Vanillin and its derivatives,potential promising antifungal agents,inhibit Aspergillus flavus spores via destroying the integrity of cell membrane rather than cell wall

Qian Li,Xiaoman Zhu

Henan Key Laboratory of Cereal and Oil Food Safety Inspection and Control,College of Food Science and Engineering,Henan University of Technology,Zhengzhou 450001,China

ABSTRACT To study the structure-antifungal activity of vanillin against Aspergillus flavus(A.flavus),the susceptibilities of A.flavus to vanillin and its isomers(o-vanillin,2-hydroxy-4-methoxybenzaldehyde(HMB),2-hydroxy-5-methoxybenzaldehyde)and the possible antifungal mechanisms have been investigated.All the four volatile aldehydes could inhibit the germination of spores,and the minimum inhibitory concentrations(MICs)of them were in this order:vanillin(200 μg/mL),o-vanillin(100 μg/mL),2-hydroxy-5-methoxybenzaldehyde(100 μg/mL),HMB(70 μg/mL).The minimum fungicidal concentrations(MFCs)of them were in this order:vanillin(240 μg/mL),o-vanillin(160 μg/mL),HMB(140 μg/mL),2-hydroxy-5-methoxybenzaldehyde(140 μg/mL).Spore size was arrested at 0 h with the treatment of the four volatile aldehydes.Effects of the four volatile aldehydes on the cell wall and cell membrane integrity of A.flavus were observed by calcofluor white(CW)staining and propidium iodide(PI)staining.The results showed that HMB exerted the strongest antifungal and fungicidal effects on the growth of A.flavus.The four volatile aldehydes had little influence on cell wall integrity after 3-hour treatment,however,they could strongly damage the cell membrane integrity.All the four volatile aldehydes could effectively prevent the growth of A.flavus on peanut seeds.The antifungal mechanisms of the four volatile aldehydes provide theoretical foundations for their development of new antifungal agents.

Keywords:Aspergillus flavus Vanillin o-Vanillin HMB 2-Hydroxy-5-methoxybenzaldehyde Antifungal mechanism

1.Introduction

Aspergillus flavus(A.flavus)is a common saprophytic fungus,which is prone to infect peanut,corn,and other food crops.A.flavus pollution has become one of the main causes of economic losses of grain and other crops and seriously damages the health of people and animals because of aspergillosis[1].Aflatoxins,especially AFB1produced by A.flavus,have potential carcinogenicity on human and animal liver[2,3].Therefore,it is urgent to control the contamination of A.flavus with safe and effective methods.

Vanillin(4-hydroxy-3-methylbenzaldehyde)is a Generally Recognized As Safe(GRAS)reagent[4],and widely exists in vanilla beans,the fruits of orchid(Vanilla planifolia,Vanilla pompona,and Vanilla tahitensis)[5].It has been reported that vanillin could inhibit Cryptococcus neoformans(C.neoformans),Botrytis cinerea,and Candida albicans[6-8].As vanillin derivatives,the inhibition effects of o-vanillin(3-methoxysalicylaldehyde),HMB(2-hydroxy-4-methoxy benzaldehyde)and 2-hydroxy-5-methoxybenzaldehyde have also been approached[9-13].o-Vanillin mainly exists in vanillin and other plant extracts,such as Pinus koraiensis fruit,Helichrysum genus,oak(Quercus)[11,14,15].o-Vanillin has already been widely applied in food,beverage,pharmacy,and cosmetics[16].Prior studies showed that o-vanillin performed good antifungal effects on A.flavus[9]and C.neoformans[10].In addition,o-vanillin could also inhibit a variety of conditional pathogens such as Aspergillus spp.,Penicillium spp.,etc.[11].As another GRAS reagent[17],HMB is a natural product from the roots of Selaginella indica[18].Notably,HMB has been reported to have activity in inhibiting the growth of A.flavus and the biosynthesis of aflatoxins[12,19].Previous studies also reported the antifungal effect of 2-hydroxy-5-methoxybenzaldehyde on many fungi,such as Aspergillus spp.,and Penicillium expansum(P.expansum)[9,13].

