatched bars), Pseudomonas sp. (herringbone bars) and Bacillus cereus (stripe bars). *P < 0.001, L. mocrides Bac6 vs. (B) subtilis; #P < 0.001, L. mocrides Bac6 vs. Pseudomonas sp.; and ; +P < 0.001, L. mocrides Bac6 vs. B. cereus.
Reduction of toxigenic fungal growth by Lysinibacillus macroides Bac6 strain
The confirmation of the reduction of toxigenic fungal growth with the most active bacterial strain Lysinibacillus macroides Bac6 was assayed on a solid medium using a dual culture method. The highest antagonistic bacterial strains exhibiting inhibition percentage (%) of fungal growth compared to control fungi were 80 and 62.5% for A. flavus f10, and F. proliferatum f30, respectively (Fig. 4). Interestingly, the bacterial strain L. macrolides Bac6 is the most potent antagonistic bacteria against the tested mycotoxigenic-producing fungal strains A. flavus f10 and F. proliferatum f30. Therefore, it was selected for further studies to illustrate its crucial role in minimizing the toxins produced by toxigenic fungi.
Using morphological, microscopic, biochemical, and molecular methods, L. macroides Bac6 was identified. The partial 16 S rRNA gene sequence resulted in 931 bp; It was compared using a BLAST search (NCBI) with complete sequences that were available in the GenBank database. Sequences obtained with those retrieved from the GenBank database were subjected to Crustal analysis using Mega Align (DNA Star) for the phylogenetic analysis. Sequenced data were inserted in GenBank, and the resulting 16 S rRNA gene sequence showed identity similarity at 100% with L. macroides AzoM1 (MK942418.1), L. macroides JB_2 (MT197307.1) and L. macroides SMV311 (MN538917.1) (S2). Therefore, from the phylogenetic analyses, it can be identified as Lysinibacillus macroides Bac6 strain which, belongs to the family Bacillaceae, order Bacillales and class Firmicutes after which the resulting sequence was entered into the genebank with the corresponding accession number OQ080068.
Evaluation of the effect of Lysinibacillus macroides Bac6 strain on fungal growth of two toxigenic strains using electron microscopic examination
The impact of the highest antagonistic bacterial strain (Lysinibacillus macroides Bac6) on the growth of toxigenic fungi was evaluated using scanning electron microscope (SEM) to evaluate the possible mode of action of Lysinibacillus macroides Bac6 (Figs. 5B and 6B) against Aspergillus flavus f10 (Fig. 5A) and Fusarium proliferatum f30 (Fig. 6A). The resulting SEM graphs showed significant bacterial colonisation and persistent adhesion around the hyphae of toxigenic mould (Fig. 5C & 6C). The colonized hyphae displayed severely malformed and limited proliferation (Figs. 5D and 6D), pitting and damaged appearance of the hyphal cell wall compared with control fungal growth. As well as, it was noticed that, there is strong inhibition of conidia formation.
Scanning electron microscope (SEM) graphing showed pores formation of the toxigenic fungal hyphae when grown with L. macroides Bac6 strain in dual culture. Therefore, it was expected that the bacterial cells had the capability to produce hydrolyzing enzymes that induced fungal cell wall hydrolysis and pore formation by chitinase. The bacterial strain (L. macroides Bac6) was tested for chitinase enzyme production. Control without bacterial colony (Fig. 7A). It showed the capability to produce chitinase, recording the appearance of a clear zone of 12 mm after 24 h (Fig. 7B). Interestingly, it showed 55 mm inhibition zone on a solid chitin medium after 3 days incubation period (Fig. 7C). Moreover, using spectrophotometer, the highest antagonistic bacterial strain showed chitinase productivity of 26.45 U/L and chitinase activity of 0.12 U/mL/min. after 5 days incubation period.
Composition of the metabolites profiling of L. macroides Bac6
The bacterial metabolites analyses were performed using Gas Chromatography/Mass Spectrometry (GC-MS), to assay the bioactive compounds produced by the antagonistic bacterial strain that may be responsible for the inhibition of the growth of toxigenic fungi. The GC-MS analysis revealed that the most common bacterial metabolites were Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro (6.69%), 9,12,15-Octadecatrienoic acid,2,3-bis[(trimethylsilyl)oxy]propyl ester, (Z,Z,Z)- (6.57%), Hexadecenoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester (1.65%), Glycerol 2-acetate 1,3-dipalmitate (1.14), Ethyl iso-allocholate (1.07%). Additionally, 2(3 H)-furanone,5-heptyldihydro- (0.81%) of the total analytes. Whereas, the bacterial metabolites Agaricic acid (0.42%), Digotoxin (0.42%), N, N’-Bis (Carbobenzyloxy)-lysine methyl(ester) (0.33%), Oleic acid (0.29%), 2-Myristynoyl pantetheine (0.26%), 2(3 H)-Furanone, 5-heptyldihydro- (0.18%) and hexadecenoic acid methyl ester (0.15%) were detected in the GC-MS analysis (Fig. 8; Table 2).
Impact of L. macroides Bac6 strain on the formation of mycotoxins
The data from HPLC for each fungal extract with their toxins and the fungal extracts with their toxins treated by L. macroides are expressed as the concentration (ppb) in Table 3. *P < 0.0001, A. flavus f10 (AFB1) + L. macroides vs. A. flavus f10 (AFB1); # P < 0.0001, A. flavus f10 (AFG2) + L. macroides vs. A. flavus f10 (AFG2); and ; +P < 0.0001, F. proliferatum f30 (FB1) + L. macroides vs. F. proliferatum f30 (FB1). The resulted data revealed that the active bacterial cells had a significant reduction for aflatoxin B1 and aflatoxin G2 which were produced by Aspergillus flavus f10 recording 99.25% and 99% inhibition, respectively. In addition, the Lysinibacillus macroides showed strong inhibition of fumonisin B1 (90% inhibition) produced by Fusarium proliferatum f30 (Fig. 9).
Multiple lines of evidence in vitro were employed to show that fumonisin and aflatoxins accumulations reflect the mycotoxigenic risk when the maize is infected with toxic Fusarium section Liseola and Aspergillus section Flavi . Similarly, Izzati et al.  shown that F. proliferatum was found on nearly all maize farms. Other lines of evidence on corn were investigated and demonstrated the high occurrence of A. flavus [49, 50], as well as Fusarium species especially Fusarium graminearum and Fusarium proliferatum were very common in all maize-cultivated areas . A. flavus, is the most risky worldwide species that is easily able to colonize corn . Contamination with mycotoxins and their producers in maize and products based on corn is mainly caused by aflatoxins, fumonisins, and their primary fungal producers . Regarding this F. proliferatum, F. verticillioides produced FB1 and FB2, with FB1 serving as the primary analogue (representing 75% of the total fumonisins). These isolates are thought to yield more than 500 g/g of FB1 . Our results revealed that the potent toxigenic fungal strains A. flavus f10 and F. proliferatum f30 which are isolated from corn samples refered that strict quarantined and proper storage practices recommended to be used with imported goods in order to minimize infection with toxigenic moulds and steer clear of hazards to animal and human health.
The results were obtained from the HPLC method was employed to verify and quantify the aflatoxins (AFB1 – AFG2), and fumonisin B1 generated by the highest concentrations of active aflatoxins and strains that produce fumonisins, respectively, namely A. flavus f10, and F. proliferatum f30. Our data revealed that, Aspergillus flavus f10 that was invading cornflakes could produce aflatoxin B1 21.239 ppb and aflatoxin G2 13.493 ppb and F. proliferatum f30 produce 9600 ppb of fumonisin B1. Compared to another previous study that stated that A. flavus isolates could produce aflatoxin B1 in the range of 0.09–50.68 ppb and aflatoxin B2 0.33–9.2 ppb isolated from Malaysian Sweet Corn . In other research, levels of Aflatoxins secreted by A. parasiticus and A. flavus isolated from silage of corn ranged 2–45 ppb and 2–100 ppb, respectively . In addition, a study performed on corn and popcorn samples monitored that A. flavus var. columnaris and A. flavus isolates could produce aflatoxins with range of 1–8 ppb . F. proliferatum and Fusarium verticillioides were investigated to accumulate FB1 and FB2 in maize kernels, which could result in a harmful or economical crop loss . It is critical to find strategies to safeguard the maize crop from fumonisin building up. In the study performed , the production of fumonisin B1 is 6.22 ± 0.89 ppb. Similarly, Mohamed et al., published that FB1 was found in all silos samples and corn markets with a range of 13.69-175.54 ppb .