Vanillin,o-vanillin,HMB and 2-hydroxy-5-methoxy benzaldehyde are isomers(C8H8O3).Although either of their antimicrobial activities has been discussed previously,to the best of our knowledge,whether this structural difference(e.g.,the relative positions of functional groups)is correlated with antifungal activity is still unclear.Therefore,we studied the sensitivity of A.flavus spores to the four volatile aldehydes and their primary antifungal mechanisms,including(1)minimum inhibitory concentration(MIC)and minimum fungicidal concentration(MFC);(2)spore size;(3)cell wall integrity;(4)cell membrane integrity;(5)growth of A.flavus on peanut seeds.This study will provide the theoretical basis for the development of potential and effective antifungal agents.

2.Methods

2.1.Fungal strain and the culture condition

Aspergillus flavus strain(CGMCC 3.6304)was purchased from China General Microbiological Culture Collection Center(CGMCC)and cultured with Sabouraud's dextrose agar(SDA)(4%glucose,1%peptone and 2%agar)at(28±2)°C.One-week-old fungal cultures were prepared to use in the following experiments.Saline was added on the SDA plate and A.flavus was gently scraped with a scraper.Spores were collected after filtration.Thereafter,the A.flavus spore suspension was diluted to a concentration of 5×105spores/mL using a hemocytometer.

2.2.Antifungal activity test

Susceptibilities of A.flavus to the volatiles were tested as the method described previously[20].In detail,spores(150 μL,5×105spores/mL)were added into a 96-well plate.The final concentration of vanillin(98%,CAS:121-33-5),o-vanillin(99%,CAS:148-53-8),HMB(98%,CAS:673-22-3)and 2-hydroxy-5-methoxybenzaldehyde(98%,CAS:672-13-9),which were purchased from Aladdin Biochemical Reagent Co.(Shanghai,China),were 0,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220 μg/mL,respectively.The plate was incubated at(28±2)°C for 72 h,the spore growth was observed under an inverted optical microscope(BM-37XBC,Shanghai BM Optical Instruments Manufacture Company Ltd.,China)at 24,48,and 72 h.When the length of the germination tube was longer than the diameter of the spore,the spore was considered as“germinated”.MIC represented as the lowest concentration where no A.flavus spore germinated at 72 h.MFC was measured as the method of Leite et al.[21]with slight modifications.For each group with a concentration higher than MIC,2 μL spores with the medium mixture were transferred to a new well containing 98 μL SDB(4%glucose,1%peptone).The plate was incubated for another 48 h at(28±2)°C.The MFC represented as the concentration where no spore germinated after 48 h.

2.3.Spore size determination

Fresh spores of A.flavus(150 μL,5×105spores/mL)were treated with the four volatile aldehydes at their respective MIC in a 96-well plate and incubated at(28±2)°C for 3 h.Then,the growth of spores was observed under an inverted optical microscope,and the mean values of spore area in the pictures(about 80 spores per picture)were calculated with Image J(1.52v,National Institutes of Health,USA).Image type:8-bit,Threshold:23&255.

2.4.Cell wall integrity test

Spores of A.flavus(5 mL,5×105spores/mL)were treated with the four volatile aldehydes at their respective MIC and incubated at(28±2)°C for 3 h.Then,the spore suspension was dropped onto the slide and stained with a drop of calcofluor white(CW)and 10 μL 10%KOH[22].Then,the samples were visualized with a fluorescence microscope(DFC 7000 T,Leica,Germany).

2.5.Cell membrane integrity test

The membrane integrity of A.flavus spores was determined according to the previous method[23].Spores of A.flavus(5 mL,5×105spores/mL)were treated with the four volatile aldehydes at their respective MICs for 3 h.Then,the spores were collected and stained with propidium iodide(PI)(1 μL/mL)for 20 min in darkness.Then,spore suspensions were washed twice with PBS and centrifuged at 8,000 g for 15 min.The centrifuged spores were ready for visualization under the fluorescence microscope and the fluorescence intensity values were measured by Images J.Image type:8-bit,Threshold:23&255.