Our current study focused on the potentiality of using a biological control agent to reduce the growth of A. flavus f10 and F. proliferatum and their toxins. Our results revealed that L. macrolides Bac6 was the best one among all the tested bacterial isolates, due to its highest efficacy as demonstrated in Fig. 3B, which interpreted the data presented in Table 1. Although, there is no significance among the effects of the four tested bacterial isolates against both tested mycotoxigenic fungi. We demonstrated that the most antagonistic is L. macrolides Bac6, as a result of its safety as demonstrated in several research papers using it as a biological agent against pathogens [41, 60, 61]. Therefore, the Lysinibacillus macrolides Bac6 was the best choice to be used as a biological control agent. The proportion of fungal growth inhibited by Lysinibacillus macroides Bac6 as compared to control fungi was 80 and 62.5% for A. flavus f10 and F. proliferatum f30, respectively. As mentioned before in a previous study Bacillus megaterium BM344-1 has the ability to reduce the toxigenic fungi growth. The inhibition ratios (%) of P. verrucosum, (A) flavus, and F. verticillioides outperformed control fungi by 66.7, 29.4, and 18.2%, respectively . Zeidan et al.  The most sensitive to yeast VOCs was found to be Penicillium followed by Aspergillus, whereas Fusarium was found to be the least sensitive. A key factor in fungus resistance is the nature of the fungal cell wall, which is affected by stressors in the microenvironment. The antagonistic effects of Bacillus volatiles as (B) subtilis, Bacillus amyloliquefaciens, Bacillus cereus, and B. megaterium towards toxigenic and phytopathogenic Penicillium and Aspergillus spp. have been illustrated [62, 64, 65].
We evaluated the underlying mechanism of the antagonistic effect of L. macrolides via electron microscopic examination, and we detected that the effect may be attributed to the production of chitinase enzyme. The tested Lysinibacillus macroides Bac6 strain in this study showed high potential production of exo-chitinase enzyme. According to the study that illustrated that Lysinibacillus spp. also produces some of these types of antimicrobial compounds, which include the development of cell wall-degrading enzymes as a biocontrol approach . Cell wall-degrading enzyme-producer Lysinibacillus can inhibit fungal hyphal development .
Bacterial metabolites analysis performed by gas chromatography − mass spectrometry (GC − MS) revealed the presence of several compounds including Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro. This compound was the highest prevalent in the L. macroides Bac6 strain metabolites. Interestingly, it was reported as a bactericidal compound which also, isolated from Bacillus tequilensis MSI45 against multidrug-resistant pathogenic Staphylococcus aureus . Moreover, it has been shown that Microcystis aeruginosa is susceptible to algicides, which is a compound was detected in Bacillus Lzh-5 . Furthermore, GC-MS analysis of L. macroides Bac6 strain metabolites revealed that the presence of these bioactive compounds namely, Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro 2(3 H)-furanone,5-heptyldihydro, 9,12,15-octadecatrienoicacid,2,3 bis[trimethylsily] propylester, (Z, Z, Z)-, agaricic acid, digotoxin, ethyl iso-allocholate, oleic acid and hexadecanoic acid methyl ester. All compounds are well-known microbial biomolecules that have strong antagonistic activities against toxigenic as well as phytopathogenic fungi    ; [62, 72] . While 9,12,15-octadecatrienoic acid, 2,3-bis[(trimethylsilyl)oxy]propyl ester, (Z,Z,Z)- (6.57%) was the second molecule found with a large peak area and has been linked to antimicrobial activity , hexadecenoic acid has recently been linked to antifungal activity . In addition, the 1-(hydroxymethyl)-1,2-ethanediyl ester (1.65%) had antifungal action , as did Glycerol 2-acetate 1,3-dipalmitate (1.14) , and Ethyl iso-allocholate (1.07%). Furthermore, 2(3 H)-furanone,5-heptyldihydro- (0.81% of total analytes) demonstrated antifungal activity. [77,78,79].
Aflatoxin and fumonisin-producing fungi share the same habitat as other microorganisms that can influence toxin production [80, 81]. Our results revealed that Lysinibacillus macroides Bac6 produced an inhibitory effect on A. flavus f10 and F. proliferatum f30 growth and their toxin production. Surprisingly, the Lysinibacillus macroides Bac6 metabolites inhibited AFB1 and AFG2 which produced by Aspergillus flavus f10, and FB1 that produced by Fusarium proliferatum f30, with 99.25, 99, and 90%, respectively. Saleh et al.  reported that aflatoxins (AFB1, AFG1, and AFG2), ochratoxin A, and FB1 production on artificial medium were completely inhibited after exposure to (A) flavus, P. verrucosum, and F. verticillioides to Bacillus megaterium BM344-1 VOCs. Moreover, Pereira et al.  study demonstrated that (B) amyloliquefaciens were significantly reducing FB1 and FB2 levels. Furthermore, it has been documented that volatiles released by B. megaterium KU143 and B. licheniformis 350-2 on un-hulled rice and maize ears prevent the production of aflatoxins by A. flavus , , respectively.
In conclusion, mycotoxins pose a serious threat to the safety of food, especially in low- and middle-income nations, incurring financial and human health consequences. Bacterial strains can be used as an eco-friendly, non-chemical, and low-cost biological control method. For the first time, we investigated the efficacy of using L. macroides Bac6 as a biological control agent against the most prevalent mycotoxigenic fungi invading corn and corn-based products through the potential impact of bacterial metabolites that significantly reduced AFB1 and AFG2 produced by A. flavus f10 and strongly inhibited fumonisin B1 produced by F. proliferatum f30. To better summarize, the methods and the resulted data of our study were demonstrated in (Fig. 10).
Materials and methods
Aspergillus flavus f10 and Fusarium proliferatum f30 strains were obtained from our previous work, in which they were isolated from yellow-corn and cornflakes, respectively . According to our published paper, prior to usage, the tested fungal isolates were cultivated and cultured on potato dextrose agar (PDA) at 28 °C. [45, 84]. Both strains were identified by molecular characteristics based on 18 S rRNA.
Analyses of mycotoxins production by HPLC technique
The fungal inoculum preparation was carried out using freshly grown fungal cultures, seven days old, at 28 °C. A 1 cm disc was removed from the borders of the fungal growth using a cork-porer and utilized as fungal inoculum according to . As an enhanced medium for mycotoxins formation, liquid yeast extract sucrose medium (YES) was employed containing (g/L); yeast extract, 1; peptone, 10; K2HPO4,1; MgSO4.7H2O, 0.5; sucrose, 30; KCl, 0.5; FeSO4.H2O, 0.01; and NaNO3, 2.0, and the pH was adjusted to 6.5. In 100 ml capacity conical flasks, 25 ml of the medium (YES) was pipetted, autoclaved, inoculated then incubated at 28 °C for two weeks and three weeks under static conditions for aflatoxins and fumonisin B1 production, respectively. For aflatoxins extraction, the homogenization occurred in the fungal broth. Using a fast speeds blender, mix 25 millilitres of chloroform solvent for a period of five minutes. The aqueous phase was subsequently filtered using Whatman filter paper No.1 after the organic phase was separated from it using a separating funnel, then dehydrated over a solution of anhydrous sodium sulphate, and dried to near dryness on a rotating evaporator. Each extract’s residuals were then reconstituted in two millilitres of chloroform then preserved in tiny brown bottles until the detection process. In contrast, the acetonitrile technique (5 ml/g of culture media) reported by  has been selected for fumonisin B1.
The mycotoxins were subjected to a quantitative estimation using high-performance liquid chromatography (HPLC) according to . In the case of aflatoxins determination, the mobile phase comprised a combination of water, acetonitrile, and methanol (55:30:15 v/v/v), whereas, for fumonisin B1, two mobile phases have been used, namely solvent A (water: acetonitrile: acetic acid (59:4:1 v/v/v)) and solvent B acetonitrile: acetic acid (99:1 v/v). Aflatoxins and fumonisin B1 were detected using a detector that detects fluorescence with wavelengths of excitation of 295 and 335 nm, respectively, and emitting wavelengths of 330 and 440 nanometers, respectively [88,89,90]. The studies were carried out using HPLC system (Agilent Technologies Series 1200, G1321A FLD with column Zorbax, the Eclipse programme + C18) located at Assuit University’s Analytical Chemistry Unit.