2.6.In vitro antifungal effect of the four volatile aldehydes on peanut seeds

Fresh peanut seeds were washed with sterile water and dried briefly,and then exposed to ultraviolet light for 20 min under laminar flow.Seven peanut seeds(total weight(3.27±0.19)g)were evenly placed in one petri dish(diameter 6 cm)as one group(nine groups in total).Five tubes containing A.flavus spores(100 μL,5×105spores/mL)were prepared,then the four volatile aldehydes were added to reach the final concentrations at their respective 1/2 MIC and MIC.The group without any volatile aldehyde was set as the control.Each peanut seed was coated with 10 μL mixture containing spore suspension and one volatile aldehyde.All peanut seeds were incubated in the incubator at(28±2)°C for 72 h,and the growth of A.flavus on the peanut seeds was observed every 24 h.

2.7.Statistical analysis

All data were presented as mean±SD and all experiments were performed in triplicate.The significant differences were determined by One-way ANOVA using Duncan's Multiple Range Test(P＜0.05).The data analysis was performed by SPSS(20.0,IBM,USA).

3.Results and discussions

3.1.The inhibition effects,MIC and MFC of the four volatile aldehydes on A.flavus spores

The inhibition effects of vanillin,o-vanillin,HMB,and 2-hydroxy-5-methoxybenzaldehyde on spore germination of A.flavus were shown in Table 1.The MIC and MFC of the four volatile aldehydes against A.flavus were shown in Table 2.HMB exerted the strongest antifungal effect among the four volatile aldehydes,and the best fungicidal effect occurred on HMB and 2-hydroxy-5-methoxybenzaldehyde.

Table 1 Inhibitory effect of the four volatile aldehydes on spore germination.

Table 2 MIC and MFC of the four volatile aldehydes on A.flavus.

Vanillin,o-vanillin,HMB,and 2-hydroxy-5-methoxy benzaldehyde share the same molecular formula and are aromatic isomers with each other(Fig.1).First of all,the poorer inhibitory and fungicidal effects of vanillin than the others may be attributed to the relative positions of hydroxyl group and aldehyde group on the benzene ring.As addressed previously,the-OH bond of ortho hydroxyl group and aldehyde group is more stretched than that of para-position,and the-OH bond is easy to break,so the reduction potential of ortho position is low,the quenching effect of free radicals is strong,hence,the antioxidant capacity is high[24].The antioxidant capacity may be positively correlated with their antifungal and fungicidal effects.For vanillin,the hydroxyl group is at the para position of the aldehyde group,while,for the other 3 volatile aldehydes,all of their hydroxyl groups are in an ortho position.Furthermore,for o-vanillin,HMB,and 2-hydroxy-5-methoxybenzaldehyde,their antifungal and fungicidal effects are different.It may be attributed to the relative position of hydroxyl group and methoxy group in the benzene ring.For o-vanillin and 2-hydroxy-5-methoxy benzaldehyde,they are in ortho and para position and for HMB,they are in meta position.In this study,in combination with both antifungal and fungicidal effects,the best inhibitory effect occurred on HMB as well.

Fig.1.Chemical formula of vanillin,o-vanillin,HMB,and 2-hydroxy-5-methoxybenzaldehyde.