Investigating the antagonistic activities of bacterial isolates against the toxigenic fungi
The antagonistic activities of the recovered bacterial isolates on the highest toxin-producing fungal strains (A. flavus F10 and F. proliferatum F30) were tested in vitro. The bacterial isolates recovered from soil were streaked with a line method at the center on PDA plates with a toxigenic strain of fungus inoculum, along with a 7-day incubation period at 28 °C for the cultures. Each treatment included three replications. The inhibitory zone was identified at the end of of the incubation time [91, 92].
Lysinibacillus macroides Bac6, the highest antagonistic bacteria, were selected for assay of the reduction of toxigenic fungal growth. Well-grown bacterial colonies which were incubated at 35 º C for 48 h were picked and further purified by streaking [93, 94]. The isolates were maintained on nutrient agar (NA) slants and stored at 4 ºC. The bacterial cultures were identified based on morphology (shape, Gram stain, spore formation and motility). L. macroides Bac6 has been identified by molecular characteristics based on 16 S rRNA .
Determination of the reduced level in toxic fungal growth by antagonistic bacteria Lysinibacillus macroides Bac6 strain
The decrease in the development of toxic fungi (A. flavus f10 and F. proliferatum f30) by Lysinibacillus macroides Bac6 strain was conducted according to the method described in [89, 92]. The interaction between the tested bacterial strains and toxigenic strains was assessed using PDA. 1 mL of bacterial suspension (107 CFU/mL) was mixed with 15 mL of molten PDA medium before solidification and poured into Petri dishes (90 mm diameter). After solidification of the medium, mycelial discs with 10 mm diameter were cut from the fungal active growing margins of 7 days old cultures and placed at the center of the agar surface. The control plates were inoculated with tested fungi but without bacteria following the same procedure. All plates were incubated at 28 °C for 72 h. The experiment was carried out in triplicates. Fungal growth inhibition was calculated by measuring the diameter of the fungal colonies using the following equation:
Where C is the diameter of the fungal colonies in control plates and.
T is the diameter of the fungal colonies in treated plates.
The alterations of fungal growth which was caused by antagonistic bacterial strains were shown and confirmed by scanning electron microscopy (SEM).
Evaluation of the impact of antagonistic bacterial strain on the Toxigenic fungal growth was evaluated by using scanning electron microscopy (SEM)
To evaluate the antagonistic capability of L. macroides Bac6 strain against the toxigenic fungi, the fungal mycelia of dual culture were examined using SEM. In brief, A 10-mm disc of mould growth boundary was removed and fixed for 2 days in 5% cool buffer glutaraldehyde. The samples were then rinsed three times (30 min each) with sodium cacodylate buffer before being post-fixed in 1% osmium tetroxide for two hours. The samples were then rinsed three times in the same buffer (30 min each) and dehydrated using an escalating ethanol gradient (30%, 50%, 70%, and 90%) for two hours and 100% ethanol for two days, before being treated with amyl acetate for a further two days. Following that, the samples were dried in a critical point drainer with liquid carbon dioxide before being attached to a metallic block with silver paint [91, 96].
Assay of chitinase activity of L. macroides Bac6 strain
Preparation of colloidal chitin
According to Hussin and Ab Majid, a colloidal of chitin was manufactured. Briefly, five grams powder of chitin were combined in a beaker with conc. Hydrochloric acid (~ 10 M HCl) 60 ml. Via a rod of glass, the prepared mixture was continuously stirred for a period of 5 min then 1 min gently stirring at a time interval 5 min for 1 h. In a 2 L conical flask, the chitin-HCl combination was subsequently processed using 2 L of cold distilled water and then it was kept under static conditions for 12 h at 4 ºC. The precipitation was gathered using crossing two distinct phases of until the colloidal chitin reached a pH of 7, the filter cloth was continuously rinsed with normal water. The produced colloid of chitin was squeezed between the filter paper (to reduce any residual moisture) and subsequently kept at 4 °C until it was used again .