3.2.Effects of the four volatile aldehydes on spore size

We have measured the size changes of spores induced by the four aldehydes.The morphology of spores in the culture medium was shown in Fig.2,and the spore areas were shown in Fig.3.The area of fresh A.flavus spores was about 22 μm2for all groups at 0 h(Fig.3).No significant difference was found among the five groups(P＞0.05).Generally,during growth,the volume of spores should first inflate before the protrusion of the tip of hyphae[25].At 3 h,in the control group,the spore area increased significantly(P＜0.05)by about 2 times in comparison with that of 0 h.After treated with vanillin,o-vanillin,HMB,and 2-hydroxy-5-methoxybenzaldehyde at their MIC for 3 h,the area of A.flavus spores was(23.85±1.77)μm2,(22.5±1.29)μm2,(22.75±2.81)μm2,and(22.42±3.55)μm2,respectively,which were similar with that of 0 h,but remarkably lower than that of the control for 3 h(49.14±4.56)μm2(P＜0.05).Latgéet al.[25]reported that during the germination of A.flavus spores,when the expanded conidia reached twice the size of dormant conidia,they started radial growth to perform a mycelial morphology(A.flavus spores germinated after 7.5 h in our lab).And during the expansion and enlargement,conidia undergo DNA replication,glycerol accumulation,and proteolytic degradation[25].This result indicated that the addition of the four volatile aldehydes could significantly inhibit the normal growth of A.flavus spores and probably maintain their state at the starting point.Pictures of the spore germination process were shown in Fig.4.

Fig.2.Morphology of A.flavus spores treated with the four volatile aldehydes at their respective MIC.Scale bar:40 μm.

Fig.3.Spore area of A.flavus treated with the four volatile aldehydes at their respective MIC.

Fig.4.Growth of A.flavus within 3 h under an inverted optical microscope.Scale bar:40 μm.

3.3.Effects of the four volatile aldehydes on cell wall integrity

Besides the internal alternation of spores,whether the surface of spores was changed due to the four volatile aldehydes is still not demonstrated.CW was used to determine the integrity of cell walls as it could combine with one main component-chitin in cell walls.As shown in Fig.5,the spores of A.flavus had no obvious difference between the control group(Fig.5a)and the four volatile aldehydes treated groups(Fig.5b-e).Bright and homogenous blue fluorescence was observed for all groups.The fluorescence intensities for either two groups were not significantly different(Table 3).Therefore,the four volatile aldehydes probably had little effect on the content of chitin.However,in our previous research,HMB could damage the integrity of A.flavus mycelia cell walls at 24 h[12].Ouyang et al.[26]reported that the blue fluorescence in the cinnamaldehyde treated group for 6 h,9 h was significantly weaker than that in the control group,suggesting that cinnamaldehyde significantly reduced chitin content of Penicillium digitatum(P.digitatum).Maybe the treatment time is still not enough to act on cell walls.Next,we will study the effect of different treatment times on spore cell walls.

Fig.5.Fluorescence emitted by A.flavus spores not treated(a),and treated with vanillin(b),o-vanillin(c),HMB(d),and 2-hydroxy-5-methoxybenzaldehyde(e)at their respective MIC after staining with CW.Scale bar:10 μm.

Table 3 Fluorescence intensity of CW and PI.

3.4.Effects of the four volatile aldehydes on cell membrane integrity

To investigate the cell membrane integrity change of spores induced by the four volatile aldehydes,PI staining was carried out.In Fig.6,the spores of untreated A.flavus showed no fluorescence at all(Fig.6a),however,for all treated groups,spores emitted bright red fluorescence(Fig.6b-e),indicating that cell membrane in the control group rather than the treated groups was intact.Quantification of fluorescence intensity was shown in Table 3,where the fluorescence intensity of the control group was set as 1.Distinctly,fluorescence intensities for all treated groups were significantly higher than that of the control.In detail,the fluorescence intensity treated with vanillin,o-vanillin,HMB,and 2-hydroxy-5-methoxybenzaldehyde at their respective MIC was about 7,8,6,and 6 times higher than that of the control,which indicated that they could destroy the integrity of cell membranes.Besides,o-vanillin had the most serious damage to cell membranes,followed by vanillin,HMB,and 2-hydroxy-5-methoxybenzaldehyde.At 3 h,the cell membrane was destroyed first,but not the cell wall,which indicated that these aldehydes had a strong affinity with the cell membrane in a short time.The increase of PI fluorescence intensity seems a common phenomenon for almost all antifungal agents,however,the extent of this increase can be varied.This is consistent with the damage of cinnamaldehyde on the membrane integrity of A.flavus spores by PI stain[27],and perillaldehyde disruption of membrane integrity of Aspergillus niger at MIC[28].Dou et al.[23]reported that the fluorescence values in P.digitatum spores treated with 0.25 and 2 μL/mL of octanal for 120 min were 1.81 times and 2 times higher than that of the control,respectively.Citronellal could inhibit the germination of P.digitatum spores by damaging the membrane integrity for 72 h[26].In combination with the result of CW,it could be deduced that in such short treatment time(3 h)for all the four aldehydes determined in this study,cell membrane might be the first and the more susceptible cellular structure,in comparison with the cell wall.

Fig.6.Fluorescence emitted by A.flavus spores not treated(a),and treated with vanillin(b),o-vanillin(c),HMB(d),and 2-hydroxy-5-methoxybenzaldehyde(e)at their respective MIC after staining with PI.Scale bar:10 μm.

3.5.Growth of A.flavus on peanut seeds

After the in vitro study,the effects of the four volatile aldehydes on A.flavus were also evaluated on peanut seeds.This investigation could confirm their antifungal effects in practice.The growth of A.flavus on peanut seeds was shown in Fig.7,the mycelia of the control group began to grow at 24 h,and no mycelium was observed for all the four volatile aldehydes treated groups at their respective 1/2 MIC at 24 h.At 72 h,mycelia grew vigorously in the control group,and all the four aldehydes treated groups at their respective 1/2 MIC exerted little inhibition on A.flavus contamination.However,all groups treated with the four aldehydes at their respective MIC showed no A.flavus infection at all.The observed contamination rate was shown in Table 4.Therefore,in addition to the susceptibility result in a 96-well plate,the four volatile aldehydes have good inhibitory effects on A.flavus both in vitro and in vivo.Similar studies also showed strong inhibition of some natural small compounds against A.flavus on crop seeds.Tang et al.[29]reported that essential oil compounds(citral and geraniol)in combination with modified atmosphere packaging could be served as a potential favorable preservation method for controlling Aspergillus spp.Ma et al.[30]reported that(E)-2-hexenal could significantly suppress A.flavus contamination in stored peanut seeds with fumigation.Further studies are required to study the content of aflatoxins(e.g.AFB1)on oil crop seeds after treatment with the four volatile aldehydes.

Fig.7.Growth of A.flavus on peanut seeds treated with the four volatile aldehydes at their respective 1/2 MIC and MIC.

4.Conclusion

In conclusion,this study revealed the different inhibitory effects of vanillin and its three isomers(o-vanillin,HMB,and 2-hydroxy-5-methoxybenzaldehyde)on A.flavus spores,which are attributed to the relative positions of functional groups,including hydroxyl,aldehyde,andmethoxy groups.The antifungal effects of them in a short treatment time were attributed to the disruption of the integrity of cell membranes rather than cell walls.All the four aldehydes exerted promising antifungal effects on A.flavus infected peanut seeds.These results indicated that vanillin,o-vanillin,HMB,and 2-hydroxy-5-methoxybenzaldehyde could be explored as potential antifungal agents.In the future,we will further explore their mechanism in depth and provide a practical way for the research and development of new antifungal agents,industrialization and practical application of A.flavus control.

Table 4 Contamination rate of A.flavus treated with the four volatile aldehydes at their respective 1/2 MIC and MIC on peanut seeds.

Author Contributions

Qian Li:Conceptualization,project administration,supervision;Xiaoman Zhu:Data curation,formal analysis,investigation,writing-original draft.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

We thank for the financial support of the Doctor Research Fund of Henan University of Technology(2019BS019),the Natural Science Research Projects of Education Department of Henan Province(21A550005),and the Innovative Funds plan of Henan University of Technology(2020ZKCJ17).