Chitinase production of L. macroides Bac6 strain on solid medium
The initial screening was carried out by adding 10 L of a 24-hour-old culture of L. macroides Bac6 (107 CFU/mL) to the middle of NA plates that contained 1% colloidal chitin, then incubating those plates at 30 °C for 5 days. Congo red (1%) was used to demonstrate the activity of the enzyme for 30 s, after which the dye was fully removed with a solution of sodium chloride (30 g L-1) until a zone of transparency established as a result of chitin hydrolysis. The test was performed in three replicates. .
Chitinase enzyme activity of L. macroides Bac6 strain on liquid medium
A 250 mL Elementary flask with 100 mL NA and 1% colloidal chitin was filled with 10 mL of a 24-hour-old cultures of bacteria. The incubation was conducted on a rotating shaking device for five days at 30 °C and 180 rpm. The supernatant without cells was obtained by centrifuging the culture broths at a speed of 120 rpm for 10 min at 4 °C.
Applying the dinitrosalicylic acid (DNS) method, the efficacy of chitinase was assessed by looking for sugars that reduced after the enzymatic process. The test was performed with the specified methodology. . After being produced in a 50 mM phosphate buffer (pH 7.0) with just one millilitre of crude extract of enzyme (cell-free culture supernatant), the substrate mixture consisted of 1% colloidal chitin. The resulting mixture had been warmed in a water bath at 37 °C for a period of one hour. By adding 3 mL of DNS reagent and boiling it in water for 20 min, the process was halted. The mixture was centrifuged at 5000 rpm for five minutes after cooling. The wavelength of the absorbance has been identified at 540 nm using the Model T60 UV-VIS spectrophotometer. The amount of chitinase activity (U/mL) was calculated as the amount of enzyme that, under normal test conditions, generated 1 mol of dinitrosalicylic acid per minute . The experiment was carried out in triplicate.
Assessment of L. macroides Bac6 metabolites using GC-MS analyses
Tryptic soy broth (TSB) was used to culture the bacterial strain. The following ingredients were present in tryptic soy broth in grammes per litre: 17.0 for pancreatic digest of casein; 3.0 for papaic digest of soybean meal; sodium chloride, 5.0; dextrose, 2.5; and dibasic potassium phosphate, 2.5; pH after sterilisation was 7.30.2. Following a 24-hour incubation period at 30 °C, the bacterial culture was centrifuged for 5 min at 6000 g before being filtered through a sterile 0.22 micron membrane filter. With petroleum ether (1:1 v/v), the bacterial metabolites were separated from the resultant supernatant.
To quantify the bacterial metabolites, the GC/MS (Model: DPC-Direct Probe Controller (DPC20451), Thermo Scientific,USA; at the Chemistry Department, college of Science at Assiut University was used. A capillary column TG-5MS with dimensions of 30 m, 0.25 mm i.d., and 1 m film thicknesses was used for the separation of fatty acid ester compounds. The temperature of the oven was initially maintained at 80 °C for 5 min, and then increased at a ramp rate of 10 °C/min to 150 °C for 10 min, 200 °C at a ramping speed of 10 °C/min (held for 10 min), and 250 °C at a ramp rate of 5 °C/min (held for 13 min). Helium was employed as a carrying gas at a flow rate of 0.5 mL/min, and the split flow was 10 mL/min. [100, 101].
Effect of Lysinibacillus macroides Bac6 on mycotoxins production
Minimization of toxins production by toxigenic fungi by using active bacterial cells was tested by growing the fungus for 10 days in coculture with the bacterial living cells (107 CFU/ml), from 24 h old culture [90, 98]. As previously mentioned, high-performance liquid chromatography (HPLC) was used to extract the poisons and quantify their amounts.
GraphPad Prism software version 5 was used to conduct statistical analysis on normally distributed data, which are represented as means standard errors of the means (SEM). One-way ANOVA was used to analyse the significant differences between the three groups, and Tukey’s posttest followed.
All the generated data and the accession number for all the tested microorganisms are available and included in the manuscript.
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Mahmoud, A.E., Kilany, A.A. & Hassan, E.A. Antifungal activity of Lysinibacillus macroides against toxigenic Aspergillus flavus and Fusarium proliferatum and analysis of its mycotoxin minimization potential. BMC Microbiol 23, 269 (2023). https://doi.org/10.1186/s12866-023-03007-4